Reviews in Fluorescence Annual volumes 2006 (Reviews in Fluorescence)

Reviews in Fluorescence 2006

Chris D. Geddes Joseph R. Lakowicz (Eds.)

Reviews in Fluorescence 2006

Springer

Chris D. Geddes Institute of Fluorescence University of Maryland Biotechnology Institute Baltimore, MD 21201 USA [email protected]

Joseph R. Lakowicz Center for Fluorescence Spectroscopy University of Maryland Baltimore, MD 21201 USA [email protected]

ISBN-10: 0-387-29342-6 ISBN-13: 978-0387-29342-4 Printed on acid-free paper. © 2006 Springer Science-hBusiness Media, Inc. All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science-f-Business Media, Inc., 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed in the United States of America. 9 8 7 6 5 4 3 2 1 springer.com

(TB/MVY)

PREFACE This is the third volume in the Reviews in Fluorescence series. To date, two volumes have been both published and well received by the scientific community. Several book reviews have also favorably described the series as an "excellent compilation of material which is well balanced from authors in both the US and Europe". Of particular mention we note the recent book review in JACS by Gary Baker, Los Alamos. In this 3rd volume we continue the tradition of publishing leading edge and timely articles from authors around the world. We hope you find this volume as useful as past volumes, which promises to be just as diverse with regard to content. Finally, in closing, we would like to thank Dr Kadir Asian for the typesetting of the entire volume and our counterparts at Springer, New York, for its timely publication.

Professor Chris D. Geddes Professor Joseph R. Lakowicz August 20*^ 2005. Baltimore, Maryland, USA.

CONTRIBUTORS AND BIOGRAPHIES Ousama M. A 'Amur. Boston University, Department of Biomedical Engineering, Boston, MA Ousama M. A'Amar is Senior Research Associate at the Biomedical Engineering Department of Boston University, MA since 2001. He received his BS in Electronics Engineering in 1989. He received his MS (1993) and PhD (1997) in automatic control and digital signal processing from the National Polytechnic Institute of Lorraine, France. His MS and PhD research work focused on optical biomedical signals; mainly Autofluorescence and InducedFluorescence for cancer diagnosis and treatment. In 1996, he received the European Diploma in Medical Lasers from the University Nancy I, France and won the Young Researcher Prize of the French Society of Medical Lasers (SFLM). He worked as: Assistant Professor at the department of Biomedical Engineering, Amman University, Jordan (1998/1999-2002/2003); Postdoctoral Research Associate at the Bioscience Division of Los Alamos National Laboratory, NM (1999-2001). He works in the field of biomedical optics and his research activities focus on optical biomedical signals and optical spectroscopy for cancer diagnosis and Photodynamic Therapy. Amit Agrawal. Emory University and Georgia Institute of Technology, Atlanta, GA Amit Agrawal is a graduate student in the third year in the Nie research group in biomedical engineering department at Georgia institute of technology and Emory University. He has a Master's degree (5 yr) in chemistry from Indian institute of technology Kanpur. His research includes ultrasensitive biological detection inside living cells and developing material and nanoparticles for use in novel cancer diagnostics schemes. His work involves nanoparticle functionalization, delivery and targeting of nanoparticles and design of novel spectroscopic and imaging instrument set ups. He is the author of several conference papers and peer reviewed journal articles. Onur AlptUrk. Department of Chemistry, Louisiana State University, Baton Rouge, LA Christopher D. Anderson. Department of Surgery, Vanderbilt University Medical Center, Nashville, TN

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Renato J. Aguilera. Department of Biological Sciences, University of Texas at El Paso., El Paso, TX Dr. Renato Aguilera obtained his Ph.D. from UC Berkeley in 1987 and was a professor at the University of California at Los Angeles from 1989 to 2002. Dr. Aguilera subsequently joined the biology department at the University of Texas at El Paso where he serves as the Director of the Biology Graduate Program and the RISE Research Scholars Program. He is also a member of the Board of Scientific Counselors of the National Institutes of Environmental Health and Safety (NIEHS). His work on the transcriptional regulation of the lymphocyte-specific Recombination Activating Genes (RAG) has been highly recognized and he has made significant contributions to others fields as well. Dr. Aguilera has many publications in high impact journals and holds a patent on an enzyme (DNase II) that is essential for engulfment-mediated DNA degradation. Most recently, Dr. Aguilera group has developed fluorescencebased assays for the rapid identification of cytotoxic and antimicrobial compounds generated by combinatorial chemistry. Egidijus Auksorius. Imperial College London, U.K. Richard K, P. Benninger. Imperial College London, U.K. Axel Bergmann. Becker&Hickl GmbH, Nahmitzer Damm, Berlin, Germany. Pieter de Beule. Imperial College London, U.K. Irving J, Bigio. Boston University, Department of Biomedical Engineering and Electrical and Computer Engineering, Boston, MA Franz Stanzel is head of the Bronchology Unit at the Asklepios Fachkliniken Munich-Gauting, Center for Respiratory Diseases and Thoracic Surgery, one of the biggest lung hospitals in Germany. He is a clinician of pulmonary medicine with a special interest on bronchology and the secretary of the Endoscopy Section of the German Society of Pneumology. Dr. Stanzel works since several years on interventional diagnostic and therapeutic procedures with the focus of lung cancer. The development of an autofluorescence bronchoscopy system together with Karl HauBinger braught early lung cancer into the center of his interest. He is an internationally accepted expert on autofluorescence bronchoscopy. Dr. Stanzel published a lot of scientific articles, papers, review articles and book chapters on bronchoscopy, interventional bronchology and fluorescent bronchoscopy Rebecca A. Bozytn. University of Maryland School of Medicine, Baltimore, MD

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John D, Brennan. Department of Chemistry, McMaster University, Hamilton, Canada John D. Brennan is an Associate Professor in the Department of Chemistry at McMaster University and holds the Canada Research Chair in Bioanalytical Chemistry. He has B.Sc, M.Sc. and Ph.D. degrees in analytical chemistry (fluorescence-based biosensors) from the University of Toronto and postdoctoral experience at the National Research Council of Canada in protein biophysics (time-resolved fluorescence). His current research primarily involves the entrapment of proteins within silica materials for the development of bioanalytical assays and devices. As part of this research, fluorescence methods are widely employed to examine the behaviour of proteins entrapped in silica. Dr Brennan has published over 80 scientific articles various aspects of protein immobilization and applications of fluorescence spectroscopy. Denis Boudreau. Department of chemistry and Centre d'optique, photonique et laser, Universite Laval, Quebec, Canada Denis Boudreau is Full Professor in the Department of Chemistry, and member of the Centre d'optique, photonique et laser (COPL) research center at Universite Laval, Quebec City, Canada. He has a B.Sc. from Universite de Sherbrooke, Canada, and a Ph.D. in analytical chemistry (plasma mass spectrometry) from the Universite de Montreal, Canada. He is the Editor of Spectrochimica Acta Electronica. Dr Boudreau has published over 40 scientific articles, papers, review articles and book chapters on various aspects of chemical trace analysis. Jan Willem Borst. MicroSpectroscopy Centre, Wageningen University, Dreijenlaan Wageningen, The Netherlands Ru-xiu Cai. Department of Chemistry, Wuhan University, Wuhan, China. Cai Ruxiu is a professor. Supervisor of PhD, Director of the Group of Molecular spectroscopy (includes fluorescence, stopped-flow fluorescence. Catalytic kinetic fluorescence) in Analytical Science center at Wuhan University, China. She has a M.S from Wuhan University. She was visiting professor at Lawrence Berkeley National Laboratory, Energy and Environment Division, U.S.A in 1997, and worked at University of Arizona. Tucson in 1990, 1992. She is the committee of Editor of the Journal of Analytical Science. Professor Cai get continually National science foundation founding for six times.and has published 150 Scientific articles, papers, review articles and book chapters on the principles and applications of fluorescence spectroscopy, UVVisible spectroscopy and kinetic Analysis. Nils Calander, Physics Department, Chalmers University of Technology, Goteborg, Sweden.

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Ravi S, Chart Department of Surgery, Vanderbilt University Medical Center, Nashville, TN Ravi S. Chari is Associate Professor of Surgery and Cancer Biology, and Chief, Division of Hepatobiliary Surgery and Liver Transplantation at Vanderbilt University in Nashville, TN. He received his MD from the University of Saskatchewan, and his surgical training at Duke University. He is secretaryelect for the Society of University Surgeons and a member of the Scientific Committee of the International Hepato-Pancreato-Biliary Association (IHPB A) and was Program Chair of the 2004 IHPBA World Congress. He is a member of the Editorial Boards of the Journal of Surgical Research, HPB, World Journal of Surgery and Surgery. Dr Chari has published 100 scientific articles, papers, review articles and book chapters on liver and biliary surgery. Herbert C. Cheung. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL. Herbert C. Cheung is Professor of Biochemistry, University of Alabama at Birmingham School of Medicine. He holds joint appointments as Adjunct Professor in the Department of Physics and Senior Scientist in the Comprehensive Cancer Center. He received a master degree in physical chemistry from Cornell University, and a bachelor's degree in chemistry and a Ph. D. in physical chemistry and physics from Rutgers University. Following a period of industrial research in polymer physics, he was a senior fellow at the Cardiovascular Research Institute, University of California San Francisco, where he began a career in fluorescence spectroscopy and in the biophysics of muscle contraction. His current work is focused on use of FRET in both equilibrium and kinetic studies to study conformational switching in molecular motors and cardiac myofilaments. Robert M, Clegg. Physics Department, University of Illinois ChampaignUrbana, Illinois. Wen-Ji Dong. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL. /aci#i/fl Z)'sowzfl. Department of Biological Sciences, Tata Institute of Fundamental Research Road, Mumbai, India. RoryR. Duncan. Centre for Integrative Physiology, University of Edinburgh Medical School, Edinburgh, UK. Christopher Dunsby. Imperial College London, U.K. Guy Duportail. Faculte' de Pharmacie, Universite, Louis Pasteur de Strasbourg, Illkirch, France.

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Daniels. Elson. Imperial College London, U.K. Jorge O. Escobedo. Department of Chemistry, Louisiana State University, Baton Rouge, LA. Carol A, Fierke. University of Michigan, Ann Arbor, MI PaulM. W, French. Imperial College London, U.K. Xiaohu Gao. Emory University and Georgia Institute of Technology, Atlanta, GA Xiaohu Gao is currently a postdoctoral fellow in the group of Dr. Shuming Nie. He earned his BS degree in chemistry from Nankai University (China), and his PhD degree in bioanalytical chemistry and nanotechnology from Indiana University - Bloomington. In the last 5 years, he published more than 20 papers, filed 4 patent applications, and delivered 15 invited talks at major conferences and academic institutions. Neil Galletly. Imperial College London, U.K. Anne Gibaud. Institut Curie, Paris, France. Jean-Frangois Gravel. Department of chemistry and Centre d'optique, photonique et laser, Universite Laval, Quebec, Canada Jean-Fran9ois Gravel is a Research Associate in the Department of Chemistry at Universite Laval, Quebec City, Canada. He has a B.Sc. in chemistry and a Ph.D. in analytical chemistry (laser spectrochemical analysis) from the Universite Laval, Canada. Dr Gravel has authored or co-authored over 15 scientific articles, papers, review articles and book chapters on laser spectrochemical analysis. Laszlo Hegyi. Imperial College London, U.K. Mark A, Hink. MicroSpectroscopy Centre, Wageningen University, Dreijenlaan Wageningen, The Netherlands Arie van Hoek. MicroSpectroscopy Centre, Wageningen University, Dreijenlaan Wageningen, The Netherlands Richard G.H. Immink. Laboratory for Biophysics, Wageningen University, Dreijenlaan, Wageningen, The Netherlands Carey K. Johnson. Department of Chemistry, University of Kansas, Lawrence, KS.

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Kyu Kwang Kim. Department of Chemistry, Louisiana State University, Baton Rouge, LA Mamata Kombrabail. Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India. G. Krishnamoorthy. Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India. G. Krishnamoorthy did his Masters in Science from University of Madras, India in 1974 and Ph.D. in Physical Biochemistry from the Tata Insititute of Fundamental Research, Mumbai, India in 1980. Subsequently he had postdoctoral research training at the Biochemistry department, Cornell University during 1981-84. Following his return to India, he joined the Faculty at the Tata Institute of Fundamental Research, Mumbai as Research Associate. At present he holds the position of Professor in the department of chemical sciences. His research interest covers application of time domain fluorescence spectroscopy to a variety of problems in macromolecular systems of biological and artificial origins. His current focus lies on the elucidation of site-specific dynamics in proteins, nucleic acids, DNA-protein complexes, cell membranes and cell interior with emphasis on correlation of dynamics and function. Peter M. P. Lanigan. Imperial College London, U.K. John Lever. Imperial College London, U.K. Wei-Chiang Lin. Department of Neuro-Engineering, Miami Children's Hospital, Miami, FL Zhi-hong Liu. Department of Chemistry, Wuhan University, Wuhan, China. Bernard Malfoy. Institut Curie, Paris, France C Mazzuca. Department of Chemical Sciences and Technologies, University of Roma Tor Vergata, Rome, Italy James McGinty. Imperial College London, U.K. YvesMely. Faculte' de Pharmacie, Universite, Louis Pasteur de Strasbourg, Illkirch, France. P.M. Krishna Mohan. Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India. Jessica Montoya. Department of Biological Sciences, University of Texas at El Paso., El Paso, TX

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Ian Munro. Imperial College London, U.K. Nabanita Nag. Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India. Mark A. A, Neil. Imperial College London, U.K. Isabella Nougalli-Tonaco. MicroSpectroscopy Centre, Wageningen University, Dreijenlaan Wageningen, The Netherlands Shunting Nie. Emory University and Georgia Institute of Technology, Atlanta, GA Shuming Nie is a Professor of Biomedical Engineering, Chemistry, Hematology, and Oncology, and also directs the program in cancer nanotechnology and bioengineering in the Winship Cancer Institute. He is the author of more than 80 peer-reviewed papers, the inventor of 12 patents, and the speaker of more than 250 invited talks and keynote lectures. After serving on the chemistry faculty at Indiana University for 8 years, he and his group moved to the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory in 2002. His research interest is primarily in the areas of biomolecular engineering and nanotechnology, with a focus on bioconjugated nanoparticles for cancer molecular imaging, molecular profiling, pharmacogenomics, and targeted therapy. Professor Nie has received many awards and honors including the Rank Prize (London, UK), the Georgia Distinguished Cancer Scholar Award, the Beckman Young Investigator Award, the National Collegiate Inventors Award, and the Distinguished Overseas Scholar Award. Professor Nie received his BS degree from Nankai University (China) in 1983, earned his MS and PhD degrees from Northwestern University (1984-1990), and did postdoctoral research both at Georgia Tech and Stanford (1991-1993). John P, Nolan. La Jolla Bioengineering Institute, La Jolla, CA John P. Nolan is a Senior Scientist and Principal Investigator at the La Jolla Bioengineering Institute, La Jolla, California. He has B.S. degrees from the University of Illinois, Urbana-Champaign in biology and chemistry and a Ph.D. in biochemistry from the Pennsylvania State University. He did post-doctoral work at Penn State and Los Alamos National Laboratory, where he was also a Technical Staff Member and Director of the NIH National Flow Cytometry Resource. Dr. Nolan's research interests are in the area of development and application of technology for the quantitative molecular analysis of biological systems.

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Jamie K. Pero. Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC. Jamie K. Pero received an Honors B.S. degree in Chemistry in 2002 from the University of Utah and is currently a Ph.D. Candidate in Analytical Chemistry in the Department of Chemistry at the University of North Carolina at Chapel Hill. She has received several honors, has participated in a wide variety of community service and humanitarian projects, and has thus far published two scientific articles. B. Pispisa. Department of Chemical Sciences and Technologies, University of Roma Tor Vergata, Rome, Italy Basilio Pispisa is Full Professor of Physical Chemistry at the University of Roma Tor Vergata (Rome, Italy). He has a doctorate degree from the University of Pisa, and spent a few years in USA, at the Polymer Research Institute of the Polytechnic Institute of Brooklyn (New York). He is fellow of the American Peptide Society, of the Biophysical Society, of the Protein Society and of the European Peptide Society. E. Shane Price. Department of Chemistry, University of Kansas, Lawrence, KS. Todd P. Primm. Department of Biological Sciences, Sam Houston State University, Hunsville, TX. T. Ramreddy. Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India. B,J, Rao. Department of Biological Sciences, Tata Institute of Fundamental Research Road, Mumbai, India Jose Requejo-Isidro. Imperial College London, U.K. Gang Ruan. Emory University and Georgia Institute of Technology, Atlanta, GA Gang Ruan is a postdoctoral research fellow in the joint Department of Biomedical Engineering of Georgia Institute of Technology (School of Engineering) and Emory University (School of Medicine). He received his PhD from the National University of Singapore. He has published 9 scientific journal articles. Dr Ruan's current research interest is biomolecular engineering and bionanotechnology. Oleksandr Rusin. Department of Chemistry, Louisiana State University, Baton Rouge, LA

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Ann Sandison. Imperial College London, U.K. Brian D. Slaughter. Department of Chemistry, University of Kansas, Lawrence, KS. Andrew M. Smith. Emory University and Georgia Institute of Technology, Atlanta, GA Andrew Smith is a third-year graduate student in the biomedical engineering department at Georgia Institute of Technology and Emory University. He obtained his BS degree from Georgia Institute of Technology. His research interest is in the areas of biomolecular engineering and nanotechnology, with a particular focus on the development of near-infrared-emitting quantum dots for molecular profiling and imaging applications. He is the author of seven publications in the last two years. Steven A. Soper. Department of Chemistry, Louisiana State University, Baton Rouge, LA Steven A. Soper, Ph.D. is currently a professor of Chemistry and Mechanical Engineering at Louisiana State University (LSU) in Baton Rouge, LA. Steve received his Ph.D. from the University of Kansas in 1989 and then, was a postdoctoral fellow at Los Alamos National Laboratory where he was involved in developing fluorescence single molecule detection for high throughput DNA sequencing. He joined the faculty at LSU in 1991 and has been working on new fluorescence detection strategies for the analysis of DNA. Pat Soutter. Imperial College London, U.K. Gordon W. Stamp. Imperial College London, U.K. L. Stella. Department of Chemical Sciences and Technologies, University of Roma Tor Vergata, Rome, Italy Robert M. Strongin. Department of Chemistry, Louisiana State University, Baton Rouge, LA Clifford Talbot. Imperial College London, U.K. Nancy L. Thompson. Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC. Nancy L. Thompson received a Ph.D. in Physics from the University of Michigan at Ann Arbor in 1982 and was then a Damon Runyon - Walter Winchell Postdoctoral Fellow in the Department of Chemistry at Stanford University. She has been a member of the Faculty of the Department of Chemistry at the University of North Carolina at Chapel Hill since 1985 where

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she currently holds the position of Professor of Chemistry. She has received several honors including a National Science Foundation Presidential Young Investigator Award, the Margaret Oakley Dayhoff Award from the Biophysical Society, a Dreyfus Teacher-Scholar Award, and the Hettleman Prize from the University of North Carolina at Chapel Hill. She has served on a variety of Editorial Boards and published numerous scientific articles in the fields of membrane biophysics and fluorescence microscopy. Richard B, Thompson. University of Maryland School of Medicine, Baltimore, MD Dr. Thompson was bom in Ohio and raised north of Chicago, Illinois. He received a B.A. in Biology from Northwestern University; while there, he began biochemical studies with E. Margoliash. He received the Ph.D. in Biochemistry from the University of Illinois in Urbana-Champaign working under the direction of Thomas O. Baldwin. He trained as a postdoctoral fellow in the laboratory of Joseph Lakowicz at the University of Maryland at Baltimore before moving to the U.S. Naval Research Laboratory as a National Research Council Associate. At the Naval Research Laboratory he began work on fluorescence-based biosensors under Paul Schoen and subsequently became a Supervisory Research Chemist under the direction of Frances Ligler; he received a Navy Special Act Award for activity related to Operation Desert Storm. He joined the faculty of the University of Maryland School of Medicine in the Department of Biochemistry and Molecular Biology where he is now Associate Professor. He serves on the Editorial Boards of the Journal of Fluorescence and the Journal of Biomedical Optics, as well as panels for the National Research Council, National Institutes of Health, National Science Foundation, and other agencies. Dina Tleugahulova. Department of Chemistry, McMaster University, Hamilton, Canada Dina Tleugahulova is Postdoctoral Fellow in the Department of Chemistry at McMaster University, Hamilton, Canada. She has a B.Sc. and M.Sc in physical chemistry from Moscow State University, Russia and a Ph.D. in biology from the University of Havana, Cuba. Dr. Tleugahulova has published scientific articles on protein separation, pharmaceutical analysis and principles and applications of fluorescence anisotropy. Khuong Truong. IMSTAR. Paris France Jay R. Unruh. Department of Chemistry, University of Kansas, Lawrence, KS. Andrew Wallace. Imperial College London, U.K. Armando Varela-Ramirez. Department of Biological Sciences, University of Texas at El Paso., El Paso, TX

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M. VenanzL Department of Chemical Sciences and Technologies, University of Roma Tor Vergata, Rome, Italy. Li Zhu. Department of Chemistry, Louisiana State University, Baton Rouge, LA. Li Zhu came to LSU in the fall of 2000 as a Ph.D. student from Nankai University in Tianjin, China. Li's dissertation work focused on developing near-IR time-resolved fluorescence detection for multiplexing applications in genomics. She received her Ph.D. in the fall of 2005 and is working at GE Global Research Center in Niskayuna, NY. Antonie J. W.G. Visser. MicroSpectroscopy Centre, Wageningen University, Dreijenlaan Wageningen, The Netherlands Nicolas Vogt. Institut Curie, Paris, France Jun Wang. Department of Chemistry, Wuhan University, Wuhan, China. Weihua Wang. Department of Chemistry, Louisiana State University, Baton Rouge, LA Xiangyang Xu, Department of Chemistry, Louisiana State University, Baton Rouge, LA

CONTENTS PREFACE CONTRIBUTORS

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1. THE HISTORY OF FRET Robert M. Clegg 1.1. INTRODUCTION 1 1.2. PRELUDE TO THE HISTORICAL BACKGROUND 3 1.2.1. The End of the Dark Ages: the Pre-Field Era 3 1.2.2. Middle Ages: Experiments That Eventually Changed Our World View 5 1.2.3. Renaissance: Enter the Theory of Electrodynamics and Fields 6 1.2.4. The Beginning of the Modem Age: The Field Surrounding an Oscillating Charge 7 1.3. FIELDS, SPECTROSCOPY AND QUANTUM MECHANICS 9 1.3.1. Fields 9 1.3.2. Quantum Mechanics and Spectroscopy 9 1.4. THE FIRST EXPERIMENTAL OBSERVATION OF ENERGY TRANSFER AT A DISTANCE - SENSITIZED LUMINESCENCE IN VAPORS 11 1.4.1. Sensitized Fluorescence 11 1.4.2. Spectroscopic and Collisional Cross-Sections in Vapors 12 1.5. THE FIRST QUANTUM MECHANICAL THEORY OF ENERGY TRANSFER 12 1.5.1. A Few of the Pre-Quantum Theories that calculated the Spectroscopic Cross-Sections of Atomic Vapors 13 1.5.2. Some Details of the Kallmann and London Paper 14 1.6. LONDON FORCES (VAN DER WAALS) AND DEB YE AND KEESOM INTERACTIONS 17 1.6.1. London Interactions: Induced-Dipole-Induced-Dipole 17 1.6.2. Keesom and Debye Interactions: Dipole-Dipole and Dipole Induced-Dipole 19 1.7. FRET BETWEEN ORGANIC CHROMOPHORES IN CONDENSED SYSTEMS 20

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CONTENTS 1.7.1. Experimental Observations of Energy Transfer in Solution .. 20 1.7.2. The Theories of J. PerrinandF. Perrin 21 1.7.3. A Derivation of the Perrins' Estimated Distances for Two Electron Oscillators in Exact Resonance 23 1.7.4. The Contribution of W. Arnold and J.R. Oppenheimer to FRET in Photosynthesis 30 1.8. FORSTER'S SEMINAL CONTRIBUTION: THE MODERN, PRACTICAL DEPICTION OF FRET (FORSTER RESONANCE ENERGY TRANSFER) 37 1.9. MATURATION OF FRET 41 1.10. EPILOGUE 42 1.11. ACKNOWLEDGMENTS 42 1.12. REFERENCES 43

2. TRICHOGIN TOPOLOGY AND ACTIVITY IN MODEL MEMBRANES AS DETERMINED BY FLUORESCENCE SPECTROSCOPY

47

B. Pispisa, L. Stella, C. Mazzuca, and M. Venanzi 2.1. INTRODUCTION 2.2. THE PROPERTIES OF THE FLUORESCENT ANALOGS 2.3. AGGREGATION IN WATER 2.4. WATER-MEMBRANE PARTITION AND AGGREGATION 2.5. BIOACTIVITY: MECHANISM OF MEMBRANE PERTURBATION 2.6. POSITION OF TRICHOGIN INTO THE MEMBRANE: TRANSLOCATION, DEPTH-DEPENDENT QUENCHING, AND DISTRIBUTION ANALYSIS 2.6.1. Peptide Translocation 2.6.2. Depth-Dependent Quenching and Peptide Distribution Analysis 2.7. PEPTIDES ORIENTATION INSIDE THE MEMBRANE 2.8. CONCLUDING REMARKS 2.9. ACKNOWLEDGMENTS 2.10. REFERENCES

3. THEORY OF METAL-FLUOROPHORE INTERACTIONS

47 48 50 53 60

61 61 64 65 67 68 68

71

Nils Calander 3.1. INTRODUCTION 3.2. SURFACE PLASMON RESONANCE 3.2.1. Plasma Oscillations 3.2.2. Surface Plasmon Resonances

71 72 72 73

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3.3. THEORY OF SURFACE PLASMON RESONANCE AT PLANAR STRUCTURES 74 3.3.1. Basic Theory 75 3.3.2. Simulations 78 3.3.3. Conclusions of surface plasmon resonance at planar structures 84 3.4. THEORY OF SURFACE-PLASMON RESONANCE OPTICAL FIELD ENHANCEMENT AT PROLATE SPHEROIDS 85 3.4.1. The Field Enhancement at Spheroids 88 3.4.2. Conclusion of Surface-Plasmon Resonance Optical-Field Enhancement at Prolate Spheroids 94 3.4.3. Solving the Maxwell's Equations in Prolate Spheroidal Coordinates 94 3.5. OPTICAL TRAPPING OF SINGLE FLUORESCENT MOLECULES BY SURFACE PLASMON RESONANCE 98 3.6. REFERENCES 104

4. CURRENT DEVELOPMENT IN THE DETERMINATION OF INTRACELLULAR NADH LEVEL

107

Zhi-hong Liu, Ru-xiu Cai, and Jun Wang 4.1. INTRODUCTION TO NADH 4.2. SIGNIFICANCE OF DETERMINING INTRACELLULAR NADH LEVEL 4.3. DETERMINATION OF INTRACELLULAR NADH LEVEL 4.3.1. Enzymatic Assays 4.3.2. Fluorometric Methods 4.3.3. Micro-Fluorescence Photometry 4.3.4. Laser Scanning Confocal Microphotographics 4.3.5. Two-Photon Excitation Micrographics 4.4. REGULATION OF INTRACELLULAR NADH LEVEL 4.4.1. Effect of Vitamins on Intracellular NADH Level 4.4.2. The Time Course of Intracellular NADH in Yeast Apoptosis 4.5. REFERENCES

107 108 110 Ill 112 116 117 117 118 118 119 123

5. PREDICTION OF THERMAL TISSUE DAMAGE USING FLUORESCENCE SPECTROSCOPY

125

Christopher D. Anderson, Wei-Chiang Lin, and Ravi S. Chari 5.1. INTRODUCTION 125 5.2. FLUORESCENCE SPECTROSCOPY TO DETECT THERMAL TISSUE DAMAGE 126 5.3. MEASUREMENTS OF FLUORESCENCE SPECTRA IN VIVO... 127

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CONTENTS 5.4. SPECTRAL CORRELATES TO THERMAL DAMAGE 130 5.5. FLUORESCENCE SPECTRA CORRELATE WITH HISTOLOGIC TISSUE DAMAGE 132 5.7. DETECTION OF ABSOLUTE CELL DEATH 133 5.8. CONCLUSIONS AND FUTURE DIRECTIONS 136 5.9. REFERENCES 137

6. DETECTION OF BIOLOGICAL THIOLS

139

Jorge O. Escobedo, Oleksandr Rusin, Weihua Wang, Onur Alpturk, Kyu Kwang Kim, Xiangyang Xu, Robert M. Strongin 6.1. INTRODUCTION 139 6.2. HOMOCYSTEINE METABOLISM 141 6.3. NEW PERSPECTIVES ON HOMOCYSTEINE'S ROLE IN DISEASE 143 6.4. OVERVIEW OF KNOWN METHODS FOR BIOLOGICAL THIOL DETECTION 144 6.5. DETECTION OF CYSTEINE AND HOMOCYSTEINE 147 6.6. HIGHLY SELECTIVE DETECTION OF CYSTEINE AND SITESPECIFIC PEPTIDE LABELING 149 6.7. HIGHLY SELECTIVE DETECTION OF HOMOCYSTEINE 151 6.8. AUTOMATED POST-COLUMN DETECTION OF CYSTEINE AND HOMOCYSTEINE 156 6.9. BIOTHIOL DETECTION BASED ON SIMPLE ARRAYS 156 6.10. CONCLUSIONS 158 6.11. ACKNOWLEDGMENT 158 6.12. REFERENCES 158

7. FLUORESCENT BRONCHOSCOPY

163

Franz Stanzel 7.1. INTRODUCTION 7.2. PHENOMENON AND TECHNIQUES 7.2.1. Drug-Induced Fluorescence 7.2.2. Autofluorescence 7.3. INVESTIGATIONS AND DATA 7.3.1. Drug-Induced Fluorescence 7.3.2. Autofluorescence Bronchoscopy 7.4. DISCUSSION 7.5 CONCLUSIONS 7.6. REFERENCES

163 165 165 165 167 167 168 169 174 175

CONTENTS

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8. QUANTUM DOTS AS FLUORESCENT LABELS FOR MOLECULAR AND CELLULAR IMAGING

181

Gang Ruan, Amit Agrawal, Andrew M. Smith, Xiaohu Gao, and Shuming Nie 8.1. INTRODUCTION 8.2. PROBE DEVELOPMENT 8.3. NOVEL OPTICAL PROPERTIES 8.4. DELIVERY OF QD PROBES INTO CELLS 8.5. APPLICATIONS IN INTRACELLULAR IMAGING 8.5.1 Cellular staining 8.5.2. Intracellular studies 8.6. ACKNOWLEDGMENT 8.7. REFERENCES

181 182 185 187 189 189 189 191 191

9. MOLECULAR ANALYSIS USING MICROPARTICLE-BASED FLOW CYTOMETRY

195

John P. Nolan 9.1. INTRODUCTION 9.2. OPTICAL MEASUREMENTS USING FLOW CYTOMETRY 9.3. SOLID PHASE ASSAYS USING MICROPARTICLES 9.4. DETECTION AND SENSOR APPLICATIONS 9.5. MOLECULAR INTERACTIONS AND FUNCTION 9.5.1. Enzyme-Substrate Interactions 9.5.2. Ligand-Receptor Interactions 9.5.3. Protein Immobilization 9.6. GENETIC ANALYSIS 9.7. SUMMARY AND FUTURE DIRECTIONS 9.8. ACKNOWLEDGEMENT 9.9. REFERENCES

195 196 197 200 202 202 205 207 208 210 210 211

10. TOTAL INTERNAL REFLECTION-FLUORESCENCE CORRELATION SPECTROSCOPY

215

Nancy L. Thompson, and Jamie K. Pero 10.1. ABSTRACT 10.2. INTRODUCTION 10.3. CONCEPTUAL BASIS AND EXPERIMENTAL DESIGN 10.4. THEORETICAL MODELS FOR DATA ANALYSIS 10.5. APPLICATIONS 10.6. FUTURE DIRECTIONS 10.7. ACKNOWLEDGEMENTS 10.8. REFERENCES

215 216 217 221 225 229 233 233

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11. FLUORESCENCE PROBES OF PROTEIN DYNAMICS AND CONFORMATIONS IN FREELY DIFFUSING MOLECULES 239 Carey K. Johnson, Brian D. Slaughter, Jay R. Unruh, and E. Shane Price 11.1. INTRODUCTION 239 11.2. FLUORESCENCE CORRELATION SPECTROSCOPY TO PROBE PROTEIN DYNAMICS 240 11.2.1. PCS Measurements of Intramolecular Dynamics 241 11.2.2. PCS Cross-Correlation Measurements 242 11.2.3. PCS of Calmodulin 243 11.3. BURST-INTEGRATED SINGLE-MOLECULE ANALYSIS 246 11.4. TIME-REVOLVED FLUORESCENCE MEASUREMENTS 248 11.4.1. CaM Conformational Substates by Ensemble Time-Resolved Fluorescence Measurements 248 11.4.2. Associated-Anisotropy Analysis to Assess the Influence of Dye-Protein Interactions 252 11.5. CONCLUSION 255 11.6. ACKNOWLEDGEMENT 255 11.7. REFERENCES 256

12. BIOLOGICAL APPLICATION OF FLIM BY TCSPC

261

Axel Bergmann, and Rory R. Duncan 12.1. INTRODUCTION 261 12.2. PHYSICAL BACKGROUND OF FLUORESCENCE LIFETIME IMAGING 262 12.2.1 Fluorescence Lifetime as a Separation Parameter 262 12.2.2. The Fluorescence Lifetime as an Indicator of the Local Environment 263 12.2.3 Fluorescence Resonance Energy Transfer 264 12.3. THE LASER SCANNING MICROSCOPE 265 12.3.1 Suppressionofout-of-focus light 265 12.3.2 Scan Rates 265 12.3.3 Two-Photon Excitation with Direct Detection 266 12.4. REQUIREMENTS FOR FLUORESCENCE LIFETIME IMAGING IN SCANNING MICROSCOPES 267 12.4.1 Efficiency 267 12.4.2 Principle of Time-Correlated Single Photon Counting 268 12.4.3 Imaging by Multi-Dimensional TCSPC 269

CONTENTS

12.5. BIOLOGICAL APPLICATION OF FLIM 12.5.1 Biological FLIM Data Acquisition 12.5.2 TCSPC-FLIM 12.5.3 FLIM Data Analysis and FRET Calculations 12.5.4 FLIM to Measure FRET In Cells 12.5.5 Dual channel FLIM 12.6. FUTURE PERSPECTIVES 12.7. REFERENCES

XXV

270 270 270 271 271 273 273 274

13. TIME-RESOLVED FLUORESCENCE ANISOTROPY APPLIED TO SILICA SOL-GEL GROWTH AND SURFACE MODIFICATION. 277 Dina Tleugabulova, and John D. Brennan 13.1. INTRODUCTION 13.1.1 Characterization of Silica Growth and Modification 13.2. SURVEY OF TRFA THEORY 13.2.1 What Is Measured in TRFA? 13.2.2 TRFA Data Analysis 13.2.3 Rotational Diffusion of Probes in Non-Interacting Environments 13.2.4 Restricted Dynamics in the Presence of Probe-Host Interactions 13.3. PARTICLE GROWTH STUDIES 13.3.1 Particle Growth in DGS Sols 13.3.2 Particle Growth in SS Sols 13.4. MONITORING SILICA SURFACE MODIFICATION 13.4.1 Background 13.4.2 Ludox 13.4.3 Monitoring Adsorption by TRFA 13.5. CONCLUSIONS AND OUTLOOK 13.6. ACKNOWLEDGEMENTS 13.7. REFERENCES

277 278 280 281 282 284 287 290 290 295 296 296 297 297 304 305 305

14. DYNAMICS OF DNA AND PROTEIN-DNA COMPLEXES VIEWED THROUGH TIME-DOMAIN FLUORESCENCE 311 Nabanita Nag, T. Ramreddy, Mamata Kombrabail, P.M. Krishna Mohan, Jacinta D'souza, B.J. Rao, Guy Duportail, Yves Mely, and G. Krishnamoorthy 14.1. INTRODUCTION 311 14.2. FLUORESCENCE PROBES FOR DNA DYNAMICS 313 14.3. PROBING DNA DYNAMICS WITH NON-SPECIFIC PROBES 316

xxvi

CONTENTS 14.3.1. DNA condensation 316 14.3.2. YOYO-1 as an indicator of DNA condensation 317 14.3.3. Structure and dynamics of condensed DNA 318 14.3.4. DNA condensation by the nucleocapsid protein probed by YOYO-1 Fluorescence 323 14.3.5. DNA Dynamics in Chromosomes from Picogreen Fluorescence 323 14.4. DNA DYNAMICS FROM SITE-SPECIFIC FLUORESCENCE PROBES 325 14.4.1. DNA dynamics in RecA-DNA filaments 326 14.4.2. Position-dependent DNA dynamics 328 14.4.3. Mismatch recognition and DNA dynamics 329 14.5. CONCLUSIONS 332 14.6. ACKNOWLEDGMENTS 332 14.7. REFERENCES 332

15. PROTEIN-PROTEIN INTERACTIONS IN VIVO: USE OF BIOSENSORS BASED ON FRET

341

Jan Willem Borst, Isabella Nougalli-Tonaco , Mark A. Hink, Arie van Hoek, Richard G.H. Immink, and Anionic J.W.G. Visser 15.1. INTRODUCTION 15.2. FRET COMBINATIONS IN CELL BIOLOGY 15.3. FRET SENSORS 15.3.1 Cameleons (Ycam) 15.3.2 Caspase sensor 15.3.3 FLAME 15.4. INTENSITY BASED FRET IMAGING 15.4.1 Confocal and wide-field FRET imaging 15.4.2 Spectral imaging 15.4.3 Acceptor photo-bleaching 15.5. FLUORESCENCE LIFETIME IMAGING MICROSCOPY (FLIM) 15.5.1 FLIM setup 15.5.2 FLIM analysis 15.6. APPLICATIONS WITH PLANT TRANSCRIPTION FACTORS 15.6.1 Sub-cellular localization via confocal microscopy 15.6.2 Molecular interaction imaging via FRET-FLIM 15.6.3 Molecular interaction imaging via FRET-FLIM 15.7. ACKNOWLEDGMENTS 15.8. REFERENCES

341 343 344 344 345 345 346 346 346 346 348 349 349 350 350 351 354 355 355

CONTENTS

xxvii

16. SPECTROSCOPY FOR THE ASSESSMENT OF MELANOMAS . 359 Ousama M. A'Amar, and Irving J. Bigio 16.1. INTRODUCTION 16.2. SKIN MELANOMA 16.3. FLUORESCENCE SPECTROSCOPY 16.3.1. Instrumentation 16.3.2. Melanoma Diagnosis by Autofluorescence 16.3.3. Melanoma Diagnosis with exogenous fluorophores 16.4. ELASTIC SCATTERING SPECTROSCOPY 16.4.1. Principles of Elastic Scattering Spectroscopy 16.4.2. Instrumentation 16.4.3. Preclinical Trials 16.4.4. Clinical Studies 16.5. CORRECTION OF FLUORESCENCE ESf TURBID MEDIA 16.6. CONCLUSIONS 16.7. REFERENCES 17. QUANTITATIVE FLUORESCENCE HYBRIDIZATION USING AUTOMATED IMAGE CYTOMETRY ON INTERPHASE NUCLEI Khuong Truong, Anne Gibaud, Nicolas Vogt, and Bernard Malfoy 17.1. INTRODUCTION 17.2. CHROMOSOME IMBALANCES IN HUMAN DISEASES 17.2.1. Cancers 17.2.2. Constitutional diseases 17.3. EXPERIMENTAL APPROACHES FOR THE IN SITU DETERMINATION OF CHROMOSOME IMBALANCES 17.3.1. Metaphase chromosomes 17.3.2. Interphase chromosomes 17.4. QUANTITATIVE FISH BY AUTOMATED IMAGE CYTOMETRY ON INTERPHASE NUCLEI 17.4.1. Chromosome 3 arms imbalances inbronchic cancers 17.4.2. Prenatal Diagnosis of Trisomy 21 17.5. CONCLUSIONS AND PERSPECTIVES 17.6. REFERENCES

359 362 363 363 363 368 369 369 371 373 374 376 378 379

387

387 388 388 388 389 389 390 390 395 395 395 397

xxviii

CONTENTS

18. IMPORTANCE OF MEASURING FREE ZINC IN CELLS

399

Rebecca A. Bozym, Richard B. Thompson, and Carol A. Fierke 18.1. INTRODUCTION 399 18.2. TSQ DERIVATIVES 402 18.3. FLUORESCENT INDICATORS BASED ON FLUORESCEIN .. 402 18.3.1. The Zinpyr family 402 18.3.2. The Zinspy Family 404 18.3.3. The ZnAFs 405 18.4. ZINC INDICATORS BY MOLECULAR PROBES 406 18.5. A ZINC INDICATOR BASED ON BENZOXAZOLE 408 18.6. LANTHANIDE CHEMOSENSORS FOR ZINC 408 18.7. EXCITED-STATE INTRAMOLECULAR PROTON TRANSFER 409 18.8. PEPTIDES AS ZINC INDICATORS 410 18.9. CARBONIC ANHYDRASE AS A BIOSENSOR FOR ZINC 411 18.10. CONCLUSION 414 18.11. REFERENCES 415

19. LASER-BASED DETECTION OF ATMOSPHERIC HALOCARBONS

421

Jean-Francois Gravel, and Denis Boudreau 19.1. ABSTRACT 421 19.2. INTRODUCTION 422 19.3. LASER INDUCED BREAKDOWN SPECTROSCOPY 423 19.4. LASER PHOTOFRAGMENTATION FRAGMENT DETECTION 425 19.5. LONG RANGE REMOTE SENSING OF HALOCARBONS BY NON-LINEAR LASER PROPAGATION 431 19.6. FUTURE DIRECTIONS 438 19.7. ACKNOWLEDGEMENTS 438 19.8. REFERENCES 438

20. FRET STUDIES OF CONFORMATIONAL TRANSITIONS IN PROTEINS

445

Herbert C. Cheung, and Wen-Ji Dong 20.1. INTRODUCTION 20.2. CALCIUM ACTIVATION OF CARDIAC MUSCLE 20.2.1. Equilibrium Conformation of Cardiac Troponin 20.3. KINETICS OF CONFORMATIONAL TRANSITIONS IN cTN

445 446 446 . 453

CONTENTS

xxix

20.4. CONFORMATION OF N-DOMAIN OF cTnC IN MYOFILAMENT 455 20.5. FRET-BASED CONSTRUCTION OF MOLECULAR MODELS 458 20.6. NUCLEOTIDE-DEPENDENT KINESIN CONFORMATIONS . 460 20.7. SUMMARY 461 20.8. ACKNOWLEDGEMENTS 462 20.9. REFERENCES 462

21. GREEN FLUORESCENT PROTEIN AS A BIOSENSOR FOR TOXIC COMPOUNDS

463

Renato J. Aguilera, Jessica Montoya, Todd P. Primm, and Armando VarelaRamirez 2L1 ABSTRACT 2L2. BRIEF OVERVIEW ON THE PROPERTIES OF GFP 21.3. GFP AS A BIOSENSOR 2L4. GFP-BASED TOXICITY ASSAYS IN MULTICELLULAR ORGANISMS 21.5. RECENT GFP-ASSAYS FOR DRUG DISCOVERY 21.6. USING THE HELA-GFP ASSAY TO DETERMINE THE CYTOTOXICITY OF ANTIBACTERIAL COMPOUNDS 21.7. LARGE-SCALE SCREENING OF COMPOUNDS ON EUKARYOTIC AND PROKARYOTIC CELLS 21.8. SUMMARY 21.9. ACKNOWLEDGEMENTS 21.10. REFERENCES

463 464 464 466 467 468 470 472 473 474

22. MULTIDIMENSIONAL FLUORESCENCE IMAGING APPLIED TO BIOLOGICAL TISSUE 477 Daniel S. Elson, Neil Galletly, Clifford Talbot, Jose Requejo-Isidro, James McGinty, Christopher Dunsby, Peter M. P. Lanigan, Ian Munro, Richard K. P. Benninger, Pieter de Beule, Egidijus Auksorius, Laszlo Hegyi, Ann Sandison, Andrew Wallace, Pat Soulier, Mark A. A. Neil, John Lever, Gordon W. Stamp, and Paul M. W. French 22.1. INTRODUCTION 22.2. FLUORESCENCE LIFETIME 22.2.1. Fluorescence lifetime of endogenous fluorophores 22.3. FLUORESCENCE LIFETIME DETERMINATION 22.3.1. Single-point measurement of fluorescence lifetime 22.3.2. Fluorescence Lifetime Imaging (FLIM) 22.3.3. Complex decay profiles and the stretched exponential ftinction

477 480 482 483 484 487 492

XXX

CONTENTS 22.3.4. Wide-field time-domain FLIM instrumentation 22.4. MULTIWELL PLATE IMAGING OF ENDOGENOUS FLUOROPHORES 22.5. FLIM MICROSCOPY OF BIOLOGICAL TISSUE 22.5.1. Cartilage 22.5.2. Artery wall and atherosclerotic plaques 22.5.3. Neoplastic tissue 22.6. TOWARDS IN VIVO IMAGING 22.6.1. Real-Time FLIM 22.6.2. Endoscopic FLIM 22.7. EMERGING TECHNOLOGY FOR FLIM AND MDFI 22.7.1. Tunable continuum source for fluorescence excitation 22.7.2. Hyperspectral FLIM instrumentation 22.8. CONCLUSIONS 22.9. ACKNOWLEDGEMENTS 22.10. REFERENCES

23. MULTIPLEXED FLUORESCENCE DETECTION FOR DNA SEQUENCING

493 495 495 497 500 501 503 504 506 508 509 514 517 518 518

525

Li Zhu, and Steven A. Soper 23.1. BACKGROUND AND RELEVANCE 525 23.1.1. What Is DNA Sequencing? 526 23.1.2. Gel Electrophoresis for DNA Sequencing 530 23.1.3. Fluorescence Detection for DNA Sequencing 535 23.2. DYE-PRIMER/DYE-TERMINATOR CHEMISTRY IN DNA SEQUENCING 536 23.3. FLUORESCENT DYES FOR DNA LABELING AND SEQUENCING 537 23.3.1. Visible Fluorescent Dyes 538 23.3.2. Near-IR Fluorescent Dyes 541 23.4. FLUORESCENCE-BASED DNA SEQUENCING STRATEGIES 546 23.4.1. Color Discrimination Methods 547 23.4.2. Lifetime Discrimination Methods 552 23.4.3. Combination of Color-Discrimination and Time-Resolved Methods 559 23.5. INSTRUMENTAL FORMATS FOR FLUORESCENCE-BASED DNA SEQUENCING 563 23.5.1. Fluorescence-scanning Detectors 565 23.5.2. Fluorescence-imaging Detectors 567

CONTENTS 23.5.3. Time-resolved Fluorescence Scanning Detectors 23.5.4. Time-resolved Fluorescence Imaging Detectors 23.6. REFERENCES INDEX

xxxi 567 569 569 589

THE HISTORY OF FRET: From conception through the labors of birth Robert M. Clegg^ 1.1. INTRODUCTION This chapter is an excursion into the historical development of energy transfer. This chapter is not concerned with a detailed review of applications, or a review of modem theoretical developments; this is available elsewhere (Van Der Meer et al, 1994; Wu and Brand, 1994; Clegg, 1996). The topic is the emergence of Forster resonance energy transfer FRET. I also examine the ideas, experiments and theories that formed the scientific backdrop that preceded and led up to FRET. FRET is a physical process whereby the excited state energy of one chromophore molecule, the "donor", can be transferred to a neighboring chromophore, the acceptor, in the ground state. This can take place whenever the two molecules are close enough, usually separated by less that 7 nm provided certain other conditions are met. FRET is one of the major experimental methods for discovering whether two molecules are in close proximity, or for determining the distance between two specific locations on macromolecules and in molecular complexes. Energy transfer is used to follow conformational changes of macromolecules, either statically or in real time. It has recently become a major experimental technique in the field of single molecules. Since the "efficiency" of energy transfer (that is, the fraction of energy absorbed by the donor that is transferred to the acceptor) is usually measured with fluorescence tools, and fluorescence is sensitive, specific and widely available, FRET has become very popular. The chromophores (donors and acceptors) that are used for accomplishing this measurement are usually attached (often covalently) to other macromolecules, such as proteins, nucleic acids, and lipids. The energy transfer can be detected relatively easily and it is often used qualitatively to signify intimate interaction

^ Robert M. Clegg, Physics Department, University of Illinois Champaign-Urbana, Illinois

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R. M. CLEGG

between two "labeled" biomolecules. Sometimes one or both of the participating chromophores occur naturally in biological macromolecules, such as tryptophan or chlorophyll. However, the number and variety of synthetic fluorescence probes available for labeling has expanded tremendously in the last several years. Several readable reviews of FRET for a general audience are readily available (Clegg, 1992; Van Der Meer et al, 1994; Clegg, 1996; Clegg, 2004a). The FRET measurement is now applied routinely with a wide variety of samples: micro structures (such as DNA and protein chips and micro/nano assay arrays), living biological cells, and even whole organisms. It is a very powerful technique, fairly simple, and can be carried out in most laboratories with their existing spectrometers and microscopes. Although the technique has been readily available and applied since the early 1950s, the use of FRET has literally exploded in the last few years, in academic research as well as industrial applications, especially in biotechnology and bioengineering. This flurry of activity has many reasons. First, FRET measures interactions and dynamics on a spatial scale that is unique. Also, our ability to produce well defined and pure macromolecules in the laboratory has increased dramatically in the last few years, and it is relatively easy to label them specifically with fluorophores. In the last several years we have developed the ability to produce hybrids of specific proteins with fluorescent proteins (for instance, GFP, YFP, CFP and RFP, respectively green-, yellow-, cyan-, and red-fluorescence proteins) that can be produced in vivo under genetic control in the living cell (and in tissue); certain pairs of these proteins can undergo FRET. These fluorescence proteins have revolutionized the field of biological fluorescence, especially the measurement of FRET, in the fluorescence microscope. A great number of excellent synthetic fluorophores are available commercially, with the required chemical groups attached for specific labeling to biomolecules. In addition there have been many instrumentation improvements and innovations that make the FRET measurement much more sensitive and convenient. These chemical, biological, and instrumentation advances have expanded tremendously the range of applications, and the ease of carrying out the experiments. In spite of the wide spread use of such a well known and useful technique, and the availability of several excellent treatise and reviews of the underlying theory, not to mention the hundreds of experimental applications published every year, little is published about the historical development of the major concepts. The historical events are not only interesting in themselves, but understanding and appreciating the major theoretical insights realized by the pioneers of energy transfer, and the scientific context in which they worked, provides insight into the mechanism, and leads to a better appreciation of the original contributions. A short history of the contributions of the Perrins and Foerster to FRET has been published recently (Clegg, 2004b). This chapter is a more extensive examination of the state of affairs and the general state of knowledge that was prevalent in physics at the time, leading up to the first observations and theoretical explanations of energy transfer.

THE HISTORY OF FRET

3

1.2. PRELUDE TO THE HISTORICAL BACKGROUND Although the practical applications of FRET started after 1950, the awareness that energy could be transferred between two atoms or molecules over distances larger than their physical collisional radii took place much earlier. The first experimental observation of energy transfer happened after 1900 and this chapter will only deal with FRET literature between 1900 and 1970. However, I will follow the thread back into the 19* century, when the ideas of an electromagnetic (EM) field and spectroscopy were being formed, and the dilemmas leading to quantum mechanics (QM) at the turn of the century were starting to appear. These theoretical concepts were essential for the observation and correct interpretation of non-radiative energy transfer. The notion of EM fields entered compellingly into the mainstream of physics only a few decades before the first observations of energy transfer. The first theoretical attempts explaining FRET were applications of this classical EM theory. And the first quantum mechanical theories of FRET were developed concurrently with the new theories of Heisenberg, Schrodinger and Dirac (Heisenberg, 1925; Dirac, 1926; Schrodinger, 1926b). So we will take a look at the historical scientific context in which the first experiments and theoretical accounts of energy transfer took place. The aim of the first part of this chapter is to indicate the scientific atmosphere in which the idea of energy transfer at a distance was bom. We start by taking a short journey through the development of the concept of electromagnetic fields, move quickly through the quantum theory of atoms and spectroscopy, and then into the first experimental and theoretical discoveries of energy transfer. This initial time frame is from 1820 to 1920. I have decidedly selected the topics, emphasizing only those concepts important for FRET. After reviewing the emergence of these physical concepts, this chapter will only deal with FRET literature between 1920 and 1960. 1.2.1. The End of the Dark Ages: the Pre-Field Era The fundamental paradigms of physics were undergoing radical changes in the 19^*^ century, and these ideas were critical for understanding that atomic (molecular) interactions could extend over distances long compared to their atomic (molecular) radii. A lively account of how the concept of a field in a void entered into physics is given in an easily readable account by Einstein and Infeld (Einstein and Infeld, 1966). The concept of electric and magnetic fields is of course now common place, but we will see it was not until Maxwell that this concept was set on firm footing. The notion that magnetism and electricity were somehow related had been suspected for some time before 1800, because of the formal similarities between static electricity and magnetism. Hans Christian Oerstead^ in 1820 was the first ^ Oerstead, a professor of Natural Philosophy in Copenhagen, received his PhD in 1799 in the medical faculty of Copenhagen; his topic dealt with Kant's philosophy.

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R. M. CLEGG

to demonstrate the interrelationship of magnetism and electricity^ In a short four page article written in Latin (Greiner, 1986) he reported that a magnet's needle held next to a current-carrying wire was deflected, and oriented itself perpendicular to the line of current^ This interaction happened at a distance, decreased in effectiveness with increasing distance, and surprisingly the force on the needle was perpendicular to the line between the wire and the magnet^ This discovery, easy to reproduce^, was the first direct demonstration of the connection between electricity (a current) and magnetism, and it was first done by accident at the end of a lecture demonstration (Whitaker, 1989a). He wanted to show that if the magnet was perpendicular to the current flow there was no effect (which was true). At the end of the lecture he inadvertently oriented the magnet parallel to the wire, and there was a pronounced deflection. It was a phenomenally significant and completely unanticipated discovery, especially since magnetism had been known from antiquity, and was conceived by many as somewhat magical with supernatural powers^ and the compass and its use for navigation had been known for a long time. Oerstead's short report instigated immense interest throughout Europe, not only in the physics community, but was also enthusiastically received by workers in all scientific (and medical) disciplines. At that time, science was not as topically separated and divided as now. Even the general public heard of, and enthusiastically discussed, his experiment. Interestingly, Oerstead wrote later that it was his interest in romanticism and the movement of romantic natural philosophy that inspired him to carry out these experiments. He was also a passionate and tireless lecturer, and this may explain some of his influence (although, apparently, some scientists of that day did not appreciate his romantic outlook, and thought of him as a lucky, amateurish, dreamy opportunist - he earned handsomely from this discovery). Whatever, one might say that this was the inauguration of a great paradigm change in physics. Two months later it was announced in Paris by Dominique Fran9ois Jean Arago^ the famous French astronomer, who had just returned from Denmark. Andre-Marie Ampere (and others, e.g. Jean-Baptiste Biot and Felix Savart) ^ Of course, this discovery could not take place before one had the ability of making currents (Voltaic piles) and had wires. Both these requirements had only been available since approximately 1800. ^ Oerstead got the idea to carry out his experiment with a galvanic circuit because it was known that deflections in a compass needle take place during lightning bursts in thunderstorms. ^ Oerstead did not determine quantitative aspects of this discovery; this was done by others soon after his discovery. ^ Interestingly, Oerstead was apparently all "thumbs" in the lab, and all his experiments had to be carried out by his (enthusiastic) students and assistants. ^ Magnets had been purported since antiquity to have healing powers. In 1780-1800 Franz Anton Mesmer, a doctor considered a charlatan by many but a medical savior by others, became a sensation "mesmerizing" his patients by passing magnets - in the appropriate mystifying setting over the location in their body of their suspected ailments (or heads, if they were mentally distressed). To be fair, he realized later that his method had to do with suggestion, and did not require magnets. His work was the forerunner of the later work of Puysesur, Braid, Charot and Freud on hypnosis. ^ Arago discovered in 1811 the rotation of the plane of polarization when polarized light passes through an optically active crystal.

THE HISTORY OF FRET

5

immediately repeated Oerstead's experiments. Ampere was adept in the manipulation (and participated in the theoretical development) of partial differential equations (he was also well educated and took part in the world of literature and philosophy), and in 1822 he soon came up with a theory of electromagnetic interactions involving currents^. He found that current-carrying wires would attract or repel each other depending on whether the currents were in the same direction or opposite. As a result of his interest in Oerstead's experiments, he suggested the possibility of a telegraph together with Jacques Babinet. This is the first mention that communication between two places could take place via electromagnetic interactions^^. I mention this, because electromagnetic communication between two locations is the basic physical event in FRET. 1.2.2. Middle Ages: Experiments That Eventually Changed Our World View In the realm of magnetism and electricity, the pictorial, intuitive representation of lines or tubes of force was introduced by Faraday in 1821 (Whitaker, 1989a) ^^ Faraday was perhaps the most thorough, dedicated, likable and honest of all experimenters. His interest in the inter-conversion and transformation of forces between different forms was a lifelong goal (this was before the concept of the conservation of energy and the equivalence of heat and energy by Julius Robert Meyer in 1842). Faraday handled his lines of force as though they were real physical entities, and not just as abstract helpful mathematical concepts^^. He pictured these lines of force as the mechanism by which electrical and magnetic substances interact with themselves and with each other; these tubes of force were so to speak for him the carriers of forces through space. He discovered the "Faraday induction" (i.e. changing magnetic fields produce circulating electrical fields), which is the basis of modem electric motors. The idea of forces at a distance had of course been a topic for a long time (e.g. Newton and gravitation). However, Faraday's ideas of tubes of force ^ Ampere's first law gives the force on a current carrying wire placed in a magnetic field; his second law is that the magnetic field B circulates around an enclosed electrical current y ( ^ ) ; that is, in vector notation, v x 5 = ^^ The electromagnetic telegraph was later implemented by Karl Friedrich Gauss and Wilhelm Edward Weber in 1833, who developed the practical telegraph; they sent the first telegraph message in Gottingen from Gauss's observatory outside the city limits to Weber's laboratory in the city. Interestingly, there was much debate among the population of Gottingen whether the wires strung over the house tops were possible health hazards because of the magnetic effects emanating from the current carrying wires. Gauss developed a system of units for measuring magnetism that was based on length, mass and time, and this is the basis for the unit system in EM called the Gaussian Units. The unit Gauss, measures the strength of a magnetic field. ^^ Actually, this concept was reported by Niccolo Cabeno, as early as 1628, and referred to as early as 1629 by Aristotelian-scholastic philosophers. ^^ Faraday had no knowledge of formal mathematics. It has been mentioned to me by a mathematical physicist that he was perhaps the supreme mathematical physicist, with an incredible spatial imagination that did not need the crutches of equations.

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R. M. CLEGG

did not sit well with many of the mathematical physicists of the day, who were used to expressing all in terms of differential equations^^. Nevertheless, he was an exceptionally capable experimenter; his experiments and graphical explanations were remarkably innovative and provided astounding, intuitive insight. His ideas set the stage for the next step important for the background for FRET, which was then carried out by Maxwell. 1.2.3. Renaissance: Enter the Theory of Electrodynamics and Fields The basic FRET phenomenon involves the electrodynamic interaction between two molecules over distances that are large compared to their diameters; and this description requires the idea of an EM field (for FRET this is a dipole interaction, which arises from a multi-pole approximation to the Coulomb interaction). Faraday did not deal with fields, but with tubes of force. It was James Clerk Maxwell who introduced the first field theory. His equations describe the EM field; the objects (electrical or magnetic) enter only through boundary conditions. The ideas and experiments of Faraday played a major role in Maxwell's theoretical development. He created a complete mathematical representation of Faraday's descriptions of electricity and magnetism (Maxwell, 1873; Simpson, 1997). He admired the work of Faraday, and read all what Faraday had written before undertaking the task of formulating his ideas in mathematics^"^. He was of the opinion that Faraday had articulated his discoveries "in terms as unambiguous as those of pure mathematics". Maxwell's accomplishment is enshrined in his famous classical equations of electrodynamics, which are familiar to all physics students (and often the cause of much sweat and toil). In addition to the concepts he borrowed from Faraday, he introduced the notion of displacement current - the circulatory magnetic field caused by a time-varying electric field (also in empty space). These equations describe all classical electrodynamic phenomena, and they are the starting point for describing energy transfer. His equations fumished the theoretical setting to predict electromagnetic radiation (e.g. the classical theory of fluorescence emission). It is a fascinating story how Maxwell wrestled with physical and mathematical analogies, experimental results and mathematical formulations in order to arrive at his equations, as well as how his equations predicted light as an electromagnetic field. He created the term electrodynamics, as this quote from him exemplifies: "The theory I propose may therefore be called a theory of the Electromagnetic Field, because it has to do with the space in the neighborhood of the electric or magnetic bodies, and it may be called a theory '^ Faraday eventually repudiated a reality that consisted of separate entities of matter (atoms) and void: "The difference between a supposed little hard particle and the powers around it, I cannot imagine". ^^ The titles of Maxwell's first two of his three main papers on EM were: "On Faraday's Lines of Force" (1855) and "On Physical Lines of Force" (1861). The first shows his respect and enthusiasm for Faraday's ideas, and the second signifies his new paradigm of the physical field concept. His third paper on this subject "A Dynamical Theory of the Electrodynamic Field" was presented in (1864).

THE HISTORY OF FRET

7

of the Dynamical Theory, because it assumes that in that space there is matter in motion, by which the observed electrodynamic phenomena are produced" (this quote is from his third paper in this series - "A Dynamical Theory of the Electrodynamic Field"). This is actually a partial description of what happens in FRET. Maxwell understood Faraday's lines of force as "a line passing through any point of space so it represents the direction of the force exerted ...". This depicts nicely the vector representation of the EM field, which is now given in every EM textbook, and is the way the orientational dependence of the interaction between a FRET pair is portrayed. The impact on the physics community and the conceptual revolutions that were initiated by Faraday and formulated by Maxwell are perhaps difficult to appreciated^. The theory of Maxwell, based on the original ideas of Faraday, turned much of physics on its head. Whereas Newton's laws conserve energy and momentum in the motions and collisions of bodies. Maxwell's field theory is concerned with the energy and momentum of the field, and does not take account of the bodies, except as boundaries. Because this concept of a field is so critical for understanding FRET, and this is a historical account, I emphasize this with two quotes. The first is from Ludwig Boltzmann^^, who, to express his admiration for Maxwell's equations, quoted Goethe: "War es ein Gott, der diese Zeichen schrieb?" - Was it a god, who wrote these expressions?". The second is from Maxwell himself^'': " In speaking of the Energy of the field, however, I wish to be understood literally. All energy is the same as mechanical [...]. The energy in electromagnetric phenomena is mechanical energy. The only question is. Where does it reside? On the old theories it resides in the electrified bodies, conducting circuits, and magnets, in the form of an unknown quality called potential energy, or the power of producing certain effects at a distance. On our theory it resides in the electromagnetic field.". This was an enormous paradigm change. The difficulty to imagine energy in a void led to the introduction of the "ether". Ether was supposed to be the inert medium through which all electric and magnetic phenomena were transmitted. This was not required by Maxwell's equations. But scientists trained in the Cartesian view of physical phenomena found the ether necessary (including Maxwell). It was difficult to imagine how the transverse undulations of light (required and predicted by Maxwell's equations and verified by Hertz) could take place without a medium. As we know, the ether was shown not to exist. (Whitaker, 1989a; Whitaker, 1989b). 1.2.4. The Beginning of the Modern Age: The Field Surrounding an Oscillating Charge The next critical step for FRET was carried out by Heinrich Hertz, with his famous Hertzian oscillating dipole. The electrodynamic field emanating from a vibrating electric dipole (the Hertzian oscillating dipole) is derived from '^ Although eventually this impact was prevailing, Maxv^^ell's EM theory was not immediately accepted, and was even highly criticized my many scientists. ^^" Vorlesungen iiber Maxwells Theorie", L. Boltzmann ^^ "A Dynamical Theory of the Electrodynamic Field" JC Maxwell (1864)

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Maxwell's equations. This is the classical theoretical basis for understanding the production of light (e.g. emission of fluorescence from atoms and molecules, as well as the absorption), and is also the starting point of the first classical descriptions of FRET. Maxwell's equations predicted the identity of electromagnetism and light (expressed in his memoirs of 1868), and even foretold the quantitative properties of light (interference, refrangibility, polarization, as well as the speed of light). This was brilliantly confirmed by the experiments of Hertz. These experiments, and his theoretical description based on Maxwell's theory, forced the skeptics in the scientific community to accept the concepts inherent in Maxwell's field equations. Hertz carried out the critical experiments in 1888, and published the theory (derived from Maxwell's equations) to explain the EM fields surrounding his electric oscillator in 1889. This derivation is given in any intermediate or advanced electrodynamics textbook. Of course. Hertz was not specifically referring to an atomic oscillator, but to a macroscopic electric oscillator. His first experiments were carried out by producing high frequency repetitive sparks in an air gap of a primary oscillating circuit (which acted as the source of the EM radiation). The electrodynamic disturbance was detected at a distance by a secondary circuit, resonant with the first, with a similar air gap. Sparks were observed in the secondary receiving circuit when it was resonant with the primary circuit. At first Hertz was primarily interested in proving the existence of propagating electromagnetic radiation (light, at frequencies of what is today radio frequencies), which was predicted by Maxwell's equations. Therefore, in the first experiments the distance between the primary and secondary circuit was long compared to the wavelength of the propagating Maxwell electromagnetic wave at that frequency of oscillation. The result was fully consistent with Maxwell's field equations, and this is of course the basis of all radio communications. Hertz's theoretical description describes the electromagnetic disturbance in the near field (much less than a wavelength of the emitted radiation), in an intermediate zone, as well as in the far zone (at distances greater than a wavelength) where the electromagnetic energy escapes and is carried away as radiation with transverse oscillations. He calculated a very graphic field-line representation of the EM field of an oscillating dipole, demonstrating how the field lines are pinched off at approximately a distance of a wavelength, at which point transverse waves in the far field are formed (E and B fields of propagating radiation are perpendicular to the direction of photon travel). Only in the far field can we think of a photon (or in the language of pre-photon concepts, a traveling light wave). In the near field (distances small compared to the wavelength, which is where FRET takes place) both transverse and longitudinal components of the EM fields are present. However, in spite of the high energy density in the near-field, no propagating EM waves are present here (no emission of energy). This corresponds to the terminology in FRET that the energy in the near field is transferred non-radiatively. Experiments carried out in the near field verified Hertz's theoretical description. The graphical and mathematical description of the oscillating electric field emanating from a Hertzian dipole, in particular in the near field, played a

THE HISTORY OF FRET

9

critical role in the understanding, and the eventual theoretical description, of FRET. The oscillating E-field in the near field of a Hertzian dipole has the same effect on a receiving oscillator as when the receiver is in ihQ far field. The only difference between the near- and far-field effects on a receiver (acceptor in FRET) is the direction of the field vector (and the intensity), which is a sum of tangential and longitudinal components in the near field, and only tangential in the far field. The oscillating electric field surrounding the Hertz oscillator is indispensable for all theoretical descriptions of FRET.

1.3. FIELDS, SPECTROSCOPY AND QUANTUM MECHANICS 1.3.1. Fields As we have seen, in a relatively short period of time the paradigm in physics that all interactions take place by mechanical contact and collisions, changed considerably (Einstein and Infeld, 1966). By 1900 the concept of a field had been generally accepted (this is only 22 years before the first report of energy transfer at a large distance between atoms in a vapor (Cario, 1922)). Nevertheless, because of the very successful dynamical gas theory (the groundwork of which was also set by Maxwell), most original interpretations of energy transfer and fluorescence quenching between gaseous atoms naturally assumed coUisional contact (mechanical interactions). 1.3.2. Quantum Mechanics and Spectroscopy At the turn of the century there was another paradigm change in physics about to take place, which is the second requirement for understanding FRET. This took place because the theory of radiation, in spite of the success of Maxwell's theory, had reached a very unsatisfactory state. This had to do with the failure to explain the dispersion (frequency dependence of the energy emission) of blackbody radiation. As is well known, in 1900 Planck solved this problem by introducing the quantum concept - energy changes in matter could only take place in well defined quantum jumps. His successful theory for explaining black body radiation was presented December 14, 1900 at the German Physical Society (Planck, 1900) ^l His reasoning centered on an ensemble oi Hertzian oscillators (the oscillators were the atomic constituents of the walls), which he proposed could only exchange energy with the radiation field in jumps of energy quanta. His famous paper was published in 1901 (Planck, 1901). This was followed by the work of Einstein (Einstein, 1905;

^^ In October of 1900 he had presented to the same society a phenomenological theory that was in agreement with black body experiments, which was a modification of previous work of his (in 1899) based on thermodynamic reasoning; but he considered this approach unsatisfactory because it contained undefined empirical constants.

10

R. M. CLEGG

Einstein, 1906), where he proposed that light itself behaved as though it were particle-like quanta of energy^^ 1.3.2.1. Conway and Ritz Up until 1907, the picture that scientists had of absorbing and emitting atoms was that each atom consisted of an electrical system with many natural periods of oscillation, all present simultaneously. In 1907 Arthur William Conway, from Dublin (Conway, 1907), proposed that the spectrum of an atom does not result from free vibrations as a whole, but that each atom produces spectral lines one at a time (that is, the actual spectrum observed at any time depends on the presence of many atoms). This is prior to the Bohr-theory of the atom, or even of the Rutherford model of an atom (1911). The idea of Conway was that in order to produce a spectral line, one electron in an atom must be in some sort of perturbed state (he had no way to describe this in detail), and that this electron is then stimulated to produce vibrations of a frequency corresponding to the observed spectral line. This disturbed state did not last indefinitely but would decay with time, emitting a fairly long train of vibrations (as was required by Maxwell's equations). This was remarkable insight considering that he did not know the later interpretations of atomic spectra (e.g. the Balmer and Rydberg series) in 1908 by Ritz (Ritz combination principle) (Ritz, 1908) who showed how the spectral lines could be interpreted by differences, taken in pairs, of certain distinct numbers. 1.3.2.2. Bohr This discussion would not be complete without mention of the critical insight of Niels Bohr (Bohr, 1913; Hettema, 1995), who integrated and selected many of the ideas that were being considered at the time (including those of Conway (Gillispie, I960)). He selected what he considered to be superior ideas from the inferior ones, and produced the paradigm of the atom (the Bohr atom) that was enormously influential and placed spectroscopy on firm ground. This is now so common-place and is even sometimes introduced in grade school; however, his synthesis of a model that could explain quantitatively the spectroscopy of many simple one-electron systems was a real eye-opener, and was critical for all that followed. Because it is so well known, I spend no time describing Bohr's ideas. Of course, extensive experimental studies in spectroscopy were carried out at that time (Pringsheim, 1928). The spectroscopic experiments stimulated the theoretical work, and provided the data for motivating and checking the theories. The extensions of these basic ideas to complex molecules could only take place after the introduction of quantum mechanics by Heisenberg in 1925 (Heisenberg, 1925) and Schrodinger '^ Although Planck had considered that energy of the field could be quantized, he resisted this because this would go against all that was known of the "continuous" Maxwellian light field; for this reason he only considered the quantized emission of energy from the oscillators, and not the absorption.

THE HISTORY OF FRET

11

in 1926 (Schrodinger, 1926b), and we will see in the next section that the new quantum mechanics was immediately applied by Kallmann and London (Kallmann and London, 1928) to explain energy transfer in vapors, and by F. Perrin for energy transfer in solution (Perrin, 1932).

1.4. THE FIRST EXPERIMENTAL OBSERVATION OF ENERGY TRANSFER AT A DISTANCE - SENSITIZED LUMINESCENCE IN VAPORS 1.4.1. Sensitized Fluorescence With the concept of EM fields, the experiments and theory of Hertz, the development of the older quantum theory, the spectroscopy data of atoms, and Bohr's theory of the atom, we have reached the point of entry for FRET. The first recorded measurements of energy transfer (observing the emission of the accepting atom) over distances larger than collision radii were made in 1922 by Carlo and Franck (Carlo, 1922; Carlo and Franck, 1922; Franck, 1922). Carlo observed emission from thallium in a mixture of mercury vapor and thallium vapor, when the vapor mixture was excited with wavelength of 253.6 nm, which can only excite the mercury atoms. This fluorescence emission from thallium was named "sensitized fluorescence". It was obviously due to the transfer of energy from the excited mercury atoms to the thallium atoms. Further experiments by many people showed sensitized fluorescence with the vapors of the alkali metals: silver, cadmium, lead, zinc and indium in the presence of mercury vapor. The importance of resonance between the energy levels of the sensitizer and the sensitized atoms was explicitly shown by fiirther experiments, especially with the later experiments of Beutler and Josephi (Beutler and Josephi, 1927; Beutler and Josephi, 1929), who studied the sensitized fluorescence of sodium vapor in the presence of mercury vapor. The sensitized fluorescence increased in intensity the smaller the energy differences between the states of the two participating atoms. This was consistent with "Franck's principle" (Franck, 1922), which had been articulated, in reference to fluorescence quenching, previous to the experiments of Carlo. This principle can be stated as: "the electronic energy of an excited atom cannot be transferred directly into kinetic energy of the colliding particles. If the excitation energy has to be taken over almost completely as internal energy of the quenching molecules, these must have some sort of excited states, which are in energy resonance with the primarily excited states", page 116 of Pringsheim's book (Pringsheim, 1949); my italics. Here we see already the entrance of "resonance" into the FRET story, which will play a central role. Sensitized fluorescence was discovered during the copious spectroscopic experiments that were carried out at different temperatures and densities with vapors of many different atoms and diatomic molecules in the first two decades of the twentieth century. A thorough discussion of literature up to 1949 can be found in Pringsheim's treatise (Pringsheim, 1949). Dynamic fluorescence

12

R. M. CLEGG

quenching (due to collisions), photochemical reactions, resonance fluorescence and molecular associations were intensively studied in the early decades of the 1900s (Wood, 1934). Many of these experiments were naturally interpreted in terms of collision theory. The number of collisions per time could be calculated simply from gas theory; and the fraction of collisions that were effective (leading to quenching, chemical reactions, or sensitized fluorescence) could then be determined. 1.4.2. Spectroscopic and Collisional Cross-Sections in Vapors The cross-section of molecular encounters (or what is equivalent, the frequency of effective collisions) gives estimates for the "spectroscopic size" of the reacting atoms. From the fraction of successful collisions, calculated by comparing the rate of successful quenching encounters to the collision rate from gas theory, one can calculate a ''spectroscopic cross-section'\ If the radius of this cross-section is smaller than the radius of the atoms or molecules (or equivalently, if the rate of collisions is smaller than calculated from the gas theory) then the conclusion is that only a certain percentage of the collisions are effective in quenching. If the spectroscopic cross section is larger than expected from the encounter radius, then it is assumed that there are interactions between the two collision partners that extend beyond their encounter radius. A larger radius of interaction than predicted from the theory of gas dynamics was found for many energy transfer measurements in the vapor. These large "spectroscopic" cross-sections constituted the first hint that many inter-atomic interactions could take place over larger distances. This discovery that energy transfer could take place over distances large compared to the encounter radii showed that hard physical collisions were not required for atoms (or molecules) to interchange energy.

1.5. THE FIRST QUANTUM MECHANICAL THEORY OF ENERGY TRANSFER A quantum mechanical theory to explain the transfer of energy between atoms at longer distances compared to collisional radii was proposed by Kallmann and London20 (Kallmann and London, 1928). This theory assumed "almost resonance" between the energy levels of the interacting atoms. Essentially this is a second order perturbation calculation to calculate the energy of interaction. I will use their notation in this paragraph. They found that provided the corresponding transitions between the energy states of the two atoms were (spectroscopically) dipole-allowed, the effective cross-section q of the two interacting atoms increases as a

-2/3

, where a is the difference between

'^This is the same F. London who proposed the quantum mechanical description of van der Waals interactions, which also involved dipole-dipole interactions, similar to FRET.

THE HISTORY OF FRET

13

the excitation energies of the two interacting systems. As cr ^ 0 , the cross section approaches a limiting value much larger than the coUisional radii. This work was the germ of the later quantum mechanical FRET theories, and is very similar to the description of London-van der Waals forces. Before discussing this paper further, we look at classical theories that were published just previous to Kallmann and London. 1.5.1. A Few of the Pre-Quantum Theories that calculated the Spectroscopic Cross-Sections of Atomic Vapors Some of the previous pre-quantum papers (Holtsmark, 1925; Mensing, 1925; Nordheim, 1926) (all referenced by Kallmann and London) dealing with "spectroscopic cross-sections" are remarkable in their insight. Mensing (Mensing, 1925) considered how intermolecular dipole-dipole interactions broaden spectra (using the Bohr-Sommerfeld atomic theory with elliptical orbitals) of atomic vapors. She derived a broadening due to dipoledipole interactions to be approximately a width of Sv^3.6\^e^/hjya^/dj\l

+ 3£^/2],

where a and £ are the long axis and

eccentricity of the atomic orbitals, eis the electron charge, and d is the distance between the two molecules. She considered dipole-dipole interactions of circulating electrons (from a semi-classical point of view, this is similar to what is done by London when the two interacting molecules are not in the same energy state). An article by Nordheim (Nordheim, 1926) presents a collision theory between atoms, whereby the atoms interact via dipole-dipole terms. Although the new quantum theory of Heisenberg (Heisenberg, 1925) and Bom and Jordon (Bom and Jordan, 1925) had been published already, Nordheim calculates the interactions classically. He justifies this choice because the results should be approximately the same, and he also wanted to by-pass the difficulties in analyzing the collisions quantum mechanically. Later, Forster, Ketskemety and Kuhn (Forster, 1951; Ketskemety, 1962; Kuhn, 1970) showed that the classical and quantum calculations arrive at the same result. Nordheim derives also higher multipole interaction energies, and uses the same classical conjugate dynamic equations of motion as Mensing. By considering collisions between the electrical multipoles that are oscillating at spectroscopic frequencies, he derives expressions for the energy transfer rates proportional to the product of the appropriate powers of the spectroscopic transition moments of the different multi-pole interactions. However, this derivation is very complex, because the rate depends on very complex averaging of the collision paths, and the velocities. The details of the theory are only applicable to vapor samples. Holtsmark's paper (Holtsmark, 1925) is a purely classical approach aimed at understanding the average deviation of the energy levels from that of the free atom values of Na-vapors, in order to understand the broadening of the spectroscopic lines. The spectral widths were known to be broader than calculated from simple hard-core collision theory according to gas theory. A

14

R. M. CLEGG

classical perturbation approach (assuming that the atoms are classical electron oscillators) was taken whereby again the interaction of the atoms was assumed to be dipole-dipole. Holtsmark calculated an interaction proportional to ijR^ for the interaction between any two atoms. Since he was interested in a random collection of atoms at a certain concentration, he integrated all interactions from the shortest distance of atomic approach {d) to infinity (including the orientation factor). This results in broadening of the lines proportional Xoye^lm\

Idt

(where e is the electron charge and m^ is the mass of the

electron); although he has assumed perfect electric oscillators, each one contributing a factor of e jm^, the interaction would also be proportional to the multiplication of the oscillator strengths. He did not assume exact resonance the total interactions were summed via a type of overlap integral. His factor d~ is the same dependence on the distance of closest approach as by Arnold and Oppenheimer (Arnold and Oppenheimer, 1950) (see below) for the rate of energy transfer in a condensed system with random acceptors. The point of discussing these early theoretical accounts of dipole-dipole interactions leading to energy transfer is to show the type of analyses invoked at this time to account for the very large effective molecular spectroscopic cross sections. These cross sections were much larger than expected from simple atomic coUisional gas theory. This could only be explained by molecular interactions at large distances. These theories were published just a few years before Kallmann and London's quantum derivation, and at the same time as J. Perrin's work on energy transfer in solution. Already at this time, it was apparent to everyone that dipole-dipole (or higher multi-pole) interactions could extend the radius of inter-atomic interactions considerably. Some of the theories showed explicitly the 1/7?^ dependence. In addition, it was shown that the spectroscopic oscillator strengths (that is the spectra) had to overlap and the orientation of the dipoles were taken into account. It is clear that the pieces are starting to fall into place. 1.5.2. Some Details of the Kallmann and London Paper The theory of Kallmann and London (Kallmann and London, 1928) dealt with energy transfer in vapors of atoms; their theoretical ideas were the basis of the later quantum mechanical theory proposed by F. Perrin (Perrin, 1932; Perrin, 1933) for energy transfer in condensed systems, which was subsequently improved and extended by Forster (Forster, 1948). The article by Kallmann and London (K&L) has many interesting aspects that are important for understanding the later theoretical treatments of FRET. Because F. Perrin essentially used a very similar theoretical approach, and his theory is outlined in the section "F. Perrin's model" below, I refer the reader to that section for a discussion of the basic ideas of the derivation. K&L assumed that the two interacting atoms have two states, but the energy levels do not have

15

THE HISTORY OF FRET

to be exactly the same. They write the differential equations derived from the Schrodinger wave mechanics pertaining to the coupled system for the case of two states (Schrodinger, 1927). The basic equations were given by originally by Schrodinger (Schrodinger, 1927), in an article titled "Energieaustausch nach der Wellenmechanik", or "Energy exchange according to wave mechanics". K&L refer to this paper (this will be important when we discuss Rabi oscillations below), and it is remarkable that Schrodinger essentially indicated the path for a solution to energy exchange between two atoms (molecules) in one of his first quantum mechanic papers. The solution for this problem, if the energy states are narrow, is oscillatory, and K&L give the probability that the system of two atoms will have transferred energy to the other, c |, as (using their notation)

\c \=-

p' -sm \^p'

P -IW^^jG* .

h ^2 is th^ interchange integral of the perturbation

W between the two states of the system, a' = a + W^^ ~ W^^ . a

is the

difference of the energy levels of the two participating molecules. They assumed for simplicity that the zero energy levels of both molecules were equal. ^ j and ^22 ^^^ ^^^ configuration integrals of the perturbation W for the two different states (the first order energy change from the perturbation). W is the dipole perturbation operator, and W = {ju^ju^)/R^ where//^ andju^ are the transition dipole moments of the two molecules; this is the usual Coulomb interaction of dipoles (the QM version of the Hertzian dipole interaction energy). They are interested in collisions between atom gas molecules, and if the time for an atom to pass by another is long, compared to the oscillation of sin^[--] term, they can take an average over this time, giving them the probability G7 that, on the average, the energy will be transferred from the first combined molecular state to the other state. This is (skipping several steps - and carrying through several substitutions in order to show the relationship between their derivation and Forster's later theory)

j/2_

1 J3'

m

=•

1+

1/2

111 ,-",

Xl2_

1/2 3 \ -

1+

R"

4(//,//.)V'o-

1+ )

R'l 0 /

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R. M. CLEGG

I have defined the variable R\={A{^ju^ju^y j ( J ^ \

in anticipation of the

normal R^ of energy transfer. Except for the factor of xjl, this is the standard expression for the efficiency of FRET. The 1/2 arises because the solution is oscillatory, and K&L have calculated the time average. In addition K&L have not integrated over a frequency spectrum of the two spectroscopic transitions, which in reality cannot be sharp lines (this would give us the overlap integral see our discussion of Arnold and Oppenheimer's derivation (Arnold and Oppenheimer, 1950)). This equation by K&L is quite remarkable - it is the first indication, except for the classical calculations given in the last section, that for two atoms separated by R the probability of energy transfer obeys a l / n + i?/i?'^ jrule. The validity of the equation depends on the ratio of the energy differences and the size of the perturbation, and I cannot delve into the reasoning concerning this ratio when calculating molecular interactions in this paper (but see the short discussion by Knox (Knox, 1996)). The important point is that already in 1928 K&L derived essentially the correct dependence on the separation of the two molecules. By integrating this equation over R they arrived at their expression for the "spectroscopic" cross section q .

2 \ + Pl \ (J J This is the effective cross-section of atomic collisions, which is significantly larger than the hard core cross sections due to dipole-dipole interactions between one excited molecule and one ground state molecule). The last equality holds for not too sharp resonance. This is the relationship q oc a'^'^given two sections previous. And it is based on an efficiency of energy transfer that varies as R'^. K&L calculate approximations to this integral, and find that the dipoledipole interaction at a distance results in an anomalously large cross section, and their equation compares well to the experimental results. The above derivation is valid if the two molecules are not exactly in resonance (that is, if the spectroscopic transitions of the two molecules are not identical). K&L derive the case for exact resonance, and decide that this would give values R \ values far too large. This is partly because one would have to account for the unavoidable frequency spread of the molecular transitions. I discuss this below. This is essentially the same as the F. Perrin derivation below, and so I refer the reader to this section on F. Perrin for a discussion of this. We will see that even though F. Perrin used the same basic quantum calculation as K&L, which is that discussed by Schrodinger in 1927 (Schrodinger, 1927), he used exact resonance and calculated the rate of energy transfer in another way. F. Perrin calculated the rate from the period of the oscillation in the sin^ [•••] term of the above

THE HISTORY OF FRET

17

solution of K&L; and this gives the wrong distance dependence of energy transfer. In summary, KcfeL had the right idea, as well as the right dependence onR.

1.6. LONDON FORCES (VAN DER WAALS) AND DEB YE AND KEESOM INTERACTIONS Before delving further into theories of FRET, we turn our attention to a closely related topic - the quantum description of van der Waals (1873) interactions (or London dispersion forces). The idea of dipole-dipole interactions at a distance between atoms and molecules was being applied by London to explain intermolecular van der Waals interactions concurrently with his work on energy transfer (previous section). The calculation of classical descriptions of dipole-dipole and dipole-induced-dipole interactions had already been introduced to explain intermolecular interactions (Keesom, 1912; Debye, 1920;Debye, 1921). There is a close connection and concurrent historical development of the theories describing London's intermolecular interactions and FRET. The major difference is that one is interested in the energy of interaction for the van der Waals forces, and in the rate of energy exchange for FRET. For normal van der Waals interactions, both interacting atoms are in the ground state^^ In FRET, one of the interacting atoms (molecules) is in an electronically excited state. The theory of London's forces is also important with regard to the first quantum theories of FRET by F. Perrin. 1.6.1. London Interactions: Induced-Dipole-Induced-Dipole Fritz London published his quantum mechanical description of these forces in 1930 (London, 1930; London, 1937). This was two years after he published the paper analyzing the transfer of energy between mercury and thallium with Kallmann (Kallmann and London, 1928). London's interaction energy is carried out by quantum mechanical second order perturbation theory. Normal London dispersion interactions involve fluctuating dipole-dipole interactions between atoms in their ground states. An excellent review of classical and quantum mechanical theories of van der Waals interactions can be found in a 1939 review of Margenau (Margenau, 1939), and good accounts are also given in books by Davydov (Davydov, 1965) and Walter Kauzmann (Kauzmann, 1957). We limit our discussion to interactions between two atoms in their ground states; although, London forces ^^ London forces usually refer to interactions between two atoms in their ground states; however, in the review by Margenau, he discusses the interaction between one atom in the excited state, and one atom in the ground state. This is essentially the same physical circumstance as FRET, except that London forces result from averaging rapidly fluctuating forces over time (and space), and one is looking at the energy of interaction, not a rate of transfer, which is what one is observing in FRET.

18

R. M. CLEGG

are summed to explain interactions between large, closely spaced macroscopic objects. The expression for the London dispersion forces between two atoms involves the product of the corresponding visible and UV oscillator strengths of the optical transition for each interacting atom, just as FRET (Forster, 1951) (see section on Forster below). Usually the London dispersion energies are expressed in terms of the polarizabilities of the two atoms; however, the polarizabilities are related to the spectroscopic oscillator strengths (Margenau, 1939; Kauzmann, 1957). The separate components of the oscillator strengths are proportional to the squares of the (spectroscopic) transition moments for each optical transition of the atoms. The full expression for the London interaction is a sum over all significantly contributing optical transitions of the two interacting atoms (see the next paragraph, and see equation 7 of Margenau (Margenau, 1939)). If both atoms are in their ground states, the London forces are always attractive^l In addition, if the interacting atoms are not spherically symmetrical, and have anisotropic polarizabilities, their relative orientation will affect the interaction (just as the orientation dependence of FRET). The simple van der Waals interaction (not taking into account retarded potentials - which are involved in the Casmir effect (Casimir, 1948)) according to London's theory decreases as R^, where R is the distance between the atoms; this is the same distance dependence as FRET between two chromophores. The interaction term in the total Hamiltonian is that of two interacting electrical dipoles, and is proportional to \/R^ . The London interaction energy is calculated using second order perturbation theory (the first order perturbation term for atoms or molecules in their ground states is zero). According to second order perturbation theory, the total energy of interaction is proportional to a sum over all higher energy states of the combined two atom system, where each term of the sum is proportional to the square of the interaction matrix element. Therefore, the total energy of interaction is proportional io\/R^, where R is the distance between the two atoms. Each component of the sum is also proportional to the product of the squares of the transition dipole moments (these are identical to the spectroscopic transition moments) for each atom between the ground state and the excited state for that term of the sum. In addition, each term of the sum is divided by the difference between the combined energies of the two ground states and combined excited states corresponding to that term of the sum. Although the sum is extended over all higher energy states, the higher energy states are usually assumed to be small, ^ However, interestingly (especially within the historical context of FRET) if one interacting partner is in the excited state, the interaction can become repulsive. If one atom is in the excited state, the atomic interaction shows resonance; that is, the interaction becomes very strong when both the electronic transitions (absorption and emission oscillator strengths) have large values at very nearly the same frequency (energy). In addition, if one of the molecules is in the excited state, the interaction energy can vary as \/R , because the energy contribution from the first order perturbation calculation is no longer guaranteed to be small. This will become important when we deal with F. Perrin's FRET theory.

THE HISTORY OF FRET

19

and are not included in calculations. This sum can be written in terms of the product of polarizabilities of both atoms. The denominator of each term is an energy difference, where the higher energy is subtracted from the lower energy. Therefore, each term contributes a negative energy component, meaning that the normal van der Waals interaction is universally attractive. Although in many respects the theories of London interactions and FRET are similar, the two theories are not identical. One major difference is that the London interactions are calculated from second order time independent perturbation theory; this is the origin of the X/R' dependence and the squares of the transition matrix terms. The y R^ dependence and square of the transition matrix terms in the Forster expression for energy transfer emerge from different reasoning (application of Fermi's Golden Rule to calculate a rate), as I will discuss later. Fermi's Golden Rule can only be applied when the interacting oscillators are dynamically incoherent; this point will be important when we discuss the Perrins' treatments of energy transfer, because they did not find a XJR^ dependence, but a l/i?^ dependence, essentially assuming a coherent interaction. However, as already mentioned, London found the correct y R^ dependence. Since the London interactions and energy transfer interactions originate from dipole-dipole interactions, it is not surprising that Kallmann and London was working concurrently on both interactions. He derived both quantum mechanical theories for the vapor state essentially simultaneously. It is remarkable that he achieved this straight away so soon after the quantum theories by Heisenberg and Schrodinger. 1.6.2. Keesom and Debye Interactions: Dipole-Dipole and Dipole-Induced-Dipole Previous to London's theory, inter-molecular interactions between molecular dipoles had already been proposed as the basis of molecular interactions. The Keesom orientation effect considered the interaction between two permanent molecular dipoles (Keesom, 1912); if the dipoles are strong, this can orient the interacting dipoles. Debye described induction forces between a permanent molecular dipole, and an induced molecular dipole (Debye, 1920; Debye, 1921); this is known as the Debye interaction or the induction interaction. For both the Debye and Keesom interations the energy varies as the inverse 6^^ power of the interatomic interactions, as do the London forces. The latter theory of London describes the van der Waals dispersion interactions as due to fluctuating oscillating induced molecular electric dipoles (London, 1930; London, 1937); that is, no permanent dipole need exist. All three interactions, dipole-dipole, dipole-induced dipole, and dipole induced - dipole induced, are usually included as components of van der Waals forces (Israelachvili, 1992). In contrast to the Keesom and Debye effects, the van der Waals interaction as described by London's dispersion forces is a pure quantum mechanical effect.

20

R. M. CLEGG

and is always present (although classical non-rigorous derivations are often given). The designation - dispersion - refers to the dispersion of light in the visible and UV spectral region (remember that the energy of van der Waals interactions can be expressed in terms of the oscillator strengths of the interacting molecules). As we will see, Forster's expression for the rate of FRET also involves the optical oscillator strengths (usually expressed in terms of the absorption and emission spectra).

1.7. FRET BETWEEN ORGANIC CHROMOPHORES IN CONDENSED SYSTEMS We've now arrived at the time when the first attempts were made to explain observations indicating energy transfer over long distances in condensed systems (in solution). The first classical description of this by J. Perrin (Perrin, 1925; Perrin, 1927) predated London's quantum mechanical derivation of energy transfer in the vapor phase, but followed Cario and Franck's studies of energy transfer in vapors. As indicated above, dipole-dipole interactions were well understood at this time, and the description of the Hertz oscillator contains all the essentials needed to explain FRET on the basis of classical models of atomic electric oscillators. The classical description of FRET in condensed matter systems, with imposed restrictions from the old quantum theory, involves the comparison of the energy escaping to the far field from a Hertzian oscillator when it is alone, to the energy escaping when another molecule or atom (the acceptor) is in the near field zone. All the concepts that have been introduced above come into play: the oscillating electric field of a Hertzian dipole (near and far field), the quantum states of the oscillators (atoms), the requirement that the two communicating oscillators be in resonance and that the orientation of the two oscillators be favorable, and the idea of competition between emission of radiation and energy transfer. All these concepts were standing by, ready to be put into place when J. Perrin approached this problem for fluorophores in condensed solvents. However, because the new quantum mechanics had just begun, some rather subtle concepts in timedependent quantum systems were not obvious. 1.7.1. Experimental Observations of Energy Transfer in Solution The experiments that led J. Perrin to attempt a theoretical interpretation of energy transfer between molecules, involved fluorescence polarization in a solution of a single chemical species of fluorophore. It had been discovered by Weigert (Weigert, 1920) and by Gaviola and Pringsheim (Gaviola and Pringsheim, 1924) that the polarization of fluorescence emission from solutions of dye molecules began to decrease rapidly when the concentration was raised to a critical value (approximately 3 mM). This happened even when the fluorescence intensity (corrected for trivial absorption of fluorescence) was still linearly increasing with concentration. A fluorescence polarization

THE HISTORY OF FRET

21

measurement determines the degree of rotational freedom of the fluorophores. If the fluorescent molecules are in a rigid environment or highly viscous solution so that they cannot rotate within the time of fluorescence decay (usually between 1-10 nanoseconds), the polarization contribution from individual molecules will have a high value (a maximum of 0.5). If the fluorescent molecules act independently, the polarization should be concentration independent. It could be shown for several well known dyes that the polarization was appreciably reduced when the molecules were on the average separated by about 50-80 Angstroms, much larger than the combined radii of the molecules. This distance was also much greater than the distance over which the excited fluorophores could diffuse within their excited state lifetimes (especially in high viscosity solvents, or solid solutions). This was the conundrum that led eventually to the discovery of FRET in condensed systems. 1.7.2. The Theories of J. Perrin and F. Perrin 1.7.2.1. J. Perrin's Model A simple classical model to explain this polarization decrease was developed by J. Perrin (for details see below) (Perrin, 1925; Perrin, 1927). He hypothesized that the transfer of the excitation energy could hop from one molecule to the other through interactions between oscillating dipoles of closely spaced molecules. According to the classical theory of electrons in a molecule and the early quantum ideas, an excited molecule will oscillate at the frequency V corresponding to the magnitude of the excitation energy (the correct view according to the Bohr model is that the energy of the emitted light, AE'^.^ = hv , is the difference between two energy levels, but it was known that the classical electron oscillator explained much of spectroscopy). Thus, he modeled the participating molecules classically as Hertzian electric dipoles (Hertz, 1888; von Hippel, 1954). As we know, close to a Hertzian dipole the oscillating electric field resembles a static dipole. Perrin assumed that if the molecules were separated by a sufficiently small distance, the energy could be transferred to the acceptor molecule non-radiatively. He called this ''transfert d'activation''. This is of course the same type of transfer considered by Mensing, Holtsmark and Nordheim (Holtsmark, 1925; Mensing, 1925; Nordheim, 1926) in their theories on atomic vapors. However, his derivation took quite a different route. According to his model of two interacting identical Hertzian dipoles, Perrin calculated that this distance is approximately XJln;, where X is the wavelength of a free electric field oscillating at the frequency of the atomic electric field, v , A = c/v where c is the speed of light. A quantitative account is given below. Because he assumed that the two molecules were identical, A is also the wavelength of the light used to excite the original donor. In J. Perrin's model the molecules had the exact same frequency of their electron oscillations; that is, the two Hertzian oscillators are in exact resonance. The two dipoles will

22

R. M. CLEGG

exchange energy similar to the resonance exchange of energy between two identical weakly interacting classical mechanical harmonic oscillators (just as two identical balls hanging on identical springs attached through a rod). Using the Hertzian dipoles as a model, and assuming exact resonance, he reasoned that if the molecules were separated by less than a critical distance (which he calculated to be XJIK ), one could detect this energy transfer between identical molecules by measuring a decrease in the polarization of the fluorescence emission, as had been determined experimentally. Because the acceptor would on the average not have parallel transition dipole as the donor, the fluorescence emission of the acceptor would be depolarized compared to the originally excited donors, and this would lead to a decrease in the measured extent of polarization. Already, we can see one problem; molecules cannot have exact resonance with each other at all times due to the uncertainty principle. In addition, their energies will also be broadened by collisions and thermal motion, and strong interactions with the solvent broaden the spectra considerably. 1.7.2.2. Where did the J. Perrin's Idea of Dipole-Dipole Interaction Come From? As discussed above, the concept of interacting dipoles had been considered in other contexts for some time; so it was natural for J. Perrin to consider this once it was clear that the interaction between chromophores took place at a distance large compared to the molecular diameters. The classical model of absorption and emission of radiation, which involves oscillating dipoles (Kauzmann, 1957; Stepanov and Gribkovskii, 1968; Heitler, 1984), was also well developed by this time. Perrin's model is an application of these ideas of dipole-dipole interaction to the case where one of the molecules is in an excited electronic state, and the other is in the ground electronic state. He reasoned correctly that this dipole-dipole near-field Coulombic interaction could lead to the transfer of the excitation energy from the donor to the acceptor, without direct mechanical interaction of the two molecules and without the emission of a photon from the donor. Unfortunately J. Perrin's model says that the transfer can take place over distances of about 1000 Angstroms, which he realized was greater by a factor of 20 than the experimental results (see below). A subtle point here is that J. Perrin proposed that the intervening solvent (e.g. water at 55 molar concentration) did not participate in the ''transfert d'activation''. This is not a trivial point when we remember that the surrounding solvent did not play a role in the energy transfer experiments or theories in the vapor phase. In other words, he assumed that the solvent acted only as a dielectric bath. 1.7.2.3. F. Perrin's Model F. Perrin (J. Perrin's son) was one of the pioneers of fluorescence (Perrin, 1929), He contributed extensively to the basic concepts. He extended J. Perrin's theory of energy transfer by developing a quantum mechanical model (Perrin,

THE HISTORY OF FRET

23

1932; Perrin, 1933) (for details see below), similar to what had been suggested for transfer of energy between different atoms in gases (Kallmann and London, 1928). However, he concluded, as had J. Perrin, that the rate of transfer takes place proportional to y R^ ; this results in energy transfer at much longer distances than found experimentally. F. Perrin also later considered collisions between the chromophores and the solvent molecules, as well as Doppler effects (Perrin, 1932; Perrin, 1933). These collisions broaden the spectrum of the absorption and emission of the molecules, which had been originally assumed by J. Perrin to be infinitely sharp in order to guarantee effective interaction. Such collisional and Doppler effects had been the subject of much spectroscopic research on vapors in the first two decades of the 19^^ century, and had also been considered by Kallmann and London in the vapor phase. The broadening of the spectra is important for the following reason (this will also play a central role in Forster's theory). The energy lost by the donor must exactly equal the energy gained by the acceptor. The probability that the energy levels of the donor and acceptor molecules will simultaneously have exactly the precise values necessary to conserve energy during the transfer is much less than one. Each molecule has only a certain probability (weighted by the spectral dispersion) of being anywhere within the small spectral distributions caused by the collisions. This decreases the probability of resonance, because the two interacting dipoles must be closer than found by J. Perrin for a successful transfer of energy to take place. F. Perrin used the known theory of spectral collisional broadening to show that in the case of collisions the new distance for energy transfer is reduced to approximately (/l/2;r)(F/r)/^

where t is

the time between collisions of the solvent with the molecule (at most« 10^^ sec), and T is the fluorescence lifetime ( « 10~^ sec ). This would reduce the distance to about 200-250 Angstroms, which was still much too long, and would lead to transfer at about 100 micromolar concentrations, instead of the experimentally determined 3-5 millimolar. In addition, the theories of the Perrins (classical and quantum mechanical) did not provide a simple means to interpret the solution experiments. This discrepancy remained a puzzle for about 20-25 years, perhaps so long due to the Second World War. 1.7.3. A Derivation of the Perrins' Estimated Distances for Two Electron Oscillators in Exact Resonance I have given in the previous sections the results of the Perrins' calculations; the following two sections are for those more quantitative aficionados who are curious how one can arrive at their answers. I only outline the basic line of attack. The reader who is not interested in these quantitative calculations can skip the next two sub-sections; but an understanding of the dynamic rates of energy transfer when the two molecules are limited to two states, and when the molecules are in exact resonance, is central for understanding why the Perrins

24

R. M. CLEGG

calculated such long distances. This will also be important when I present the major contributions of Forster. 1.7.3.1. The Classical Derivation of J. Perrinfor two Hertzian Oscillators in Exact Resonance - with a Pinch of Old Quantum Theory The electric field Ej^ in the near field zone surrounding a "donor" oscillating dipole has the same form as the field of a static dipole. This is

nR Where n is the index of refraction, R is the vector from the dipole (assume to be a point dipole) to the point of observation, and ju^ is the dipole moment. The arrows indicate a vector quantity, and the carrot signifies a unit vector. If another dipole (the acceptor //^) is placed in the E^ -field, the energy of interaction E is

n R where the last equality is because in J. Perrin's theory the donor and acceptor dipoles are assumed to be identical, K is the orientation factor,

'^ = [/>./iz,-3(/i.-^)(A>-^)]Using a bit of Planck's old quantum theory, we can set the energy of interaction equal to a frequency (corresponding to the energy of interaction) and therefore calculate the time period of oscillation. E = KLi^ n^R^ = ho) ^ fi T la

r~

I

int

/

int

, or , r '

'

= filE int

I

= fin^Rj KI/ la

11^

In other words, the rate of transfer k^ is k^ = l/r.^^ = Kf/ jfinR!' . From the theory of a Hertzian oscillating dipole, we know that an isolated quantized Hertzian dipole radiates its energy with a time constant of r = 3hc/a/jul

, where co is the frequency of oscillation of the donor Hertzian

oscillator, c is the speed of light.

THE HISTORY OF FRET

25

Now we simply find the distance R^ where the natural decay time of the oscillator is equal to the time that the energy is transferred, r^ = r.^^ ^^; this gives , R'=

3KC^

. . A' « . 0 U ' ;

3K:

= n (o

[In)

n

or R^ « 0.2A . So we see that according to this model, the energy transfer would take place over a distance approximately that of 1/5 the wavelength of light radiating from the oscillating dipole oscillator. This would be about 100 nm, which is much too large. The reason for the \j R^ dependence, which leads to such a large value oiR^, is the assumption of exact resonance. We should be clear what this means. Exact resonance between any two individual molecular oscillators is required in order to conserve energy. However, in an ensemble of molecules in solution, there is a distribution of energies, and the width of this distribution must be taken into account correctly (this was done in gases by Kallmann and London, and by Mensing, Nordheim and Holtsmark, by considering the broadening effects of collisions and Doppler shifts). The critical role that this broadening plays will become clear in our discussion below of Forster's first theory. In the following section I show that this is also the major reason that F. Perrin's quantum mechanical derivation arrived at a distance that was too large. However, we do not want to lose sight of the historical context. The basic model of J. Perrin was correct, and he used all the ideas prevalent at the time: the Maxwellian electromagnetic field of an oscillating Hertzian dipole, the decay constant of a quantized Hertzian dipole (semiclassical description), the Bohr condition of quantum energy jumps and the condition of resonance between the two Hertzian dipoles exchanging energy. When J. Perrin first developed his theory (Perrin, 1925), the two new quantum mechanic theories of Heisenberg (Heisenberg, 1925) and Schrodinger (Schrodinger, 1926b; Schrodinger, 1926a) were just being developed. 1.7.3.2. The Quantum Mechanical Derivation ofF. Perrin with Exact Resonance This section is a resume of the quantum mechanical theory of F. Perrin (Perrin, 1932; Perrin, 1933). It is beyond the topic of this chapter to go into details, and a full understanding of this section requires some acquaintance with

^ The designation R was first given by Kallmann and London in their 1928 publication, and even earlier by Holtsmark and Mensing; the same expression was then used by J. and F. Perrin, as well as Forster.

26

R. M. CLEGG

the quantum theory of two states. But it is important to show his approach, because it is the first detailed quantum mechanical description of energy transfer in solution. He draws on the ideas of Kallmann and London (Kallmann and London, 1928), but he develops a theory applicable for solution studies. He arrived at the same estimate as the classical derivation by J. Perrin; that is, energy transfer over distances of R^ « 0.2A, where the wavelength is that of the fluorescence of the donor. This distance is far too long, and the reasons for this are revealing from a historical point of view. F. Perrin (Perrin, 1933) derived the rate of energy transfer between two identical molecules, each with very narrow energy states. The two molecules are only considered to have two states - the ground state and the excited state and they are in resonance. A similar derivation was given later by Forster (Forster, 1965a), where he used this theory to illustrate exciton theory with a molecular dimer. Forster mentions that this oscillatory transfer rate for resonance between the two monomers of an exciton dimer would be difficult to measure if it took place exactly by this mechanism. Indeed, it is, and to observe optical resonance oscillations requires time resolution far beyond that available in Forster's time. In a normal FRET mechanism - i.e. Forster transfer - the transfer takes place between a single level of the donor to either a continuum, or many closely spaced states, in the acceptor; and the theory for this is different and usually uses the Fermi Golden Rule. The reader must consult F. Perrin's paper for details. Similar accounts of two interacting molecules with two states are available in QM textbooks (Pauling and Wilson, 1935; Davydov, 1965; Landau and Lifshitz, 1965; Schiff, 1968; Cohen-Tannoudji et al., 1977). A very good discussion of many aspects of two-state systems, with and without coupling to the environment, can be found in the textbook by Scully and Zubairy (Scully and Zubairy, 1997). Our short derivation follows Davydov's book (Davydov, 1965), but the derivations of other texts are identical. As mentioned above, the derivation of the basic equations is based on the work of Kallmann and London (Kallmann and London, 1928). We consider one of the two molecules to be in an excited state, and one to be in the ground state; therefore, the first order perturbation to the energies does not go to zero, as in the London interactions between two ground state molecules (see the section on London's theory). The two wavefunctions (including their time dependence) of the whole system 4^, and 4^^ (including both molecules) are sums of products of the stationary slates of each of the molecules ^ ( l ) and |^(2) (1 and 2 designate the different molecules)

* i ' , = - r K ( 0 n ( 2 ) + n(0v'„(2)}e"

V2

THE HISTORY OF FRET

27

Subscripts n and 0 designate which molecule is in the excited state n or the ground state 0 . The molecules are identical, and only one can be in an excited state. £", and E^ are the energies of the two states (formed from the linear combination of the products of the atomic orbitals) for which we have to solve. First order perturbation theory gives the correction to the energies of the combined system (the zero order energy of the two-molecule system, with one molecule in the excited state, is E^ + E^) to be 2

2

K le^/RjD

R

is the Coulomb perturbation between the two molecules.

Substituting the expressions for the wavefunctions and the perturbation, we get 2

AE^{R) = -AE^{R) = U{n\P\0)\'K(l,2)

,

K

where \n) and |o) designate that the corresponding molecule is in the n* excited state or in the ground state. /^(l,2) is the geometric orientation factor between the dipole moments and \{n\r\ 0)f is the square of the dipole transition matrix element. In terms of the oscillator strength of the 0 -> n transition, \(^n\r\o)\^ =hf^j2ju^o)

(Forster, 1951; Kauzmann, 1957; Stepanov and

Gribkovskii, 1968). This derivation is very similar to the quantum mechanical theory of London's van der Waals forces; however, the London interaction between two ground state molecules varies asl/i?^ (from the second order perturbation). In the case where one molecule is in the excited state and we are at exact resonance, the energy of interaction varies as l/i?^ (because in this case we must use the first order perturbation). The excitation energy of the two states Tj andT^ is distributed at any moment over both molecules; that is, the separate molecules do not have well-defined energies at any time. Now we calculate the rate of energy exchange between the two molecules according to F. Perrin. Using the calculated energies and wavefunctions, we can write (leaving out all the details) a superposition of the states ^ ^ and ^^ • r = - ^ [ * , + ^ J = K ( l ) l ^ „ ( 2 ) c o s ( v O + n ( l ) l ^ „ ( 2 ) s i n ( w ) } e ' " "^^ V2

v = e'l,K{\,2)l(2n,0}R')

28

R. M. CLEGG

This is a valid solution to the Schrodinger equation of the whole system, r can be substituted into the Schrodinger equation for the combined system, and the coefficients (the time dependent cosine and sine terms) in the above equation can easily be derived. So the time it takes to completely transfer the energy from 1 —> 2 (assuming that the energy is solely in molecule " 1 " at time zero) is ITT T:

it TicoR^ ^'

The rate of energy transfer in this case is therefore

K=- T = ^lAjicoR ^ ^ ^ ^ =h\^\{nm\ R

-(1,2) =h V . {R)\

We see that the rate of transfer is proportional to the energy splitting of the two exciton energy levels —[A^^ (i^)|, which is proportional to the square of h the transition moment |(«|^|o)| . F. Perrin's quantum rate of transfer of energy shows the same XJR^ dependence as J. Perrin's classical derivation. Again, the reason is that we have assumed exact resonance of the two oscillators at all times, and infinitely sharp energy levels have been assumed. In addition he chose to look at the dynamics of the oscillating term, which Kallmann and London did not. If we use the well known expression for the Einstein rate coefficient in terms of the oscillator strength, which is the natural rate of fluorescence emission k^ competing with the energy transfer, and set the rate of emission equal to the rate of energy transfer derived by F. Perrin, k^ - k^^, we can calculate the distance where half the energy will be transferred within the excited state lifetime. This gives 7?^^ = 0.19A , which is again the same as was obtained from the calculation of two classical oscillators in exact resonance; this is much too large to explain the distances of interaction measured in solution. F. Perrin calculates R^ - ^IIX . F. Perrin was aware that this distance was too large, and he suspected that broadening of the spectra could lead to shorter R^ distances. As we learned in the section "F. Perrin's Model", he invoked collisional broadening (by the solvent), which did decrease R^ to approximately 25 nm. This was still much too long. We now know that collisional and Doppler broadening is miniscule compared to broadening caused by specific interactions with the solvent (such as polar effects). The oscillation of this system between two interacting two-state molecules with sharp energy levels, where the two systems are in exact resonance, behaves

THE HISTORY OF FRET

29

essentially the same as what is known as Rabi oscillators. Such resonant oscillators are named after Rabi (Rabi, 1936; Rabi, 1937) and were originally obtained by Giittinger (Giittinger, 1931) to describe a spin subjected to a timedependent magnetic field. In the optical realm, this is called an optical Rabi oscillation (Cohen-Tannoudji et ah, 1977; Allen and Eberly, 1987; Loudon, 2000). The oscillatory exchange between the two states of an atom in resonance with an optical field (photons) near the resonance irequency is called optical nutation (taking over the terminology of spin nutation in magnetic resonance). The frequency is essentially the interaction energy divided by h, ( 4 / / ? ) | A £ {R)\.

These are the solutions for the Bloch equations (Bloch, 1946)

describing the time dependent interaction of a two state quantum system with a weak perturbation of an oscillating electromagnetic field in exact resonance with the molecular system (Allen and Eberly, 1987). In the case of an externally applied optical field (light), the oscillatory energy exchange is between the twostate molecule and the electromagnetic field. In F. Perrin's case the electromagnetic interaction is from the near field of the other identical molecule, and the energy exchange is between the two molecules. In general, if multiple discreet eigenstates are well isolated from all the other levels of the unperturbed Hamiltonian, then the transitions between the two levels are superpositions of Rabi oscillators. When the number of coupled states becomes very large (which is the normal case for fluorophores in solution) the different Rabi oscillations with different frequencies and amplitudes interfere, and then the system evolves with the normal irreversible character. The important point is that the interacting molecules of F. Perrin were limited to only two states, and the two molecular oscillators were exactly the same and in exact resonance. The result is valid for the case of identical molecules where the width in energy levels of the two states, ^^ ( l ) ^ ^ (2) and y/^{\)\f/^{2)

are less than

2|A£'J

, which is the energy splitting caused by the

perturbation. This results in a coherent interaction, and that is responsible for the prediction of the FRET interaction at much longer distances. However, if coherence is not lost during the interaction between two atoms or molecules with discreet eigenstates, the distance dependence of the rate of energy transfer would b e l / i ? \ as calculated by J. and F. Perrin. This would happen, for instance, in a vacuum between two isolated atoms. This is not an irreversible transfer of energy from a donor to an acceptor, because in this idealized case (for solution) there is an oscillatory "back and forth" transfer. See two papers by Robinson and Frosh(Robinson and Frosch, 1962; Robinson and Frosch, 1963) and references there-in for more detailed discussion of the introduction of irreversibility with multiple states, which leads to the normal exponential decay,. If the perturbation of the two state system is not resonant, a more involved calculation shows that the probability for complete transfer is less than one. But the system still oscillates, and still shows the If R^ dependence of the oscillation frequency (Cohen-Tannoudji et al, 1977).

30

R. M. CLEGG

1.7.4. The Contribution of W. Arnold and J.R. Oppenheimer to FRET in Photosynthesis It is not commonly known that Oppenheimer reported the theory of FRET (with energy transfer at the correct distances) in 1941 (Oppenheimer, 1941). Perhaps even less well known (except in the field of photosynthesis) is that Oppenheimer's contribution to FRET, together with Arnold, led to a major advance in our understanding of photosynthesis. Although the full description of Arnold and Oppenheimer's contribution was not published until 1950 (Arnold and Oppenheimer, 1950) (probably because of his work in Los Alamos; in 1950 he was at the Advanced Institute at Princeton). Even though this latter publication is after the contributions of Forster (Oppenheimer's earlier abstract was not known by Forster at the time he developed his theory) it is clear that Oppenheimer had the correct solution in 1941; so I will consider this first. 1,7.4.1. Oppenheimer's Short Abstract of 1941 At the American Physical Society in 1941, a paper was presented by J. R. Oppenheimer, entitled "Internal Conversion in Photosynthesis" (Oppenheimer, 1941). As we know, Arnold had gone to Oppenheimer in 1940 consult about this problem (Arnold, 1991), and this short abstract was the result of their work together. Dutton wrote a historical account of sensitized photosynthesis, with a discussion of Arnold and Oppenheimer's contributions (Dutton, 1997)). In order to account for the rate of photosynthesis when light was absorbed by certain dyes (which absorbed where chlorophyll absorbed little), the energy must be transferred to chlorophyll, where it can then be transported to the photosynthetic reaction sites. In the abstract (Oppenheimer, 1941) Oppenheimer points out that the high efficiency of this transport of energy cannot be due to light emission and re-absorption (the probability for this is too small). However, the energy transfer could be enhanced if the chlorophyll molecules are much closer than the wavelength of the chlorophyll fluorescence (near field of a Hertzian dipole). In this abstract, the ratio of the number of quanta transferred to the number of quanta emitted as fluorescence is given as naXjdt, where d is the closest distance of approach between the chlorophyll molecules, n is the chlorophyll concentration, a is the absorption coefficient, and 2n% is the wavelength of light in water. This is for the case of chlorophyll molecules located randomly in space, and he has already integrated the rate of energy transfer over all molecules from d to infinity (see the next section). Although in this abstract Oppenheimer does not show the solution for the rate of transfer between only two molecules, the XJd^ result is obviously the result of integrating \j/ from d to infinity. So it is clear that he had the correct equation. He also assumed a quantum yield of one for isolated chlorophyll molecules.

THE HISTORY OF FRET

31

There is a very interesting sentence in this abstract: "This transfer gives a large scale model of the internal conversion of nuclear gamma-rays.". Oppenheimer was well versed in the theory of internal conversion in nuclear physics - the non-radiative transfer of energy between a radioactive nucleus and tightly bound electrons, which is a process that competes with the emission of gamma rays. Very probably Oppenheimer already had the solution applicable to FRET at his fingertips. Arnold and Oppenheimer discuss this in more detail in their subsequent 1950 paper (Arnold and Oppenheimer, 1950). I will explain the remarkable analogy between FRET and nuclear internal conversion in the next section. It is not surprising that this abstract was not noticed by many researchers, and certainly not by those interested in chemical and biological systems. The reasons are many: the war, the shortness of the abstract, the audience where it was presented, and the fact that he did not present a general expression for energy transfer - he only presented the integration over the distribution of acceptors that was relevant specifically to his particular problem. He became interested in this topic through his contact with William Arnold, an expert and pioneer in photosynthesis (Knox, 1996). There is no record of Oppenheimer's talk, other than this abstract, and I have not found anyone who heard Oppenheimer's presentation. At this time he was in the California Institute of Technology and Berkeley, and had not yet started to work on the war effort in Los Alamos. In an article by Arnold (Arnold, 1991), he recounts how Oppenheimer came to know about the photosynthesis problem^"^. Emerson had told Arnold of the experiments indicating the transfer of energy from phycocyanin to chlorophyll. Arnold did some experiments to verify this, and then went to Berkeley in 1940 where Oppenheimer was at the time, and consulted with him about the apparent energy transfer. As soon as Oppenheimer heard about the problem, he realized the analogy with internal conversion in radioactive nuclei, and the connection to gamma rays - only the length scale was different by 10"^ (see the end of the next section). Arnold had known Oppenheimer since 1935 when he went to Berkley to audit Oppenheimer's course on quantum mechanics. We now turn our attention to the later paper by Arnold and Oppenheimer (A&O) from 1950 (Arnold and Oppenheimer, 1950). 1.7.4.2. Arnold and Oppenheimer's Derivation of the Rate of Energy Transfer of 1950. In 1950, A&O published the work (Arnold and Oppenheimer, 1950) that was alluded to in the abstract of 194 PI In this paper, they provided a mechanism of energy transfer from phycocyanin (which is one of the accessory dyes in plants, in addition to e.g. carotene, xanthophylls and phycoerythrin) to chlorophyll in the blue green algae. The major question was "whether or not any of the light energy absorbed by these accessory pigments is used by the

^"^ I thank Robert S. Knox for reminding me of this history. ^^ This was Oppenheimer's last official scientific publication.

32

R. M. CLEGG

plant to reduce carbon dioxide" (all quotes in this section are from the A&O paper). It had been shown by Emerson and Lewis (Emerson and Lewis, 1942) that almost all of the energy absorbed by phycocyanin in Chroococus is used with an efficiency of approximately one in photosynthesis; the efficiency of photosynthesis was essentially as high as if the photons had been absorbed by chlorophyll. For our purposes we concentrate on A&O's contributions to the theory for energy transfer. A&O consider three ways the energy could be transferred: 1) by direct collision, 2) by trivial emission and reabsorption of fluorescence by another molecule, and 3) by "internal conversion, or the resonance transfer of energy from one oscillator to another in resonance with it, and lying within the quasistatic rather than the wave zone field of the former". Direct collision only happens for distances on the order of atomic dimensions, and does not require resonance; in addition, the concentration of acceptors is too small, their mobility is highly restricted and the spatial separation is much too great for collisions to occur in the excited state lifetime. Trivial absorption-reabsorption happens only for distances larger than the wavelength of the emitted light, and in the far-field zone. In this case, they calculate the percentage of light transferred is only roughly FanR = 10"^ which is much too small (F is the fluorescence quantum yield, <j is the absorption cross section of the dye, n is the concentration of chlorophyll - the acceptor - and R is the dimension of the cell). Therefore, they consider transfer in the near field of Hertzian dipole radiation, for which "the electric field of an oscillator (emitting primarily electric dipole radiation), which in the wave zone falls off linearly with the inverse distance from the emitter, increases, as the emitter approaches within distances small compared to a wave length, as the inverse cube of the distance". He also makes the important point, which is often not appreciated, that the energy transfer efficiency can be much greater that the fluorescence efficiency if the two molecules are close enough. This was very important for photosynthesis because it was known that the in vitro fluorescence quantum efficiency of the accessory pigments was often much smaller than the quantum efficiency of the energy that was transferred to the photosynthetic unit. A&O then embark on a succinct two page derivation of the rate of energy transfer. We cannot give this derivation justice, but only skim the important points for this FRET history. The reader is referred to their paper (which is terse; it is not particularly easy to read unless you are versed in EM calculations). First they calculate the fluorescence yield of the Hertzian dipole by considering the Poynting vector, using the EM vector and scalar potentials (Greiner, 1986) at large distances (far field zone) from the donor oscillator; then they calculate the rate of absorption of the acceptor dipole that is in the near field zone of the donor oscillator, using again the vector and scalar potential of a Hertzian dipole, and using the expression for the absorption coefficient in terms of the electric transition dipole moment M .

THE HISTORY OF FRET

33

The electric field in this near zone is (keeping his notation) E =

2C, sin Invt

3(5-r)r

Inv/ a is the amplitude of the vector potential A oscillation^^ C^ is the speed of light in the medium (water), v is the (spectroscopic) oscillator frequency, r is the distance from the oscillator to an acceptor, and h is Planck's constant. For some readers, the vector potential may be unfamiliar - it is a potential used in EM calculations with Maxwell equations (actually first introduced by Maxwell), which is an analogue of the scalar electric potential ^ ( r ) (which Oppenheimer also uses)^\ The expression in square brackets represents the orientation factor (the direction of the E-field in vector notation). The rate of absorption of an acceptor molecule in the near zone of the donor E-field is calculated as [clM'[a'

-^^{a-ry

jr^'^l^hW

,2

M is the square of the transition dipole moment of the acceptor. Here Arnold and Oppenheimer have assumed that the acceptor molecule, bathed in the EM field of the near zone of the donor Hertzian dipole, will absorb energy according to the same rate equations (Fermi's Golden Rule) as if the acceptor molecules were bathed in the EM field of radiation (light). The expression in square brackets is a^ times the usual K^ orientation factor of FRET. This rate of energy transfer is the same as derived by Forster (see below), but in terms of the vector potential squared and it has been assumed that the fluorescence quantum yield of the donor is one (you just have to multiply the equation by the quantum yield). The equation for the rate of energy transfer (which requires knowing an expression for 5 ) is to be compared to the expression of Forster (see later). Next, they average over all angles, giving the following equation for the rate of energy transfer to a randomly oriented acceptor at a distance of r (this averages the orientational factor, to give K^ = 2/3).

^^ The oscillating vector potential of a Hertzian dipole at position r is A = (2a/r)cos2;rv(< - r/ C ) ^^ As a reminder for those who are familiar: the magnetic field in terms of the vector potential A is B = S/ X A , and the electric field in terms of the scalar potential ^ and the vector potential is

E = -v M'j^. is not taken into account, because the aggregate in the two phases exhibit a different size and, possibly, structure (see below), ascribable to the different environment. Therefore, Mj^ and M\, species can not be related by a simple partition equilibrium. Furthermore, the prime refers to the species in the membrane, K and K' to the equilibrium

B. PISPISA ETAL.

56

•*-

"

r \ s .

o o G

5^

^*^ 5-

^"^^ r

1—

10

Time (ns)

Time (ns) Figure 2.8. Fluorescence decay curves of FIO (50 \M) in the presence of 2 mM ePC/cholesterol = 315 nm. The dashed line represents the fit to the experimental data by a ( l : l ) ; ; i _ = 265, ?ie triple exponential function, with two lifetimes fixed at 5.6 and 0.87 ns, as those observed in pure water, and the third one left free (x = 4.6). By contrast, the solid line represents the fit to the data by a four exponential function, in which two lifetimes were still maintained fixed, and the other two were left free for the global fit of the fluorescence decay (x = 0.95). The residuals clearly show how the four exponendal fiinction (a) is definitely better than the triple one (b).

constant of aggregation in water and membrane, respectively, and IC to the equilibrium constant of the water-membrane partition, linked to the change in the overall standard free energy of transfer by the following expression:^^'^^ AGO,, = .RXinKp = A G 0 , „ „ +

AGO,,^^^,

(6)

In Eq (6), AG^g^iy is the change in the standard free energy of solvation of the peptide and membrane upon partitioning, and AG^j^n that arising from both perturbation of the peptide conformation caused by the bilayer, and perturbation of the lipid structure upon partitioning and binding processes.^^'^^" '^^ No coulombic term is present because the peptides examined have no

TRICHOGIN TOPOLOGY AND ACTIVITY

57

b o

0.5a

O

.25-

"v^-0-

d

1

1

50

100

—€>

[F10](^iM)

Figure 2.9. Pre-exponential factors from fluorescence decay curves of FIO in 2 mM ePC/cholesterol (1:1), corresponding to the following lifetimes: 0.87 ns (a), 2.2 ns (b), 5.6 ns (c) and 7.0 ns (d); see text.

charged residues. Owing to the composite nature of both free energy changes, it is quite difficult to estimate AG^g^j^ and AG^jj^yg^. The sole quantity that can be confidently evaluated is the total standard free energy change of transfer, i.e. AG^^j. = -1.8 kcal -mol'^ as obtained from the value of IC reported below. Interestingly, the value of AG^^j. is much smaller than that of, say, the transfer of hydrocarbons from water to an organic solvent. For instance, for nC|QHJ2 at 25^C, AG^jj ~ - 11 kcal-mol"^.^^ In our case, however, there are contributors of opposite sign that partially balance, giving rise to a relatively small value of AG^^j.. For example, contributors with positive sign are the variation in conformational free energy and, possibly, in desolvation free energy of the peptide upon transferring from water to the membrane. We next examined each equilibrium reported in the foregoing scheme, separately.^^ The results are shown in Figures 2.10 and 2.11. The main inference to be drawn from these Figures is 3-fold. Firstly, the water-membrane partition equilibrium is characterized by a constant value of the fraction of the monomer associated to the membrane, within the 5-10"^ - 65*10"^ range of the total peptide/lipid molar ratio explored (data not shown). This implies the occurrence of a true equilibrium, whose constant is Kp =(1.3 ± 0.3)10^. Secondly, the results referring to aggregation in water are similar to those obtained in pure water and already reported in Figure 2.5, all data being satisfactorily fitted by a single curve, as illustrated in Figure 2.10. Thirdly, the curve describing the aggregation equilibrium in membrane (Figure 2.11) shows

B. PISPISA ETAL.

58

that this process is much less favored and much more cooperative than that in water. The best fit of these latter data by eq (5) gives n' = 8.0 ± 3 and K' = 154 ± 8, implying that the average size of the aggregate in membrane is definitely larger than in water. This very likely reflects the peptide state of being ordered and constrained.

.o '•0 o




i'

\

It-"-A'-

•'

- * /

\\

/ 71 JB*

8 CO

\*V*A *

1 0.2 E

•/..' n •;

,1

1 10

, 1 15

\ 1 J 20 R'(A)

Figure 2.14. Fluorescence quenching profiles as a function of the distance from the bilayer center, R' (A), generated by the distribution analysis of the membrane-inserted FIO peptide, at 0.5 |iM (a), 3.5 |iM (b) and 10.8 ^M (c), in 2 mM labeled liposomes aqueous solution Q^^y^ = 290, X^^ = 315 nm). The distribution appears doubly peaked, as explicitly shown by the fitting of curve c, because a single gaussian is unable to fit all experimental data (see text). In all cases, the second peak is much closer to the center of the bilayer. A schematic representation of a lipid molecule is reported at the bottom of the Figure, in which the possible quencher positions along the stearoyl chain are indicated by a full circle.

Table 2.1. Results of translocation experiments of FIO inside the three labeled liposomes, expressed as quenching efficiency for the fluorene donor (E) and intensity increase (FVF ') for the NBD acceptor. ^ Liposomes o-L i-L s-L

E = 1 - (F/Fo) (donor) 0.19 ±0.02 0.23 ± 0.03 0.34 ±0.03

FVFJ (acceptor) 2.0 ±0.2 1.9 ±0.2 1.9 ±0.2

a. All data were obtained using a peptide concentration of 0.5 |LIM

64

B.PISPISA^r^i:.

2.6.2. Depth-Dependent Quenching and Peptide Distribution Analysis A detailed information on the position of a fluorophore within a membrane can be obtained by the method of depth-dependent quenching,^^ based on exploiting quenchers covalently bound to the phospholipid acyl chains. By varying the quencher position along the lipid acyl chain, the depth of a fluorophore within a membrane can be explored. The experiments were carried out with FIO at three concentrations, i.e. 0.5, 3.5 and 10.8 \xM, covering the whole activity range, corresponding to the release of membrane-entrapped carboxyfluorescein of 0%, 50% and 100%, respectively. The results are illustrated in Figure 2.14, where the profiles generated by the distribution analysis of the membrane-inserted peptide are reported as a function of the distance from the bilayer center, R' (A). The inferences to be drawn from the data of this Figure are the following, i) In all cases, the distribution appears doubly peaked, because a single gaussian is inadequate to fit all experimental data. The second peak, explicitly shown only in the case of curve c, for the sake of clarity, is much closer to the center of the bilayer; ii) the ratio of the area under the peaks was estimated to be approximately 11 (curve a), 7 (b) and 4 (c), indicating that the amount of the peptide deeply buried into the membrane increases as FIO concentration increases; iii) at 0.5 \xM, the highest decrease in fluorescence intensity is caused by the liposome having the quencher in position 7, which is near the polar headgroups; iv) at both 3.5 and 10.8 |iM (curves b and c in Figure 2.14), i.e. at concentrations high enough to determine liposome leakage, the relative quenching efficiency that increases significantly is that corresponding to the liposome with the deepest quencher, according to the ratio of the area under the peaks, which is almost halved or quartered, respectively. These results once againg indicate that the peptide becomes depeely buried in the bilayer upon increasing the membrane-bound peptide/lipid molar ratio, r. An independent confirmation of the foregoing results was obtained by performing similar experiments, but with different labeled lipids. As r rises, the fluorophore is becoming, on the average, more accessible to the quencher positioned close to the center of the bilayer. This is shown in Figure 2.15, where the ratio of fluorescence intensities, measured in the presence of liposomes containing stearic acids labeled in position 5 or 16 of the acyl chain, is reported. As may be seen, the F(16)/F(5) ratio decreases with a non-linear trend as the membrane-bound peptide/lipid molar ratio increases, because of the concomitant variation of the two intensities, the first increasing and the other one decreasing. The same effect is observed with A3, despite the fact that in this peptide the fluorescent label is close to the N-terminus rather than to the Cterminus, as in FIO. This implies that the increase in the relative efficiency of the quencher located deeply in the bilayer is the same, irrespective of the direction taken by the peptide in going inside the membrane.

TRICHOGIN TOPOLOGY AND ACTIVITY

65

F(16)/F(5) 1

30

^ 40 10 r

Figure 2.15. Relative quenching of F10 by liposomes containing stearic acid labeled on the 5* or 16* position by doxyl moiety, expressed as the ratio of fluorescence intensities of the peptide interacting with the two samples, as a function of the membrane-bound peptide/lipid molar ratio, r. [Lipid] = 2 mM; X^^ = 265,, XQYYI ~ 315 nm.

2.7. PEPTIDES ORIENTATION INSIDE THE MEMBRANE The results reported in Section 2.6.2 prove that trichogin changes its position in the lipid bilayer as the membrane-bound peptide/lipid molar ratio rises. At low r values, the peptides lie close to the polar headgroups region, but as r increases, reaching a value corresponding to a peptide concentration able to cause membrane leakage, the quenching efficiency of the doxyl group positioned approximately in the middle of the bilayer increases significantly. Therefore, under these conditions, the peptide in the membrane experiences both a gathering (Figure 2.12) and a diving-like process (Figure 2.15). The change in orientation is thus strictly related to aggregation, as further illustrated in Figure 2.16, where both the fluorescence quenching data of F10 in lipids labeled with a quencher on the 5**^ or 16*^ position and the fraction of membrane-bound aggregate are plotted as a function of the membrane-bound peptide/lipid molar ratio. The {I(5)-I(16)}/[F10] quantity is, in fact, linearly related to the fraction of deeply buried peptide, representing the difference between the fluorescence intensities of F10 in the lipids labeled with a quencher on the 5* or 16* position, as obtained from the data of Figure 2.15, normalized by the peptide concentration in the membrane. A similar trend was observed for A3 analog, too. From the results, it appears that there is a strict relationship between the two sets of data, suggesting that FIO, and hence trichogin, populates two states only, i.e. a monomeric, surface bound and inactive form, and a buried, aggregated state, responsible for membrane leakage. There is,

B. PISPISA ETAL.

66

0

o

Figure 2.16. Dependence of the fraction of membrane-bound FIO aggregate (full symbols) and of the same peptide deeply buried into the bilayer (empty symbols) on the membrane-bound peptide/lipid molar ratio, r. The {I(5)-I(16)}/[F10] quantity is linearly related to the fraction of deeply buried peptide, [FIO] representing the peptide concentration in the membrane (see text).

therefore, a threshold for the transition to a transmembrane orientation, and the interconversion between the two states is controlled by the membrane-bound peptide/lipid molar ratio. A concentration-induced orientational transition, like that above described, was already reported for a few other antibiotic peptides,^^ as well as for the antifungal polyene nystatin.^^ This phenomenon can be explained by considering that a perturbation of the surface tension of the membrane arises from the binding of the peptide to the surface,^ and that at high peptide concentration excluded volume effects come also into play.^"^ As a result, at fixed lipid concentration, a transmembrane orientation becomes thermodinamically favored as the amount of peptide increases. The features of the transmembrane arrangement depend on the charge state of the peptide. For neutral or weakly charged peptides, such as alamethicin, a full insertion into the lipid bilayer is feasible, while for highly charged peptides, such as magainin, the insertion in the apolar region of the membrane is unfavored. In the latter case, an increase in peptide concentration leads to the formation of bilayer defects, the so-called "toroidal pores", which is another way in which a relaxation process for the accumulated surface tension is thought to occur.2 Accordingly, the foregoing results indicate that a high value of r forces the neutral lipopeptide trichogin to go deeply inside the bilayer.

TRICHOGIN TOPOLOGY AND ACTIVITY

67

An earlier study^^ - in which both a trichogin analog containing the quencher aminoacid TOAC and liposomes labeled with phosphatidylcholine analogs, bearing the fluorophore BODIPY at different positions along the acyl chain, were used - apparently contradicts this conclusion. Because the fluorescence quenching was found to be independent of the position of TOAC in the peptide and of the peptide/lipid ratio, the idea was that trichogin was lying along the membrane surface, at all concentrations examined. However, it has been conclusively shown"^^ that the BODIPY group attached to phospholipids exhibits a clear tendency to reside in the polar headgroups region of the bilayer, irrespective of its position along the acyl chain. Therefore, we are highly inclined to think that, using this fluorophore, in no case a transition in peptide orientation could be detected. This idea is supported by the lack of any clear dependence of fluorescence quenching on BODIPY position along the lipid acyl chain (Figure 2.2 of ref 45), indicating that the fluorescent lipid analog employed makes it impossible to determine the actual peptide position inside the membrane. By contrast, the data presented here have not such limitation,^^ because the use of doxyl labeled lipids and fatty acids is a very well established method for determining the membrane position of fluorescent probes.^^'^^ It must be also mentioned that some EPR results'*^ support the hypothesis put forward in ref. 45. However, the 0.1 mM peptide concentration used in this study is definitely higher than that normally needed for antimicrobial and membrane perturbing activity, and too high to make the EPR data and those reported here worthy of comparison, also in view of the complex interplay between the strongly concentration-dependent aggregation and membrane-peptide partition phenomena.'*^

2.8. CONCLUDING REMARKS From the data set considered here, four major conclusions can be drawn. Firstly, the analogs of trichogin GA IV investigated exhibit structural features and bioactivity very similar to that of the natural peptide, and undergo a monomer-aggregate equilibrium both in water and model membranes. The aggregates in the two phases differ, however, in size and, possibly, structure. Secondly, fluorescence quenching measurements, carried out using water soluble quenchers and quenchers positioned in the membrane at different depths, indicate that at low membrane-bound peptide/lipid molar ratio (r) trichogin lies close to the region of polar headgroups, while, as r increases until membrane leakage occurs, a cooperative transition takes place, leading to an arrangement that sees the peptide deeply buried into the bilayer. Thirdly, the transitions from a surface to a transmembrane topology and from monomers to oligomers are very likely concerted, in the sense that a transmemrane arrangement makes enough room for the peptide to aggregate. Fourthly, Forster

TOAC = 4-amino-4 carboxy-2,2,6,6-tetramethylpiperidino-l-oxyl; BODIPY = 4,4-difluoro-4bora-3a,4a- diaza-S-indacene).

68

B. PISPIS A ETAL.

energy transfer measurements indicate that, on the average, trichogin is equally distributed between the outer and inner leaflet of the membrane. The mechanism of trichogin action can be then envisaged as a two-state transition controlled by peptide concentration. One state is the monomeric, surface bound and inactive peptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. Since trichogin suffers of hydrophobic mismatch, a complex supramolecular structure is likely to form when the peptide is buried into the bilayer, "^^ an hypothesis which is under investigation.

2.9. ACKNOWLEDGMENTS We thank Prof. C. Toniolo for kindly providing the trichogin analogs, and the Ministry of University and Research for financial support.

2.10. REFERENCES 1. N. Sewald, and H.-D. Jakubke, Peptides .Chemistry and Biology (Wiley-Vch, Weinheim, 2002). 2. Y. Shai, Mode of action of membrane active antimicrobial peptides, Biopolymers 66, 236-248 (2002). 3. R. M. Epand, and H. J. Vogei, Diversity of antimicrobial peptides and their mechanisms of action, Biochim. Biophys. Acta 1462, 11-28 (1999). 4. K. Matsuzaki, Molecular mechanisms of membrane perturbation by antimicrobial peptides, in Development of Novel Antimicrobial Agents: Emerging Strategies, edited by K. Lohner (Horizon Scientific Press, Wymondham, 2002), pp. 167-181. 5. L. Yang, T. A. Harroun, T. M. Weiss, L. Ding, and H. W. Huang, Barrel stave or toroidal model? A case study on melittin pores, Biophys. J. 81, 1475-1485 (2001). 6. F. Y. Chen., M. T. Lee, and H. W. Huang, Sigmoidal concentration dependence of antimicrobial peptide activities: a case study on alamethicin, Biophys. J. 82, 908-914 (2002). 7. C. Toniolo, M. Crisma, F. Formaggio, C. Peggion, R, F. Epand, and R. M. Epand, Lipopeptaibols, a novel family of membrane active, antimicrobial peptides, Cell. Mol Life Sci. 58, 1179-1188(2001). 8. B. Pispisa, L. Stella, M. Venanzi, A. Palleschi, F. Marchiori, A. Polese, A., and C. Toniolo, A spectroscopic and molecular mechanics investigation on a series of Aib-based linear peptides and a peptide template, both containing tryptophan and a nitroxide derivatives as probes, Biopolymers 53, 169-181 (2000). 9. B. Pispisa, L. Stella, M. Venanzi, A. Palleschi, C. Viappiani, A. Polese, F. Formaggio, and C. Toniolo, Quenching mechanisms in bichromophoric, 3io-helical Aib-based peptides, modulated by chain-length dependent topologies, Macromolecules 33, 906-915 (2000). 10. P. Scrimin, P. Tecilla, U. Tonellato, A. Veronese, M. Crisma, F. Formaggio, and C. Toniolo, Zinc(II) as an allosteric regulator of liposomal membrane permeability induced by synthetic template-assembled tripodal polypeptides., Chem. Eur. J. 8, 2753-2763 (2002). 11. A. D. Milov, Y. D. Tsvetkov, F. Formaggio, M. Crisma, C. Toniolo, and J. Raap, Selfassembling and membrane modifying properties of a lipopeptaibol studied by CW-ESR and PELDOR spectroscopies, J. Pept. Sci. 9, 690-700 (2003). 12. B. Pispisa, A. Palleschi, C. Mazzuca, L. Stella, A. Valeri, M. Venanzi, F. Formaggio, C. Toniolo, and Q. B. Broxterman, The versatility of combining FRET measurements and molecular mechanics results for determining the structural features of ordered peptides in solution, J. Fluoresc. 12, 213-217 (2002). 13. C. Toniolo, A. Polese, F. Formaggio, M. Crisma, and J. Kamphuis, Circular dichroism spectrum of a peptide 3io-helix, J. Am. Chem. Soc. 118, 2744-2745 (1996).

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14. B. Pispisa, C. Mazzuca, A. Palleschi, L. Stella, M. Venanzi, F. Formaggio, C. Toniolo, and Q. B. Broxterman, Structural features and conformational equilibria of 3io-helical peptides in solution by spectroscopic and molecular mechanics studies, Biopolymers (Biospectroscopy) 67, 247-250 (2002). 15. W. C. Wimley, and S. H. White, Experimentally determined hydrophobicity scale for proteins at membrane interfaces, Nat. Struct. Biol 3, 842-848 (1996). 16. S. H. White, and W. C. Wimley, Hydrophobic interactions of peptides with membrane interfaces, Biochim. Biophys. Acta 1376, 339-352 (1998). 17. L. Stella, C. Mazzuca, M. Venanzi, A. Palleschi, M. Didone, F. Formaggio, C. Toniolo, and B. Pispisa, Aggregation and water-membrane partition as major determinants of the activity of the antibiotic peptide trichogin GA IV, Biophys. J. 86, 936-945 (2004). 18. V. Rizzo, S. Stankowski, and G. Schwarz, Alamethicin incorporation in lipid bilayers: a thermodynamic study. Biochemistry 26, 2751-2759 (1987). 19. M. Castanho, and M. Prieto, Filipin fluorescence quenching by spin-labeled probes: studies in acqueous solution and in a membrane model system, Biophys. J. 69, 155-168 (1995). 20. A. S. Ladokhin, and S. H. White, Alphas and taus of tryptophan fluorescence in membranes, Biophys. J. 81, 1825-1827 (2001). 21. H. W. Huang, Action of antimicrobial peptides: two-state model. Biochemistry 39, 8347-8352 (2000). 22. S. H. White, and W. C. Wimley, Membrane protein folding and stability: physical principles, Amu Rev. Biophys. Biomol. Struct. 28, 319-365 (1999). 23. T. J. Mcintosh, A. Vidal, and S. A. Simon, The energetics of peptide-lipid interactions: modulation by interfacial dipoles and Cholesterol, in Peptide-Lipid Interactions, edited by T. J. Mcintosh, and S. A. Simon (Academic Press, San Diego 2002), pp. 309-338. 24. C. J. Russell, T. E. Thogeirsson, and Y. -K. Shin, The membrane affinities of the aliphatic amino acid side chains in an alpha-helical context are independent of membrane immersion depth. Biochemistry 38, 337-346 (1999). 25. C. Tanford, The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed. (Wiley, New York, 1980). 26. B. Pispisa, M. Venanzi, L. Stella, A. Palleschi, A., and G. Zanotti, Photophysical and structural features of covalently bound peptide-protoporphyrin-peptide compounds carrying naphtalene chromophores, J. Phys. Chem. B 38, 8172-8179 (1999). 27. L. Stella, M. Venanzi, M. Carafa, E. Maccaroni, M. E. Straccamore, G. Zanotti, A. Palleschi, and B. Pispisa, Structural features of model glycopeptides in solution and in membrane phase: a spectroscopic and molecular mechanics investigation, Biopolymers 64,44-56 (2002) 28. M. Schumann, M., Dathe, M., Wieprecht, T., Beyermann, M., and M. Bienert, The tendency of magainin to associate upon binding to phospholipid bilayers. Biochemistry 36, 4345-4351 (1997). 29. J. Strahilevitz, A. Mor, P. Nicolas, and Y. Shai, Spectrum of antimicrobial activity and assembly of dermaseptin-b and its precursor form in phospholipid membranes. Biochemistry 33, 1095110960(1994). 30. B. Pispisa, C. Mazzuca, A. Palleschi, L. Stella, M. Venanzi, M. Wakselman, J. -P. Mazaleyrat, M. Rainaldi, F. Formaggio, and C. Toniolo, A combined spectroscopic and theoretical study of a series of conformational^ restricted hexapeptides carrying a rigid binaphthyl-nitroxide donor-acceptor pair, Chem. Eur. J. 9, 2-11 (2003). 31. G. R. Jones, and A. R. Cossins, Physical methods of study, in Liposomes: a Practical Approach, edited by R. R. C. New (Oxford University Press, Oxford., 1990) pp. 183-220. 32. A. R. Curran, and D. M. Engelman, Sequence motifs, polar interactions and conformational changes in helical membrane proteins, Curr Op. Struct. Biol. 13, 412-417 (2003). 33. K. Matsuzaki, O. Murase, N. Fujii, and K. Miyajima, Translocation of a channel forming antimicrobial peptide, magainin 2, across lipid bilayers by forming a pore, Biochemistry 34, 6521-6526(1995). 34. W. C. Wimley, and S. H. White, Determining the membrane topology of peptides by fluorescence quenching. Biochemistry 39, 161-170 (2000). 35. E. London, and A. S. Ladokhin, Measuring the depth of amino acid residues in membraneinserted peptides by fluorescence quenching, in Peptide-Lipid Interactions, edited by T.J. Mcintosh, and S.A Simon (Academic Press, San Diego 2002) pp. 89-115.

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36. A. S. Ladokhin, Distribution analysis of depth-dependent fluorescence quenching in membranes: a practical guide, Meth. Enzymol. 278, 462-473 (1997). 37. A. S. Ladokhin, Analysis of protein and peptide penetration into membranes by depthdependent fluorescence quenching: theoretical considerations, Biophys. J. 76, 946-955 (1999). 38. J.C. Mclntyre, and R.G. Sleight, Fluorescence assay for phospholipid membrane asimmetry, Biochemistry 30, 11819-11827 (1991). 39. L. J. Lis, M. McAlister, N. L. Fuller, R. P. Rand, and V. A. Parsegian, Measurement of the lateral compressibility of several phospholipid bilayers, Biophys. J. 37, 667-672 (1982). 40. S. Mazeres, V. Schram, J. F. Tocanne, and A. Lopez, 7-Nitrobenz-2-oxa-l,3-diazole-4-yllabeled phospholipids in lipid membranes: differences in fluorescence behavior, Biophys. J. 71, 327-335 (1996). 41. D. E. Wolf, A. P. Winiski, A. E. Ting, K. M. Bocian, and R. E. Pagano, Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer. Biochemistry 31, 2865-2873 (1992). 42. M. Lee, F. Chen, and H. W. Huang, Energetics of pore formation induced by membrane active peptides. Biochemistry 43, 3590-3599 (2004). 43. A. Coutinho, and M. Prieto, Cooperative partition model of nystatin interaction with phospholipid vesicles, Biophys. J. 84, 3061-3078 (2003). 44. M. Zuckermann, and T. Heimburg, Insertion and pore formation driven by adsorption of proteins onto lipid bilayer membrane-water interfaces, Biophys. J. 81, 2458-2472 (2001). 45. R. F. Epand, R. M. Epand, V. Monaco, S. Stoia, F. Formaggio, M. Crisma, and C. Toniolo, The antimicrobial peptide trichogin and its interaction with phospholipid membranes, Eur. J. Biochem. 266, 1021-1028 (1999). 46. R. D. Kaiser, and E. London, Determination of the depth of BODIPY probes in model membranes by parallax analysis of fluorescence quenching, Biochim. Biophys. Acta 1375, 1322(1998). 47. C. Mazzuca, L. Stella, M. Venanzi, F. Formaggio, C. Toniolo, and B. Pispisa, Mechanism of membrane activity of the antibiotic trichogin GA IV: a two-state transition controlled by peptide concentration, Biophys. J. 88, 3411-3421 (2005). 48. V. Monaco, F. Formaggio, M. Crisma, C. Toniolo, P. Hanson, and G. L. Millhauser, Orientation and immersion depth of a helical lipopeptaibol in membranes using TO AC as an ESR probe, Biopolymers 50,239-253(1999). 49. M. R. R. de Planque, and J. A. Killian, Protein-lipid interactions studied with designed transmembrane peptides: role of hydrophobic matching and interfacial anchoring, Mol Membr Biol. 20, 271-284 (2003).

THEORY OF METAL-FLUOROPHORE INTERACTIONS Nils Calander This chapter is mainly a review of research done by the author, concerning theory of surface plasmon resonance interaction with fluorophores. Surface plasmon coupled emission (SPCE) is studied theoretically and compared to experiment. Surface plasmon resonance optical field enhancement is investigated at elongated particles by solving the Maxwell's equations with the use of spheroidal vector wave functions. Finally, the theoretical possibility of trapping fluorophores by optical gradient forces at surface plasmon enhanced hot spots is examined.

3.1. INTRODUCTION Surface plasmon resonance (SPR) is a phenomenon that may occur at metal interfaces. It is also the name of a method that is used to investigate biomolecular binding to surfaces ^"'*. It relies on the absorption of optical energy by surface plasmons and is very sensitive to the optical properties near the surface. The opposite to optical absorption by excitation of surface plasmons is that surface plasmon energy couple out into an optical field. This is called surface plasmon coupled emission (SPCE) and has recently ^"^ been shown to have a potential for enhanced detectability of fluorophores near a planar surface. The radiation is concentrated at specific angles and polarizations making it possible to collect a larger proportion of the emitted photons. The Raman cross-section can be greatly enhanced by surface plasmons. This is called surface enhanced Raman scattering (SERS). The method has been refined and found uses in the life sciences. It has been found that the Raman signal can be increased several orders of magnitude ^^"^"^ by surface plasmon Physics Department, Chalmers University of Technology, SE-412 96 Goteborg, Sweden e-mail: [email protected] 71

72

N. CALANDER

resonance at colloidal metal particles, preferentially silver or gold. This has made Raman spectroscopy of single molecules possible. It has been suggested that the strong optical field gradient may attract molecules to the hot spots by gradient forces ^^, in a way enhancing the Raman spectroscopy. The opposite of the optical field enhancement at hot spots is that the radiation out from the hot spot is also enhanced, i.e. radiative enhancement. An excited fluorophore nearby couple out its energy more efficiently by the surface plasmons, an antenna or transformer action, resulting in radiative decay enhancement. This technique is sometimes called radiative decay engineering (RDE) ^'^'^^-2^^ which has a number of advantages in fluorescence spectroscopy. The quantum yield of a fluorophore is greatly enhanced. The fluorescence signal is also enhanced, as is the time before bleaching of the fluorophore. These advantages may make weak intrinsic fluorescence of biomolecules come into play and tagging by fluorescent labels may be unnecessary. Also, the radiative decay enhancement may make the optical trapping of fluorophores possible.

3.2. SURFACE PLASMON RESONANCE 3.2.1. Plasma Oscillations Interacting charged particles may show plasma oscillations where the restoring force is electrical, as illustrated in Figure 3.1. If the free electrons in the metal are displaced from the lattice of positively charged ions an electrical restoring force tries to restore neutrality. Since the electrons are rather free an oscillating behavior results at the so called plasma frequency.

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Figure 3.1. a): A weight hanging in a spring. Oscillations are due to the inertia of the weight and restoring forces in the spring, b): Assuming the electrons moving as a whole in the metal compared to the fixed positive background of ions, displacements of the electrons cause electrical restoring forces turning the metal back to the homogenously neutral state. These restoring forces make the electrons oscillate, i.e. plasma oscillations.

THEORY OF METAL-FLUOROPHORE INTERACTIONS

73

3.2.2. Surface Plasmon Resonances The plasma oscillations near a surface may interact with the electromagnetic field from the charged particles in such a way that the plasma oscillations are localized to the surface. These oscillations are called surface plasmons and are illustrated in Figure 3.2. Phenomenologically, the metal can at optical frequencies be seen as having a permittivity of negative real part. The imaginary part is due to losses. In the quantum theory language the plasma oscillations are quantized in the energy quanta hcop where cOp is the oscillation frequency, and are called plasmons. The surface plasma oscillations are quantized in units of surface plasmons. The quantized theory is not needed for explaining the surface plasmon resonance effects in this chapter, and is therefore not discussed further. The theoretical background of the surface plasma oscillations in this chapter only relies on the Maxwell's equations and the macroscopic behavior of the metal in terms of permittivity, believed to be valid down to feature sizes of below a nanometer. The Maxwell's equations are solved separately in the metallic and non-metallic domains and joined together by appropriate boundary conditions. The boundary conditions are that the normal component of the electric field times the permittivity and the in-plane component of the electric field are continuous at the interface. The magnetic field is also continuous. The permittivity versus wavelength is shown in Figure 3.3 for silver and gold.

Figure 3.2. Surface plasma oscillations at a plane and spherical interface between two media, one of positive and one of negative permittivity. The plasma oscillation is a solution to the Maxwell's equations with the appropriate boundary conditions. Two evanescent plane waves in the two media are matched at the interface. The waves are traveling or standing in the plane and exponentially decaying away from the interface. The electromagnetic field is accompanied by oscillating surface charges at the interface. A condition for surface plasma oscillations at the plane interface is that £1+82 < 0 .

74

N. CALANDER Gold

Silver • Ima^inbry 0

r^ ii^^jiiiiiiii'iHtmi,

.}...•....7

-20

i RealV^ -40 -60 on 0.2

i 0.4

i

i

0.6 0.8 X{\irr\)

'1

1 1.2

0.2

0.4

0.6 0.8 A,{^m)

1

1.2

Figure 3.3. The permittivity (dielectric function) of silver and gold. The real parts are negative due to plasma oscillations. The imaginary part is due to electric losses in the metal. Data from Reference

3.3. THEORY OF SURFACE PLASMON RESONANCE AT PLANAR STRUCTURES Surface plasmon resonance at planar structures is studied by solving the Maxwell's equations using Fresnel theory. The electromagnetic fields from oscillating dipoles are expressed as integrals over plane waves in order to fit into the Fresnel scheme. In this way the interaction of fluorophores with planar structures possessing surface plasmons is studied. In particular surface plasmon coupled emission is theoretically investigated. This section is mainly a review of Reference ^. Fluorescence spectroscopy is an important method for high sensitivity detection and analysis in many fields of biotechnology. One way of improving the sensitivity and selectivity is by resonance enhancement and modification of the fluorescence signal. In recent publications ^'^'^^ experimental studies of surface plasmon-coupled directional emission (SPCE) are reported. Surface plasmons in a thin metal layer, excited by fluorophores, can radiate into a glass prism at sharply defined angles determined by the emission wavelength and the optical properties of the respective layers and the glass. SPCE is related to surface plasmon resonance (SPR) \ a well-known and used phenomenon in which absorption of light takes place at specific angles of incidence, which are sensitive to the optical properties near the metal surface. SPCE is in a sense the opposite; emission is detected rather than absorption. The directional property of SPCE can be used to observe the emission selectively. Coupling to the surface plasmons and the collection of the emission can be made much more efficient than with standard methods. Only fluorophores close to the surface contribute to the SPCE, which means that

THEORY OF METAL-FLUOROPHORE INTERACTIONS

75

background emission is highly rejected. These properties of SPCE will surely find numerous applications in biotechnology and chemistry. One aim of this chapter is to show that a theoretical / simulation approach to SPCE is a useful tool in order to design experimental setups and structures, fine-tune measurements, predict results, explain experimental findings, and to show promise for further refinement of SPCE. Quantities simulated are SPCE angles, power levels, decay enhancements and decay times. In this chapter a theoretical simulation approach for SPCE is compared to experimental results. Theoretical explanations of some experimental findings thus become evident from these simulations. 3.3.1. Basic Theory The radiation from a dipole can be decomposed into an integral over plane waves, see Figure 3.4. This is a 2-dimensional Fourier transform. The decomposition is also called the Weyl identity ^^. The electromagnetic field is also divided into a p- and an s-polarized part. Fresnel theory ^^'^^, i.e. the theory of refraction of plane waves in a dielectric planar structure, can then be applied, see Figure 3.5. Proper matching of the plane waves has to be done at the boundaries and also at the dipole. A pre-integration (pre-summation) in azimuth (cp) is done by using Bessel integrals, see Reference ^^, which means that only one-dimensional numerical integration (in 0) has to be done in order to calculate the electromagnetic fields at any point. The method can also be explained in terms of the Sommerfeld identity ^^. No integration is needed for calculation of the far-fields. Parseval's equation is used for the power flow.

iillliilililsli;-;""' ''"••"Jlilillliillliii

Figure 3.4. The electromagnetic field from an oscillating dipole is fit into the Fresnel's scheme by decomposing it into an integral (or sum) of plane waves in the two main directions of the planar structure. The evanescent waves, i.e. non-propagating plane waves or plane waves with imaginary normal components of the wave-vector have to be included. When incorporated into the layered structure (Figure 3.5), appropriate matching of the electromagnetic fields at the surface indicated by the straight vertical line above, has to be done, with the assumption of including an electromagnetic

N. CALANDER

76

2 ; 3

k,

K

k2

W§m

N

1 k3

K { 14 \

\ k^

Figure 3.5. The Maxwell's equations for the electromagnetic fields are solved by using Fresnel's theory of plane wave propagation in a planar dielectric structure. Two plane waves with mirrored wave-vectors are assumed to propagate in each homogenous layer and boundary-matched to the plane waves in the neighboring layers. The in-plane components of all couples of plane wave's wave-vectors in all layers are the same (Snell's refraction law). Appropriate matching of the electromagnetic fields has to be done at all boundaries. When including an oscillating dipole in one of the layers as a radiating source, appropriate matching of the electromagnetic fields also has to be done at the dipole.

Here some equations used in the simulations are given. The quantities in the equations are defined in

Table 3.1. The integrals for the electromagnetic fields at an arbitrary position in the layered structure, from a dipole normal to the plane are: 1 3

^

Y\^

H = - ^ c p , j d n ^ _ p ^ j , (k„n^p)(ae'^«"'^ +be •^»"'^) 47C

4nnn d

0

0

(1)

-y/nd-np

V" oo, and that V is finite everywhere. The solution outside the spheroid is V = E[^+AQ,(^)]II

(11)

where (12) ^„^(^o)-^Q.(^o)

Q>(^) = | l n

-1 is a Legendre function of the second kind ^-1

The field enhancement y at the top or bottom of the spheroid (r| = ±1, § = ^o)is

Ttop

''-t y -O' NH

o U

,A^^SH

HO'

KILI

NH2

L-homocysteine 1

OH

OyJ ^^ ^ / v . ^"^^^^^^c^

^ NH2

1

Hcys* A

Protein B

1

Y^^oo Proteinv„ „

"^^^^^^0

s-s ^ ^ •

coo^ 1

c

Figure 6.2. We are interested in the following: how might homocysteine chemistry fundamentally differ from that of other biothiols? Can its unique chemistry serve as the basis for selective spectroscopic detection methods? Can its fundamental chemistry help explain its role in disease? Chemical hypotheses based on free (A) and protein homocysteinylated materials (B,C): (A) The geometrically-favored intramolecular alpha-H abstraction coupled electron transfer by Hey thiyl to afford an alpha amino-carbon-centered radical. The same process in Cys and GSH proceeds via less readily formed 4- and 9-membered ring transition states, respectively. This analysis was proposed initially by Zhao et al and recently shown by us to promote highly selective Hcy-induced colorimetric and fluorescence signals. This allows us to selectively detect Hey over other thiols'*'' and additionally to propose new studies of Hcy's biochemical roles {vide infra). (B) Proposed stabilization of the S-centered radical cation of a disulfide-conjugated Hey. This radical cation has been postulated by Armstrong in the case of homocystine. Its favored formation compared to the analogous radical cation of cystine was reported in 1981. Armstrong noted that the amino lone pairs would stabilize the cation; more recently, Butterworth presented strong evidence that the analogous process occurs in C-terminal methionine residues, stabilized (and promoted) by the carboxylate anion. This latter Met-derived species is strongly implicated in Alzheimer's and lipid peroxidation. Hcy-conjugated proteins have not yet been studied for S-centered radical formation, to the best of our knowledge. (C) Armstrong also proposed that perthiyl (RS-S*) radicals may form more readily from homocystine than cystine and related disulfides. Hcy-conjugated proteins have not yet been studied perthiyl radical formation, to the best of our knowledge. We thus propose that structures/processes depicted in B and C may cause free radical damage to peptide backbones. Importantly, Hey is found physiologically as mainly the protein bound disulfide cysteine residue conjugate. The processes depicted in B and C are assisted by the amino and/or the carboxylate moieties in Hey. Protein-bound Hey is commonly disulfide-linked. It would thus not be necessary (as in the case for methionine) to be specifically bound at a terminus. This is because the amino and carboxylate are free to assist in these processes in a Cys residue-Hcy disulfide.

DETECTION OF BIOLOGICAL THIOLS

141

6.2. HOMOCYSTEINE METABOLISM The universal methylating agent S-adenosylmethionine (SAM) is synthesized from methionine and ATP (Figure 6.3). SAM is essential for one carbon metabolism. Demethylation of SAM produces S-adenosyl homocysteine (SAH). This reaction is followed by the enzymatic hydrolysis of SAH by Sadenosyl homocysteine hydrolyase (SAHH) to afford adenosine and Hey. A transsulfuration pathway leading from Hey to Cys begins at this point. Hey reacts with serine via Cystathionine Beta-Synthase (CBS), the vitamin B6-dependent enzyme, to afford cystathionine. Cystathionine reacts to form cysteine, a source of glutathione, sulfate and sulfite.^ Homocysteine may also be methylated, released into the extracellular medium or deaminated. Folate-dependent Hey methylation to methionine can be promoted by methionine synthase or via betaine homocysteine methylase.^ When Hey metabolism is disrupted, export of Hey from the cellular to the extracelluar medium becomes imbalanced. Lowered Hey cellular levels produce elevated export levels, resulting in more Hey in plasma and urine. Higher Hey levels in plasma and urine are thus directly related to lower methionine synthase activity and folate or vitamin Bu deficiency. Hyperhomocysteinemia is the condition where plasma Hey concentration exceeds 12-15 fiM.^ It has thus been proposed that vitamin or folate therapy may be useful for hyperhomocysteinemia-related disorders. Hey is found in several forms after being released into plasma. The sum of all these forms is the plasma total homocysteine level. Oxidation to disulfides in plasma is coupled to O2 reduction, leading to oxidative stress. Reactive oxygen species (ROS) levels can be diminished by peroxidases. Hyperhomocysteinemia appears to inhibit the expression of peroxidases.^ Nitric oxide (NO) released by endothelial cells can react with Hey to furnish S-nitrosohomocysteine (SNOHO), a strong antiplatelet and vasodilator agent. The consequence of nitrosylation is the repression of peroxide production and therefore inhibition of ROS formation.^ When present at hyperhomocysteinemic levels. Hey is not significantly affected by this mechanism. Low-density lipoprotein oxidized by ROS suppresses endothelial nitric oxide synthase expression.^ Hey has been cited as an agent involved in lowering NO availability upon its nitrosylation.^ NO is a neurotransmitter and involved in muscle relaxation and microphage cytotoxicity.^ Importantly, Hey impairs endothelial cell function in the absence of NO. Although the mechanism is not perfectly understood, it is believed that the direct action of homocysteine on endothelial cells could either involve enhanced oxidative stress or result from a direct effect of the oxidation products of homocysteine.^ The impairment of endothelial cells in hyperhomocysteinemics is believed to be an origin of cardiovascular diseases. It is believed that Hey switches

J. O. ESCOBEDO ETAL.

142

endothelial cell phenotype from anticoagulant to procoagulant. Some studies show that homocysteine-mediated cardiovascular risk can be as strong as that of hyperlipidemia.^^'^^"^^ It has been reported that high homocysteine levels were detected in up to 20 % of people suffering from heart disease. Based on the effect of homocysteine on endothelial cells, studies have potentially linked hyperhomocysteinemia to Raynaud's syndrome. Since blood vessels carry oxygen to the brain and heart, oxidative stress generated by hyperhomocysteinemia may cause brain damage and Alzheimer's disease. The overexpression of glutathione peroxidases can occur in Alzheimer patients, linking the disease to oxidative stress in the brain. In addition, elevated levels of plasma homocysteine have been detected under the same conditions. Further evidence for the role of oxidative stress is that antioxidant supplement delays the Alzheimer's-related complications. Increased risks of birth defects,^^"^^ and renal failure^^ are other diseases also related to hyperhomocysteinemia. Hyperhomocysteinemia and associated disorders may be treatable by vitamin therapy. In fact, diagnosis may help prevent neural tube defect pregnancies, ischemic heart disease and strokes, and possibly colon cancer. The risk for heart disease can be reduced up to 40 %.^^ Again, nutritional folate deficiency is often attributed to hyperhomocysteinemia. Folic acid supplementation is recommended against many of these and related conditions. Triphosphate

C H - C H 2 - S - O - C - C H N H 2 — ^ H 2 N - C H C - O H — • ^ Glutathione NH2

CH2 OH Cystathionine

CH2 SH Cysteine

Figure 6.3. Some of the pathways involved in homocysteine metabolism.

DETECTION OF BIOLOGICAL THIOLS

143

6.3. NEW PERSPECTIVES ON HOMOCYSTEINE'S ROLE IN DISEASE After many years of study, it is still not yet known if homocysteine causes cardiovascular and other disease, is a consequence of them, or is just a biomarker.^^ Our work addresses elucidating unique chemistry of homocysteine (Figure 6.2) . We plan to build upon our recent finding that proves homocysteine forms an alpha amino-carbon-centered radical at room temperature and neutral pH more readily compared to all other amino acids tested to date."^^ The chemistry of biological thiols is complex. This has prompted investigators to assume that homocysteine at elevated levels causes oxidative stress.^^ Jacobsen has pointed out that cysteine, however, undergoes similar chemistry and is present at higher (20-30 fold) concentrations than homocysteine but is not often associated with oxidative damage."^^ Jacobsen has proposed the "molecular target hypothesis," suggesting that the study of Hey should focus on elucidating its interactions with specific biomolecules. Hey promotes detachment-mediated programmed cell death, and contributes to the development of atherosclerosis in hyperhomocysteinemia. Hey decreases the expression of a wide range of antioxidant enzymes.^^ Hey inhibits glutathione peroxidase activity.^"^ Homocysteine disrupts folding in the endoplasmic reticulum.^^ Protein homocysteinylation is now under intensive study. Conjugation to bovine serum albumin (BSA), for instance, was shown to occur via a thiolate anion and disulfide exchange reactions.^^ Most (ca. 70 %) plasma homocysteine is protein-bound. Armstrong (Figure 6.2, B and C) has stated that homocystine (Hey disulfide) may, for example, be more prone to perthiyl radical formation (due to favorable, specific homocystine pj^a effects) or radical cation formation at sulfur (due to homocystine-specific favorable geometry allowing for stabilization by the amino lone pairs).^^ This allows us to propose homocystine, mixed Hcydisulfide or Hcy-protein conjugates as potential specific sites of free radical damage induction (Figure 6.2). Earlier research involving methionine residues as sites of free radical damage initiation may be relevant to Hey and disease. Posttranslational modifications of proteins are known to be initiated by disulfide radical cations.^^ Additionally, evidence exists showing that the oxidation of methionine to the radical cation may result in amyloid and prion-induced disease with backbone scission, aggregate formation and/or lipid peroxidation.^^ Recently, new studies involving the "apoE" knockout mouse model of atherosclerosis were reported. The aopE KG mouse develops atherosclerosis spontaneously. When the animals are on a high methionine diet (low folate diet, or both), the progression of atherosclerosis accelerates dramatically. Thus, the apoE KG mouse with hyperhomocysteinemia is highly prone to developing atherosclerosis. These findings have greatly bolstered the field of Hey research. ^^

144

J. O. ESCOBEDO ETAL.

There is growing interest in studying metabolites of hyperhomocystei nemia.^^ This prompts us to extend our prior work in characterizing the products of Hey carbon-centered-radical formation."^^ It is well-known that such radicals form NH3 and several other radical termination and disproportionation products.^^ Due to the ready ability of Hey to form carbon-centered-radicals,"^^ we propose that the formation of NH3 and other toxins may play a role in Hcyrelated disease. For instance, hyperammonemia is associated with Alzheimer's: however, no link between Hey and ammonia formation has yet been studied. This may be of particular importance in homocystinuric patients. They often have tHcy (total plasma) levels approaching 500 jaM. Many homocystinurics, if left untreated, have mental retardation, often severe.^^ The study of the extent of ammonia formation and other byproducts is planned. The thorough identification of products and their yields will be studied via the treatment of Hey with a variety of common oxidants at room temperature in neutral media. 6.4. OVERVIEW OF KNOWN METHODS FOR BIOLOGICAL THIOL DETECTION There are numerous procedures for detecting thiols, which mainly include chromatographic separations, immuno- and enzymatic assays, electrochemical, mass spectrometric and flow injection technology. Electrochemical detection is complicated by interference from oxidizable impurities.^'* Electrochemical detection of thiols by CE is hampered by the need for precision electrode alignment and isolation of the detector from the separation voltage. Amperometric post-column detection of cysteine and homocysteine also can suffer from low selectivity and high background current as cysteine exhibits irreversible oxidation requiring positive overpotential.^^ Small volumes in the separation capillaries in CE require that the detector be placed in-line to minimize line-broadening. Good sensitivity often requires dual electrode configurations. The stability of the detection cell components is another concern, depending on the type of analysis. Mercury and mercury amalgam electrodes have been widely used for thiols but have severe limitations including toxicity and poor stability.^^ Chemically modified electrodes require complex preparation, can exhibit poor stability and need controlled working conditions.

o

o

BTHZC

NPM

CH3

mBrB •••"••-'

,1'-thiocarl 1,1'-thiocarbonyl diimidazole

5-iodoacetamidofluorescein

Figure 6.4. Representative thiol derivatizing agents.

DETECTION OF BIOLOGICAL THIOLS

145

Fluorescence polarization immunoassays (FPIA) and enzyme immunoassays (EIA) are very useful but have shown some (inter-laboratory) imprecision (vide infra).^^ Enzymes are unstable (low shelf life compared to common organic reagents) and relatively expensive, making enzyme-based assays less attractive in spite of high specificity. Radioimmunoassays involve toxic substances. STE (Substrate-TrappingEnzyme) technology necessitates a batch chromatography step and exhibits low precision.^^ Mass spectrometry coupled to HPLC requires complex and expensive equipment. GC-MS also uses cumbersome equipment and requires tedious procedures not suitable for routine diagnostic applications. Gas chromatography-electron capture detection and flame photometry detection require tedious sample preparations and/or high operating temperatures.^^ Trap & Release Membrane Introduction Mass Spectrometry (T&R MIMS) requires time-consuming derivatizations and sophisticated instrumentation rendering it unsuitable for routine analysis."^^ The determination of specific thiols is often carried out in conjunction with HPLC separations.^ Thiol derivatizing agents often contain electrophilic alkylating groups for reaction with sufhydril moieties. They include iodoacetamides/^ maleimides,"^^ and monobromobimanes (mBrB)."^^ They are non-selective among the thiols. Other interferences are of concern as well. For instance, iodoacetamides can react with histidine, tyrosine and methionine."^^ Other reagents such as l,r-thiocarbonyl diimidazole can derivatize cysteine and penicillamine; however, derivatives can only be detected at short wavelength.'^'^ Thiol derivatization conditions can lead to several other problems. Excess derivatization agents must often be removed from the reaction mixture. In addition, conditions can be time consuming and complex. For instance, the reaction with OPA (orthophthalaldehyde) is pH-dependent. No adduct formation takes place at pH less than pH 9."^^ In some cases, the derivatives are prone to unwanted further reactions. For instance, the products of isothiocyanates and succinimidyl esters with biological thiols have limited stabilities and undergo further reactions with neighboring amines to afford thioureas. Maleimide-based derivatization agents have similar problems. Although maleimides are inert to the interferences of iodoacetamides, amines have been reported to crosslink the derivatized products. A related observation has been reported concerning the detection of penicillamine via NBD-F."^^ The possible instability of NBD-thiols leading to S-N migrations has been proposed as an explanation (Figure 6.5). NBD-F has been used successfully via precolumn derivatization; however, the sample labeling/pretreatment process takes 2 h."^^ Terminal cysteine residues converted to S-carbamoylmethyl-cysteine during the standard alkylation step with iodoacetamide afforded new insight into the difficulties of some current labeling procedures. Cyclization produced a ring structure that corresponded to a loss of 17 Da in the mass spectrum, by loss of ammonia. This modification was reported."^^ Other findings showed that every peptide known to have a cysteine at the iV-terminus was modified.

146

J. O. ESCOBEDO ETAL.

affording a lowering in m/z value of 17 Da. About 50% of the peptides concerned are lost, causing a large decrease in mass spectrometer sensitivity."^^ Some thiol-chromophore/fluorophore derivatives also are sensitive to light and hydrolysis. The OPA-Hcy adduct is stable only in dark.^^ Monobromobimane is well-known to produce fluorescent hydrolysis products.^^ When thiols are derivatized with certain maleimides, hydrolysis peaks are encountered at the beginning and at the end of chromatographic elution.^^ Hexaiodoplatinate, on the other hand, produces no hydrolysis products.^^ However, hexaiodoplatine exhibits a broad reactivity: thioethers, thiazolidines and ascorbic acids are among the reported interferences. Some derivatization agents themselves are prone to instability. lodiacetamides"^^ are unstable to light. In addition, monobromobimane is known to be photosensitive and unstable in water. ^"^ The instability of certain maleimides in aqueous conditions necessitates the use of water-miscible organic cosolvents.^^ The most widely used universal thiol derivatizing agent in clinical studies is monobromobimane.^*' In addition to problems with fluorescent hydrolysis byproducts formed during pre-column derivatization (vide supra), there have been older reports describing the need to remove excess monobromobimane (e.g., by Sepharose-SH treatment prior to HPLC analyses).^^ Researchers have also noted that levels of fluorescent impurities in monobromobimane vary significantly from batch to batch.^^ Monobromobimane is stable in wateracetonitrile but at temperatures < -20 °C . Solutions must be replaced after a few days.^^ Thiol and sulfide quantitation kits are available. The procedure necessitates an enzymatic reaction to release the thiols followed by their determination by Ellman's reagent. However, enzymes are expensive and fragile.

N02 S-N migration

P +

HS

NH2 , N ^ ^

NH2

NO2

Figure 6.5. NBD-F adduct S-N migrations.

Refsum, Ueland and co-workers, as well as several others, have made pioneering advances in developing HPLC assays towards homocysteine and biological thiol analysis. These methods are used in large-scale population studies which have been ongoing for two decades. Refsum and Ueland addressed the issues of removing excess monobromobimane and other

DETECTION OF BIOLOGICAL THIOLS

147

(unknown) fluorescent impurities prior to HPLC analyses in 1989.^^ They used a chromatographic "heart-cut," with a column switching device and two-solvent delivery system. They later^^ ingeniously devised an automated system (which is still used today^^) requiring less tedium, instrumental manipulation and wear as compared to their 1989 method. The improved method requires very precise pH control. It is based on the fact that the retention times of the impurity peaks due to monobromobimane are pH-sensitive. At pH 3.50, homocysteine and cysteine peaks are overlapped by an unknown fluorescent impurity peak (apparently a common monobromobimane reaction hydrolysis product). At pH 3.80, homocysteine and glutathione peaks overlap. At pH 3.65, the homocysteine peak is resolved; however, nitric acid is added to the mobile phase to attain separation of homocysteine from an unknown latereluting peak. Elevated (800 JLIM) amounts of monobromobimane and added EDTA must be used in order to obtain maximal fluorescence yields of cysteine and cysteinylglycine. Dithioethryrytol is added to increase the fluorescence of all thiols.^^ Blood is collected and centrifuged at 2000 x g at 0-2 °C. An autosampler is filled with 60 samples (20 h analysis tune) and the derivatization reagents. The vials and most reagent reservoirs are stored in chilled racks at 0 °C. The sample processor collects the sample and reagents into a vial where derivatization is carried out. The monobromobimane derivatization is quenched by addition of glacial acetic acid after 3 min.^^ As stated by Refsum and Ueland. et al. in their comprehensive 2004 review (which is co-authored by a group of the world's leading experts in this field)^^ many analytical methods afford results that do not fulfill baseline bias and imprecision criteria. Interlaboratorv. inter and intrapatient errors in homocysteine and biological thiol assays persist. Specifically: "the different t(total) Hey measurements give comparable results, but the variation among methods and laboratories are considerable. Ideally, from the known biological inter- and intraindividual variation in tHcy, the bias should be < 10 %. and the imprecision no higher than 5 %. but many methods do not fulfill these criteria."^^ They state that there is a significant need for standardization, despite recent advances.^^ While HPLC monitoring with a universal thiol derivatizing reagent affords simultaneous monitoring of thiols, this technique is "not suited for many labs," according to Refsum et al. It requires "skilled staff and (is) labor intensive, and throughput may be low."^^ The commercial immunoassays (e.g., the fluorescence polarization immunoassay run on Abbott's Imx and AxSYM platforms^^) are easier to use than HPLC but can only monitor one analvte and utilize relatively fragile biological materials. 6.5. DETECTION OF CYSTEINE AND HOMOCYSTEINE We have reported prior progress towards the colorimetric and fluorimetric detection of mono- and oligosaccharides. Our studies featured new functionalized xanthenes which we found arose in situ from ring-opened

J. O. ESCOBEDO ETAL.

148

450

500

Wavelength (nm)

Figure 6.6. UV-Vis spectra of 6 (2.5 x 10"^ M) and cysteine (4 x 10'^ M - 8 x l o ' M) in 0.1 M carbonate butYer pH 9.5 at room temperature. Each spectrum was acquired after 5 min. As the concentration of cysteine increases, a red shitY from 480 nm to 505 nm is observed. Reprinted with permission from J. Am. Chem. Soc, 126(2), 438-439 (2004). Copyright 2004 American Chemical Society.

resorcinarenes and related materials. Our interest in biological thiols arose initially from the reports that cysteine interferes with known sialic acid (an important saccharide cell-surface residue) determinations.^"* Our studies of the possible interference of cysteine with xanthene dye-based detection of sugars led us to find that our sugar detection methods did not suffer interference from cysteine. During the course of our saccharide sensing work, we discovered novel methods for Hey and Cys detection. Upon addition of cysteine or homocysteine (1.0 X 10"^ M) to a solution of 6 previously used as a synthetic intermediate for a zinc sensor^^ (1.0 x 10'^ M, H2O, pH 9.5), a solution color change from bright yellow to brownish-orange is observed. Similar color changes are observed on Ci8-bonded silica. UV-Vis absorbance changes of cysteine-6 solutions, readily monitored in the 10"^-10"^ M cysteine concentration range, exhibit a 25 nm red shift (Figure 6.6). Addition of cysteine to solutions of 6 results in monitorable fluorescence quenching (Figure 6.7). The reaction leads to thiazolidine (Cys 7a, Hey 7b heterocycle, not shown) adducts. Their structures have been unambiguously confirmed by ^H NMR and MS. UV-Vis spectra of solutions containing 6 and other common thiols (L-methionine, mercaptoethanol, glutathione), other amino acids (L-glutamine, L-serine, glycine, L-glutamic acid), and amines (D-glucosamine hydrochloride and CH3NH2 (8 x 10'"^ M, pH 9.5) confirm selectivity of 6 for cysteine and homocysteine. At most a 15 % change in absorbance at 480 nm is observed in response to the aforementioned analytes, even when they are applied in 10-fold molar excess relative to cysteine."*^

DETECTION OF BIOLOGICAL THIOLS

149

c 0)

o c o w o o 3

450

500

550

600

650

Wavelength (nm) Figure 6.7. Fluorescence emission spectra of solutions of 6 (1.0 x 10^ M) and cysteine excited at 460 nm. Inset: FO represents the maximum fluorescence intensity in the absence of analyte and F represents the corresponding intensity in the presence of analyte. Reprinted with permission from J. Am. Chem. Soc, 126(2), 438-439 (2004). Copyright 2004 American Chemical Society.

Solutions containing 6 and bovine serum albumin or urease also exhibit only small absorbance (less than 15 %) decreases and no wavelength shifts. Compound 6 can be used to readily detect cysteine and homocysteine in the range of their physiological levels (healthy plasma total homocysteine concentrations are ca. less than 12-15 |aM; cysteine concentrations are typically 20-30 times that of homocysteine). Again, interference from amines, amino acids and certain thiols and proteins is minimal. Importantly, we have demonstrated that both Cys and Hey can be detected in human blood plasma within range of their physiological levels."^^ 6.6. HIGHLY SELECTIVE DETECTION OF CYSTEINE AND SITESPECIFIC PEPTIDE LABELING Respective solutions of 6 containing identical concentrations of Cys and Hey exhibit similar spectrophotometric changes."^^ However, (Figure 6.8), we have found that 6 can be used in combination with other aldehydes such as 4(dimethylamino)-cinnamaldehyde (DMA) to afford selective detection of cysteine. To the best of our knowledge this is the first example of such a high degree of direct Cys selectivity over other biological thiols, without using any separations or biological materials. Since the DMA signal is at 400 nm and responsive only to Cys, one may also determine Hey indirectly by differences.

J. O. ESCOBEDO ETAL.

150

• L-Cys

1.2 n

• DL-Hcy 1.1 -

i 0.9

• I

•

E

•

•

0.8

i

•

•

#

0.7 0.6 n^ 0.0000

0.0005

0.0010

0.0015

Figure 6.8. UV-Vis titration data from mixtures of 4-(dimethylamino)-cinnamaldehyde (DMA, 3 x 10^ M) and L-cysteine and homocysteine in 0.1 M carbonate buffer (pH 9.5) monitored at 400 nm at room temperature. Concentrations of the amino acids were increased from 0 to 1.7 x 10'^ M. The error bars are based on three runs. This shows that DMA affords reproducible selectivity for Cys.

using non-selective absorbance changes at longer wavelengths promoted by both Cys and Hey in the presence, for example, of 6. Increasingly more electrophilic a,P-unsaturated aldehydes such as cinnamaldehyde and 4-nitrocinnamaldehyde afford successively poorer selectivity for Cys over Hey. The electron donating dimethylamino group of DMA renders the dye less electrophilic, resulting in enhanced Cys selectivity. Presumably, the enhaced reactivity towards Cys is a result of more facile 5- vs. 6-membered ring (as expected for Hey) thiazolidine formation. There is current interest in the site-specific labeling of peptides and proteins. One approach involves the fluorescent tagging of existing or preengineered A/^-terminal cysteine residues.^^ The fluorophores used include fluorescein or NIR dyes with appended thioester groups. Pretreatment with Omethylhydroxylamine is needed to free the cysteine residues that exist as carbonyl (thiazolidine) adducts.^^ The reactions may require the use of GdmCl to proceed effectively; however, this impacts protein folding. These ligations also typically require thiol co-factors such as thiophenol or benzylmercaptan. They are irreversible. They are subject to thioester hydrolytic instability and side-reactions.^^ Thus, our aldehyde-appended-dye techniques should find use in forming reversible and strong, covalent site-specific fluorescent labels. Figure 6.9 exhibits this approach towards the selective and facile labeling/detection with simple dipeptide models.

DETECTION OF BIOLOGICAL THIOLS

320

370

151

420

470

520

Wavelength (nm)

Figure 6.9. UV-Vis spectra of 4-(dimethylamino)-cinnamaldehyde (left, 3 x 10 M, 400 nm) and 6 (right, 3 X 10'^ M, 480-500 nm) with varying concentrations of the dipeptide cys-gly (4 x 10"^ M - 8 X 10^ M) in 0.1 M carbonate buffer pH 9.5 at room temperature. Each spectrum was acquired after 5 min. Spectrophotometric changes were insignificant for gly-gly (at most < 3 % absorbance changes), as expected, under the same conditions, for each of the two dye solutions.

6.7. HIGHLY SELECTIVE DETECTION OF HOMOCYSTEINE Fluorone black (8) is a commercially available xanthene dye. Upon addition of 1 equiv Hey (1.0 x 10'^ M final cone.) to a solution of 70 % MeOH:H20 (MeOH is used for solubility) at pH 7.3 and containing 8 (1.0 x 10" ^ M), a change (increase) in absorbance at 510 nm is observed. A similar, but smaller absorbance change is observed for Cys, GSH and «-propanethiol. Other amino acids (glycine, alanine, serine, methionine, glutamine, lysine, arginine, threonine) or the disulfide homocystine do not promote a detectable absorbance change.'^'^

We investigated the effect of PPha on this process, initially in order to mimic the conditions used to prepare biological samples for analysis via disulfide reduction (bound thiol liberation). When PPhs (45 equiv to dye) is present in the dye solution, an absorbance change only occurs in the presence of homocysteine. No change is observed for Cys or any other biological thiols. If a 30-fold molar excess of Cys (to Hey) is added to solution of Hey (at levels approximating both the homocysteine risk level in plasma and the proportion of Cys to Hey), 8 (1.0 x 10"^ M) and PPhs (45 equiv), no change in the

J. O. ESCOBEDO ETAL.

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The other major factor stimulating microparticle-based molecular analysis is the development of optically encoded microparticles for multiplexed analysis. While duplex and triplex assays had been demonstrated using different sized beads, the introduction of microsphere sets bearing different amounts of dye that gave discrete levels of fluorescence^^'^^ increased the multiplexing capacity by more than an order of magnitude, and potentially much more. Different intensities of a single dye can allow approximately ten different beads to be distinguished. Size (light scatter) can be used to increase this number by a factor of two to four, while the addition of another dye with as many as ten discrete intensities of a second color expands the multiplex set to nearly 100. Sets of optically encoded microparticles, sometime referred to as suspension arrays, thus constitute a variant of the popular microarray technology, in which the surface on which an assay is performed is defined not by a physical spot on a two dimensional surface, but by the features of the encoding elements it exhibits. Pioneered by Luminex Corp (Austin, TX), encoded microspheres, assay kits, and dedicated microsphere reading instruments are now available from a number of commercial sources. An attractive feature of this platform is its open nature, in that beads and assay kits from any manufacturer can in principle be used with any flow cytometer with the appropriate detection capabilities. While the most popular application for microsphere-based flow cytometry is the ELISA-like sandwich immunoassay, the number of other potential applications is quite large.

200

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9.4. DETECTION AND SENSOR APPLICATIONS A key step in realizing the potential of multiplexed analysis using encoded microparticle arrays is the development of quantitative single bead assays for the analysis of molecular interactions and functions. As mentioned above, the first and still the most popular multiplexed microparticle application is the sandwich immunoassay, in which a soluble analyte is captured by an immobilized antibody and detected with a second, labeled, antibody resulting in the formation of an antibody-analyte-antibody complex on the particle surface and an increase in signal from the microsphere. This approach, which is analogous to the widely used ELISA assay, has advantages over that method in that it can be performed with fewer or no wash steps and, more importantly, can be used to measure multiple analytes in a single sample using encoded particles. A recent example of this approach applied to infectious disease detection is presented in Figure 9.4. To detect and differentiate between circulating influenza virus types, monoclonal antibodies to influenza types A and B immobilized on different sized beads captured virus particles from a sample. The antibody-bound virus was then detected with a cocktail of fluorescence-labeled polyclonal antibodies against the A and B type viruses. Using this approach, the two virus types could be uniquely identified with a limit of detection of less than 1 ng of viral protein (Figure 9.5), more than 100times less than required by two commercial point of care kits'^. This approach has also been adapted to fluorescently encoded beads^^ and is being adapted to detect influenza virus sub-types as well. This immunoassay sandwich format has also been applied to the detection of other pathogens''^'^^.

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While the antibody sandwich format is well established, there is a great deal of interest in developing assay chemistries that are simpler, faster, and more sensitive. Towards this end, the specific binding of receptors by proteins has been exploited for the detection of bacterial toxins. Cholera toxin, a multisubunit protein secreted by the pathogenic bacteria Vibrio cholerae, binds to specific glycolipid receptors on mammalian cells in order to gain entry to the cell. This receptor, the ganglioside Gml, can move laterally within the plane of the cell membrane and is aggregated by the pentavalent cholera toxin. This aggregation forms the basis of a very sensitive FRET-based assay for cholera toxin^^"^^. Fluorescently labeled analogues of Gml were synthesized with a fluorescence donor (BODIPY-TMR) or acceptor (BODIPY-TR) and were incorporated at low mole fractions into artificial membrane vesicles (liposomes), which were then assembled onto the surface of glass microspheres. Upon excitation at 514 nm in a flow cytometer, the supported bilayer membranes containing the fluorescent Gml analogues exhibited significant BODIPY-TMR fluorescence, but low levels of BODIPY-TR fluorescence, as that dye is inefficiently excited at 514 nm. Upon addition of cholera toxin (or its B subunit, which contains the receptor binding activity) the GMl analogues were brought into proximity such that energy transfer between BODIPY-TMR and BODIPY-TR occurs, resulting in a decrease in the fluorescence of the donor and an increase in the fluorescence of the acceptor (Figure 9.6). The ratio of the acceptor to donor fluorescence was proportional to the amount of cholera toxin present, and it was possible to detect less than 50 pM with only a five minute incubation (Figure 9.7). This sensitivity is comparable to the traditional ELISA immunoassay, but is much simpler as it involves only a single reagent

202

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Wavefength (nm) Figure 9.6. Receptor aggregation-induced FRET. Cholera toxin at the indicated concentrations was added to microsphere-supported bilayers bearing BODIPY-TMR and BODIPY-TR GMl analogues, and the emission spectra measured by spectrofluorimetry (excitation wavelength: 530 nm).Reprinted with permission from reference 17.

(the receptor-bearing microspheres) and no washing or other sample processing steps. Adapted for other receptors using different-sized beads, this assay approach could in principle be used for single step detection of multiple analytes. 9.5. MOLECULAR INTERACTIONS AND FUNCTION The features of sensitive, homogeneous detection have been applied to the analysis of molecular interactions and function for a range of different systems. Here, I present some examples that illustrate some of the key approaches and their advantages relative to other methods. 9.5.1 Enzyme-Substrate Interactions The interactions between enzymes and substrates play key roles in health and disease, and high resolution methods for their analysis are key to understanding of basic mechanisms as well as for the development of potential therapeutic agents. The structure specific nuclease human flap endonuclease-1 (FEN-1) recognizes and cleaves a particular DNA structure characterized by a double stranded character with a single stranded 5' flap. This flap structure is thought to be an intermediate in DNA replication and repair, and mutations in FEN-1 have been associated with defects in DNA replication associated with

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certain types of cancer^^. Thus, the mechanisms of action of this enzyme are of importance in understanding human disease. The traditional approach to measuring FEN-1 activity involves the generation of a synthetic oligonucleotide substrate with a radioactive label at the 5' end of the flap strand, and using gel electrophoresis to resolve the intact substrate from the shorter, cleaved product. This approach is slow and labor intensive, and not well suited to kinetic analysis and mechanistic studies of enzyme structure and function. To develop an improved approach for these types of studies we replaced the radioactive label with a fluorophore, added a biotin to one of the ends, and immobilized the flap substrate on the surface of a streptavidin microsphere (Figure 9.8). When the enzyme FEN-1 was added in the presence of its essential cofactor Mg^^, the flap strand bearing the fluorophore was cleaved, releasing the fluorophore and resulting in a decrease in microsphere fluorescence. Because the flow cytometer can resolve free from microspherebound fluorophore, it was possible to measure this decrease continuously, without the need for a wash step. Apart from the homogeneous assay format, which enables continuous kinetic resolution, the microsphere based approach has important advantages in sensitivity of other methods. In a typical assay, approximately 50,000 substrate molecules were immobilized per bead, which represents a fairly bright signal

204

J. P. NOLAN

Figure 9. 8. Schematic of the flap DNA substrate. The synthetic substrate composed of a biotinylated template strand, fluorescence-labeled flap strand, and invading adjacent strand, is attached to a streptavidin microsphere.

for the flow cytometer. However, at the microsphere concentration used, typically 500,000 per ml, this translates to a solution substrate concentration of ~ 10 pM, 2-3 orders of magnitude less than is practical with radioisotope labeling. This allowed us to provide enzyme in excess to perform pre-steady state, single turnover kinetic analysis which are particularly powerful kinetic approaches. This combination of sensitivity and continuous kinetic resolution allowed us to easily perform kinetic analysis that would have been difficult using radioisotope methods. For example, we could readily determine the enzyme concentration dependence of the overall reaction time course (Figure 9.9A). We could evaluate the cleavage step independently from the binding step by allowing the enzyme and substrate to bind in the absence of Mg^^, and the start the reaction by the addition of Mg^^. These studies were performed using a specially-constructed stopped flow cytometer that was able to mix reagents and initiate measurements in less than 0.3 seconds, allowing accurate estimation of the cleavage rate constant (Figure 9.9B). To examine the binding step in detail, we took advantage of a poly-his epitope tag at the amino terminus of the FEN-1 enzyme to immobilize it on the surface of Ni^^-chelate beads (Figure 9.10A). In the absence of Mg^^, it was possible to measure the kinetics of substrate binding to the immobilized enzyme (Figure 9.1 OB). Addition of Mg^^ resulted in cleavage of the substrate and loss of fluorescence from the microsphere. We could then use these independent measurements of binding and cleavage in combination with the overall reaction time course and a kinetic model of the reaction mechanism to estimate the rate constants for enzyme-substrate association, dissociation, and cleavage^^\ Apart from providing detailed information about the FEN-1 reaction mechanisms, the microsphere-based assay approach also provided a convenient tool to investigate the role of individual amino acid residues in FEN-1 function^^"^"^

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TRFA FOR SOL-GEL STRUCTURE ELUCIDATION

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where k is the Boltzmann constant. As neutral and anionic fluorescent probes do not bind to the silica surface and thus are free to diffuse in the water-filled pores, the viscosity of silica sols and gels can be measured from the correlation time (j) of these probes in the entrapped state.^^ The TRFA decay of R6G in water at 295K (Figure 13.2) gives ^ = 0.16 ± 0.01 ns, ro= 0.38 and XR^ = l.^^'^^The TQ value is in excellent agreement with the steady-state anisotropy of R6G in glassy frozen solvents ^^ whereas ^ corresponds to the radius of the R6G sphere of 0.56 nm,^^ in agreement with previous reports.^^

13.2.3.2 Viscosity Measurements In the absence of interactions with silica surfaces, the diffusion of anionic and neutral fluorescent probes can be affected not only by the sol microviscosity, but also by collisions with other molecules dispersed in sol-gel pores (molecular crowding). The described model of rigid sphere provides theoretical support to study these aspects by TRFA. The TRFA decay of the anionic probe 8-hydroxyl-l,3,6-trisulfonated pyrene (pyranine) entrapped in a sodium silicate sol prior gelation (Table 13.1) shows unexpectedly high molecular mobility in the densely polymerized and aged silica network.^^ The decay is monoexponential, indicating that pyranine samples a single, average fluid environment with no effects from distribution between different environments. The 60% increase in the average rotational correlation time of pyranine upon aging of the SS hydrogel is likely related to shrinkage of the silica network leading to a higher solvent viscosity. These findings have been recently supported by NMR/EPR studies of the rotational mobility of neutral and negatively charged small solutes in TMOS sol-gels. ^^ This puts in serious doubt the existence of presumed "viscosity domains",^^' ^^ questioned earlier by Geddes,^^ and supports the view that the sol-gel interior is composed of a rigid silica network with relatively uncrowded aqueous pores, in which small molecules can rotate freely. Since the binding of a probe to the silica surface severely restricts its rotation, the use of the rigid sphere model in the presence of probe-host interactions can be misleading.^^ The binding of cationic probes to silica can be altered in the presence of alcohols or at extreme pH values to generate a fraction of free, silica-unbound probe. The free probe molecules diffuse to solvent-rich regions with a characteristic short correlation time^^' ^^ that can be used for the viscosity measurement. In this approach, the second correlation time reflects the diffusion of the silica-bound probe. As only one time component is available for the viscosity calculation, the presence of multiple viscosity domains, if they exist, might be overlooked. While the presence of multiple viscosity domains in silica remains an open question, such domains have been detected in aqueous solutions of watersoluble polymers.^^' ^^' ^^ Polymer solutions are good host systems for testing the rotational diffusion of small and large molecules in the absence of rigid

286

TLEUGABULOVA ETAL.

geometrical confinement as found in the silica sol-gel network. The TRFA decay of R6G in diluted aqueous solutions of poly(ethylene oxide) (PEO) is monoexponential, but shows the statistical significant contributions from two correlation times upon increasing the polymer concentration to 30 wt % (see Table 13.1) r{t) = P^ Gxpi-t/(^) + P2 expH/ 2.5 nm) leads to significant residual anisotropy r^> 0 (Figure 13.2) and thus adds the third term to Eq. (13), addressed as a hidden or non-decaying 4.28„30

component " r(t) = y^i exp(-/l(k) + P2 exp(-//^2) + ^oo = n)/i exp(-^/^) + ro/2 exp(-^/^2) + ^'b (15) where g = rjr^ is the fractional contribution from the non-decaying component, fx'^fi^g^ 1 • If the NPs are normally distributed around R > 2.5 nm, the upper part of the normal particle distribution is hidden in the non-decaying component, whereas the lower part of the distribution, which will no longer be normally distributed, but rather will be a truncated distribution, is averaged as ^ . Thus, caution must be used when interpreting the average size of silica NPs based on the value of ^ in cases where g ^ 0, as under these conditions one cannot assume an underlying normal distribution of particle sizes, and indeed a highly asymmetric particle distribution could exist.

13.2.4.3 R6G Mobility in Porous Monolithic Silica In the absence of surface modification, it is possible to have essentially all R6G bound to the silica surface and thus the (j>\ component in Eq. (15) disappears (/l = 0). This situation has been seen in the TRFA decays of R6G in aqueous silica sols, but is more prevalent in gelled sodium silicate samples. On the other hand, in TMOS sols and gels, f\ ~ 0.7^^ due to the presence of methanol, highlighting the importance of removing alcohol co-solvents. Gelation of aqueous sols leads to the formation of a continuous network of macroscopic dimensions immersed in a liquid medium exhibiting no steadystate flow.^^ From this definition, it is obvious that most R6G molecules entrapped in the sol-gel compartments of an aqueous silica gel should be bound to the walls of the rigid silica network, rather than to discrete NPs (Figure 13.2).^^ Assuming that this rigid network is immobile, Eq. (11) can be rewritten KO = fioJQ exp(-^/^^,,) + r^

(16)

As R6G binds rigidly to the silica surface,^ ^ the local motion is absent (fioc = 0) and thus r(t) = Too. Hence, the residual anisotropy reflects not only the binding

290

TLEUGABULOVA ETAL.

of R6G to large silica NPs (Eq. 15), but also senses the binding to the silica network. Importantly, the ability to achieve a situation where r(t) = r^ rules out any possibility for local probe motion owing to "wobbling" or exchange equilibria.^^ Hence, this probe is ideally suited for studies of silica particle growth.

13.3. PARTICLE GROWTH STUDIES 13.3.1 Particle Growth in DGS Sols Most theoretical models of the growth of silica particle have been developed using TEOS as a silica precursor and propose the nucleation of primary silica NPs (-' 20 Si atoms)^^ and their fast evolution into secondary NPs and higher order aggregates. However, the notion that the primary NPs may actually exist in solution as discrete, stable structures has been viewed with skepticism for many years, mainly because of the extremely fast kinetics of the silica growth in water and the analytical limitations to follow such fast processes occurring on the nanoscale.^^ Prior work by Geddes and Birch demonstrated that the real-time growth of silica NPs in an aqueous SS sol is too fast (- 7 s) to follow the initial stages of nucleation and silica particle growth.^^ Primary silica NPs could be detected in TMOS sols,^^ where their growth occurs over a very limited range, from ~ 0.8 to 1.1 nm in radius, however the later stages in silica particle formation could not be directly observed by TRFA since particles did not grow beyond 1.1 nm. As part of our research into entrapment of proteins within silica, it became apparent that conventional alkoxysilane precursors, such as TEOS and TMOS, were not ideal for formation of bioglasses. As a result, our group developed the biocompatible silane precursor diglycerylsilane (DGS, Figure 13.1), which releases the protein-compatible solute glycerol as a byproduct of hydrolysis. A somewhat unexpected benefit of DGS was the ability of this precursor to undergo rapid hydrolysis (< 1 min) in aqueous solvent over a broad pH range (pH 5-11),^ removing the need to use multiple pH regimes to initiate hydrolysis and condensation. More importantly, the glycerol evolved in the initial hydrolysis reaction acts to suppress silica condensation reactions that nonnally occur very rapidly at neutral pH in an aqueous environment, thus slowing the rate of formation of the silica NPs. This precursor therefore appeared to be ideal to prove the existence of primary particles, and the overall validity of the nanoparticle metrology approach. Figure 13.3 shows TRFA decays for R6G-DGS collected after one hour following DGS dissolution and the corresponding residual plots from the fits to Eq. (15). Figure 13.4 shows the fractions of R6G fluorescence in DGS sols associated with ^7, ^2 and g as a component ^ , indicating that the DGS sol is mainly composed of primary silica NPs. With time, ^ is increased to 3.7 ns (t = 50 min) and subsequently to 7.45 ns (/ = 150 min). AFM imaging of the diluted DGS sol shows individual particles that are close to spherical in shape and

TRFA FOR SOL-GEL STRUCTURE ELUCIDATION

291

compact with smooth interfaces (Figure 13.5A). The phase image (Figure 13.5B) shows that the particles contain a dense nucleus (white zone) and a less dense outer shell (dark layer). The radius of the dense nuclei is in the range of 1-2 nm, according to the particle height on the substrate surface, which minimizes tipconvolution effects. The dark outer shell is likely composed of oligosiloxanes and glycerol molecules, which are dispersed in water in the initial sol, but become adsorbed to the particle surface after drying the sample for AFM imaging. These layers would be expected to provide steric barriers to NP aggregation during imaging, but, on the other hand, make the imaging process difficult. The DGS sample sticks to the AFM probe and is dragged across the surface producing streaks. When the silicon substrate containing the DGS sample is heated for 20 min at 70°C, the dark layers around the NPs disappear (Figure 13.6B), indicative of condensation cure. Figures 13.5 and 13.6 clearly show predominance of individual spherical NPs of different sizes in the diluted DGS sol, but provide no evidence for large aggregates. Thus, the AFM data confirm the TRFA data, and provide direct evidence for existence of stable sols composed essentially of primary silica NPs. As the silica NPs are of spherical shape (Figures 13.5 and 13.6), one can estimate the average particle radius from ^ using Eq. (14). Since ;; = 0.894 x 10"^ Pa s and 7 = 298 K, the average particle radius isR = 0.9 nm in the DGS sol (0.3 wt % SiOi, pH 9.2) 11 min after DGS dissolution. Higher silica concentrations in the initial sol lead to a larger size of the particle nuclei (Figure 13.7). For the next 20 min after DGS dissolution, the initial size of the nuclei continuously increases, whereas the fraction of ^ does not change (Figure 13.4) indicating no further nucleation. The fraction of primary NPs remains essentially constant for at least 3 weeks,^^ while the average radius of these NPs attains a maximum value of ~ 2.8 nm after 7 days, after which the NPs contract slightly in size, owing to continued syneresis. The particle size measurements are consistent with the formation of stable primary NPs in the DGS sol that do not undergo significant aggregation or further growth into secondary NPs. The TRFA decays collected at pH 8.2 show significantly different parameters than those collected at pH 9.2 (Figure 13.3c). In 40 min after DGS dissolution, the fraction of ^ decreases rapidly to zero. Both the initial and maximum ^ values are considerably higher (12 and 20 ns, respectively) than those measured for R6G-DGS at pH 9.2 (Table 13.3), pointing to a larger particle size at pH 8.2. The fraction of ^ initially increases and then decreases (Figure 13.4B). Unlike the situation at pH 9.2, the decreased fractions of ^i and ^ correlate to time-dependent increases in the fraction of the non-decaying component, g. During the first 15 min, g and /^ increase simultaneously at the expense of a decreased/i value. After 15 min, g continues to increase, b u t ^ decreases. Overall, this behavior is consistent with more rapid formation and subsequent aggregation of primary NPs as the pH value approaches neutrality.

292

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6.5

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Figure 13.3. TRFA decays and residual plots for R6G-DGS in 20 mM borate buffer. (A) 0.3 wt% Si02, pH 9.2; (B) 0.3 wt% SiOa, pH 9.2; (C) 3 wt% Si02, pH 8.2 and (D) 3 wt % SiOs, pH 7. Measurements were performed 1 h after DGS dissolution. R6G, l^M; X^x ~ 495 nm; A-em = 551 nm; T = 298 K. The starting point (t = 6.5 ns) corresponds to the prompt response. Reprinted with permission from Ref 31, Copyright 2004 American Chemical Society.

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Currently, there are two extreme models describing silica particle growth: one model is a "growth-only" model ^^'^^ based on LaMer's synthesis of monodisperse colloidal particles by homogeneous precipitation.^^ This model neglects aggregation and holds that a narrow distribution of colloids can be achieved by a brief "burst" of nucleation, followed by diffusion-limited growth. Growth is defined as the addition of soluble species directly to the particle surface. Van Blaaderen et al?^ proposed a related model whereby nucleation is

294

TLEUGABULO VA ETAL.

Table 13.3. TRFA decay parameters for R6G-DGS in 20 mM borate buffer, pH 9.2. Decays were recorded in - 10 min-time intervals following DGS dissolution and fitted to Eg. (15). T= 298 K;r = 3.89 ± 0.02 ns; ^ < 0.003.^" time after DGS dissolution

0 11 min 20min 29 min 38 min 47 min 56 min 150 min

^7, ns

0.16 0.13 0.21 0.16 0.15 0.17 0.11 0.17

+ 0.01 ±0.06 ±0.08 ±0.05 ±0.06 ±0.07 ±0.08 ±0.03

(^2, ns

0.69 ±0.17 1.83 ±0.20 2.81 ±0.12 4.34 ±0.16 3.03 ± 0.20 3.66 ± 0.05 7.45 ± 0.07

ro

1 0.41 0.32 0.10 0.06 0.07 0.04 0.01

0.380 0.336 0.330 0.359 0.346 0.355 0.365 0.356

_IR_

1.02 0.96 1.00 1.00 0.96 0.98 1.05 1.01

controlled by the aggregation of soluble species, but subsequent growth is controlled by the surface reaction. The other extreme model describing how the particle size distribution matures is an "aggregation-only" model.^^'^"^ Much recent evidence provided by ^^Si-NMR, conductometry and photon correlation spectroscopy of the Stober synthesis process supports the aggregation model.^^ Our study of DGS growth at different pH values showed that in the case of DGS, there is no contradiction between these two models; by setting up different initial conditions of the sol, such as the silica concentration and the pH, the particle growth can be driven by one mechanism or the other. Under conditions of low silica concentration (< 0.5 wt % Si02) and at pH 9.2, the only route available for consumption of the monomeric DGS is nucleation; the

Figure 13.5. Height (A) and phase contrast (B) images of the DGS sol dispersed on a silicon surface. SiOz, 0.2 wt %; pH 9.2. Image field, 500 nm. Reprinted with permission from Ref 31, Copyright 2004 American Chemical Society.

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nucleation stage is brief and ends up with the formation of « 1 nm radius nuclei, which grow slowly with time until reaching a stable size on the 2-3 nm scale. Upon increasing the silica concentration to 1 wt % Si02, there is clearly an increase in the initial radius of nuclei formed, which subsequently grow to a larger size (> 4.5 nm radius) or aggregate with time. At pH 8.2, particle formation is much faster and thus the nucleation and aggregation occur simultaneously in the early stage of silica formation. Hence, primary silica particles form rapidly in the early stages of silica formation and further growth occurs by aggregation of these particles. This results in the formation of larger secondary silica structures in the sol. These results highlight the potential for TRFA analysis, since the method is amenable to in situ studies under a variety of solution conditions. 13.3.2 Particle Growth in SS Sols In protein entrapment studies, the SS sol is prepared by acidification of SS via reaction with Dowex resin.^^ The R6G dye added to the freshly made SS sol (< 3 wt% Si02) exhibits a high steady-state anisotropy indicating the presence of already grown NPs and aggregates.^^ This corroborates previous findings of Geddes and Birch that the real-time growth of silica NPs in aqueous SS sols is too fast to follow the initial stages of nucleation and silica particle growth.^^ The anisotropy value is even higher (r = 0.35) if the SS sol is submitted to conditions that promote fast silica particle growth and aggregation.^^' ^^ The R6G dye dispersed in such sols exhibit no fluorescence depolarization and r(t) ^ ro (Figure 13.2), indicating complete restriction of R6G diffusion. As noted

Figure 13.6. Height (A) and phase contrast (B) images of DGS-based NPs dispersed on the silicon substrate and heated at 70° C for 20 min. SiOi, 0.2 wt %; pH 9.2. Image field, 500 nm. Reprinted with permission from Ref 31, Copyright 2004 American Chemical Society.

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X 2.5 E =

2

X X

X

X X X

>K

2 o o o o ^ ^ ^ • • • •

o

X

•§1.5

•

re Q.

0.5^

25

50

75 time, min

100

125

150

Figure 13.7. Silica particle radius (± 0.1 nm) in DGS sols (pH 9.2) as a function of time following DGS dissolution. SiOz: 0.3 wt% ( • ) , 0.5 wt% Si02 (0) and 0.8 wt%(*). Reprinted with permission from Ref 31, Copyright 2004 American Chemical Society.

above, this constitutes the strongest experimental evidence for the rigid bindingof R6G to the silica surface supported by theoretical considerations^^ and corroborated by several independent studies of rhodamine adsorption onto silica surfaces by second harmonic generation in combination with another spectroscopic methods. ' ' ' These studies show that the R6G dye, spincoated on the surface of fused silica or embedded in layered silicate films, penetrates into the interlayer spaces of silicates and orients with the molecular plane of the xanthene ring almost parallel to the silica surface.

13.4. MONITORING SILICA SURFACE MODIFICATION 13.4.1 Background High residual anisotropics in the TRFA decays of R6G in aqueous silica sols reduce the fraction of anisotropy available to reconvolute ^i and ^ from the decay, and thus obscure information on the ps-ns dynamics. However, the high residual anisotropics are an advantage when TRFA is used for monitoring the modification of silica surfaces. One of the commonly used practices to modify particle surface properties is to have molecules with specific properties physically adsorbed or covalently attached to the NP surface. The choice of target molecules for the adsorption studies is determined by their relevance in suppressing imr ! J rij to the silica surface (APTES, tertiary amines), ^^ promoting native-like protein function (GLTES, MLTES),^^' ^^ stabilizing colloidal dispersions (PEO)^^ or modeling biological systems (peptides).^^ It is therefore important that we have the experimental ability to quantitatively detect molecular adsorption and

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discuss it in the context of colloidal properties based on particle surface conditions. Some of the intrinsic properties of colloidal silica make it difficult to employ as a sorbent in adsorption studies. In water, the silica surface continually evolves due to the dissolution of silicate species and reduction of the available surface area. The silica dissolution affects the surface charge density calculated from potentiometric titrations, especially when the volume fraction of the silica NPs is low. The silica NPs may flocculate as a result of a decreased surface potential (by increasing pH), increased concentration of electrolyte (counterions) or due to a reduced degree of hydration. In any silica suspension formed via nucleation and growth processes one never has a truly monodisperse system. The polydispersity of silica sols has been demonstrated by SAXS^^ and dynamic light scattering,^^ which measure the hydrodynamic radius of solvated particles. A difference between the average radius of solvated particles and the radius of vacuum-dried particles measured by TEM points toward the existence of superficial gel layers or "surface steric barriers" on the NP surfaces composed of poly silicates and bound cations. All these features of colloidal silica cause it to deviate from the ideal model of a solid surface, and complicate the interpretation of adsorption phenomena in colloidal silica systems.

13.4.2 Ludox Ludox AM-30 dispersion (30 wt%, pH = 8.9) consists of stable silica NPs (mean radius of 6 nm) whose negative charge is mostly independent of pH due to substitution of a small portion of tetravalent silicium ions by trivalent aluminum ions (Figure 13.8).^'^"^'^^ Ludox NPs are normally distributed around an average radius R = 6 nm (TEM).^ These properties makes Ludox a good model of colloidal silica as a class, given that the silica surface charge does not depend on the synthesis procedure used to obtain the NPs.^ However, some challenges still remain. Despite the fact that vacuum-dried Ludox NPs appear as perfect spheres under the electron microscope, they are highly hydrated in water and, as discussed above, exhibit gel-like layers on their surfaces. ^^ Although such layers contribute to the stabilization of Ludox against flocculation, on the other hand, they lead to an abnormally high concentration of surface-exposed silanol groups.^ As a result, the actual number of the adsorption sites on Ludox cannot be accurately estimated from the particle dimensions. Even so, Ludox is an excellent model system to allow investigation of silica surface modification and biomolecule:silica interactions. 13.4.3 Monitoring Adsorption by TRFA 13.4.3.1 Conditions for Monitoring Adsorption One important requirement for the use of TRFA in adsorption studies is the absence of interactions between the target molecules and the R6G dye. In order

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Nrtf

V

^!k

OH

^^K

Na*0"

^^^

/

•

•

Figure 13.8. Surface of Ludox particle. Thick line denotes the particle surface. Reprinted with permission from Ref 51, Copyright 2005 American Chemical Society.

to prove this, the emission parameters of R6G (emission spectra, quantum yield, fluorescence lifetime, TRPA decay) must be measured in aqueous solutions containing different amounts of the dissolved modifier (in the absence of Ludox). A characteristic lifetime of r ~ 4 ns, a correlation time of ^ < 0.2 ns and the absence of residual anisotropy in the decay are good indicators of the absence of interactions between the modifier and R6G. The adsorption of nonfluorescent targets onto Ludox NPs is measured by competitive adsorption of R6G, which is added to the modified Ludox sol immediately after the modification has been done. The TRFA decay of R6G in the modified Ludox is modeled as the mixture of rigid spheres (Eq. 15). The coating of the Ludox surface by target molecules leads to a decreased fraction of R6G bound to large Ludox NPs (g) and an increased fraction of free, unbound dye (/i), as compared to the TRFA parameters of R6G in the plain Ludox suspension. This situation is shown schematically in Figure 13.9, where addition of a surface modifying agent leads to blockage of the silica surface, and hence a higher proportion of free R6G in solution. It is worth noting that the use of R6G as the adsorption marker is conceptually different from what is commonly done in another analytical methods.^^'^^'^^'^^ Usually, the adsorption marker is added in sufficient amounts to create surface saturation and then a target is added. The adsorbed target displaces the adsorption marker from the particle surface and thus the adsorption is monitored as a decrease of the signal due to the displacement of the dye to solution. In contrast, TRFA uses extremely low concentrations of the fluorescent probe, below one R6G molecule per silica particle. Even so, statistically, the adsorption of surface modifiers will lead to a shift in the R6G binding equilibrium in a manner that depends on both the affinity of the modifier for the surface and the concentration of modifier in solution.^^' ^^' ^^ One issue with using Ludox for adsorption studies is that the adsorption of target molecules onto Ludox NPs usually decreases the repulsive energy barrier

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large Ludox NP

small Ludox NP

299

R6G

PEO

Figure 13.9. Schematic representation of competitive adsorption of R6G and PEO onto Ludox NPs. As more PEO chains bind to Ludox, less silica surface is available for R6G adsorption, leading to an increase in free dye. This alters the relative contribution of each decay parameter ifufi and g) depending on the extent of surface modification.

existing between plain NPs. This decrease may be due to the reduced particle surface charge, ^^ a lower degree of hydration^ or both. If the repulsive potential between modified Ludox NPs is low, but their surface is not completely covered by target molecules, the NPs collide, form Si-O-Si bonds and flocculate. Since the flocculation affects the optical transparency of the sample, the concentration of a modifier in the Ludox sample for the TRFA measurement must remain below the critical flocculation concentration. Table 13.4. TRFA decay parameters for R6G in PEO-modified Ludox suspensions.^^ (f>2,ris (l>hns Target fi 3 wt% Si02 0.55 kD PEO, 1 wt% 0.55 kD PEO, 30 wt% lOkDPEO, 1 wt% lOkDPEO, 30wt% 100kDPEO,0.1 wt% lOOkDPEO, 1 wt%

0.16 ±0.01 0.16 ±0.03 0.55 ± 0.02 0.20 ± 0.06 0.37 ± 0.04 0.12 ±0.03 0.24 ±0.16

5.6 ±0.6 2.7 ±0.3 2.8 ±0.4 4.1 ±0.3 2.5 ± 0.3 4.4 ± 0.6 3.5 ±0.6

0.01 0.09 0.47 0.02 0.19 0.04 0.05

0.79 0.52 0.18 0.68 0.38 0.78 0.54

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TLEUGABULOVA ETAL.

13.4.3.2 Adsorption of Polymers The first system we examined by TRFA was the interaction between PEO and silica. PEO can undergo hydrogen bonding and hydrophobic interactions with siHca, which are similar to those found in proteins and other biological materials. Additionally, PEO is known to stabilize colloidal dispersions,^^ act as an antifouling agent on membranes,^^ resist the adsorption of proteins and increase the intracellular uptake of modified NPs. ^^ Furthermore, sol-gel derived silica doped with PEO and other polymers has been shown to provide enhanced activity for entrapped enzymes^'*"^^ and to alter the dynamics of entrapped biomolecules."^^' ^^ These aspects make the incorporation of PEO chains into silica-based nanocomposites the basis of a general methodology for the entrapment of bioactive molecules into ceramics and glasses,^^ and make such materials useful for biomedical and environmental immunochromatography and immunosensing.^ Table 13.4 shows the decays of R6G in unmodified and PEO modified Ludox samples. The first point to note is that under appropriate conditions, the fraction of free dye present in unmodified silica systems, given by/i, is on the order of 1%. Thus, 99% of the R6G is bound to the silica surface. A second key point is that the decays of R6G in the PEO-modified Ludox exhibit a concentration-dependent increase in /i and a corresponding decrease in the g value. However, a high PEO concentration (30 wt%) is required cause substantial changes in the/i and g values. An important aspect of TRFA measurements is the ability to assess fractional surface coverage using the TRFA parameters. Considering the 10 kDa PEO samples, the g value gradually decreased from 0.8 (plain Ludox) to 0.38 (30 wt % PEO), with the fractional fluorescence of the nanosecond component increasing from 20% to 43% and that of the picosecond component increasing from ~ 1 % to 19%. Since the emission properties of R6G do not change upon adsorption, the total fluorescence from the nanosecond and residual anisotropy components (1-fi) corresponds to the fraction of bound dye. Thus, the fraction of rigidly bound dye is reduced from -99% to 81% upon adsorption of the highest concentration of 10 kDa PEO to the Ludox suspension, indicative of a surface coverage of 18%. Using this approach, the extent of blockage of dye adsorption can be calculated as a function of the molecular weight and concentration of added polymer. As shown in Figure 13.10, more significant changes in the fraction of free dye were obtained after introduction of low molecular weight PEO to the Ludox samples. More importantly, the higher fraction of free, silicaunbound R6G as a result of the silica coverage by the modifiers, indicates a higher degree of Ludox modification by these agents. The majority of the changes occurred at low polymer concentrations in the case of 0.55 kDa PEO. These data clearly show that the lower molecular weight polymer is able to form close packed layers that block the adsorption of the dye to the silica surface, and thus suggest that the smaller polymer chains more thoroughly coat the silica NP surface. This is not unexpected, as the larger polymer would lose

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10

15

301

20

35

Wt% of Polymer Figure 13.10. Fractional contributions from free probe to the total anisotropy decay of R6G in polymer-doped Ludox sols (3 wt % SiOz) determined from TRFA measurements. ( • ) 10 kDa PEG modified Ludox; ( • ) 0.55 kD PEO modified Ludox. Reprinted with permission from Ref 36, Copyright 2004 American Chemical Society.

more degrees of freedom relative to a smaller polymer if all segments adsorbed to the silica surface. 13.4.3.3 Adsorption of Covalent Modifiers One area where anisotropy measurements are particularly useful is for monitoring the modification of silica by organosilanes species. Adsorption of neutral sugarsilanes, cationic aminopropylsilane species and cationic buffer diallyldimethylammonium chloride (DADMAC) to the surface of Ludox NPs was examined by steady-state anisotropy and TRFA.^^ However, relatively low levels of modifier were used to avoid flocculation of the Ludox. Maximum concentrations of the various modifiers were 2.5 wt% for GLTES, 2.0 wt% for MLTES, 0.3 wt% for DADMAC and 0.1 wt% for APTES. Although flocculation was an issue, it was possible to examine samples containing up to 5 Table 13.5. TRFA decay parameters for R6G in organosilane-modified Ludox suspensions.^^ (l>2,ns ^7, ns Target fi Neutral: Cationic:

GLTES, 2.5 wt% MLTES, 2 wt% APTES, 0.005 wt% APTES, 0.01 wt% DADMAC, 0.3 wt%

0.15 + 0.02 0.16 ±0.01 0.20 ±0.01 0.17 ±0.01 0.19 ±0.01

2.3 ±0.5 2.1 ±0.2 2.2 ± 0.9 1.7 ±0.9 2.0 ± 0.4

0.22 0.16 0.73 0.80 0.55

0.49 0.54 0.18 0.09 0.14

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TLEUGABULOVA ETAL.

wt% GLTES or MLTES, since flocculation of the aqueous Ludox sol (3 wt% Si02) required 15 min, providing enough time to carry out the measurement of R6G steady-state anisotropy.^^ Steady-state anisotropy values dropped from 0.336 (unmodified Ludox) to 0.117 (GLTES-Ludox) and 0.09 (MLTES-Ludox), and further increases in the concentration of silane agents has little effect on R6G anisotropy, with minimum values approaching 0.087 and 0.078, respectively. Even lower final steady-state anisotropy values were obtained from cationic modifiers, with values of 0.061 being obtained for 0.3 wt% DADMAC and 0.020 being obtained for 0.1 wt% APTES. If we attribute this decrease to a higher fraction of free, silica-unbound R6G as a result of the silica coverage by the modifiers, the lower anisotropy values should correspond to a higher degree of Ludox modification by these agents. Table 13.5 shows the TRFA parameters obtained for the R6G-Ludox system at the highest level of each modifier. As was the case with PEO, the presence of the modifier led to marked decreases in the g value and correspondingly higher/i values. Such results are consistent with the covalent binding of silane coupling agents to the silica surface, although hydrogen bonding may contribute to the binding. Consistent with the steady-state anisotropy results, the observed increases in/i and decreases in g followed the trend of APTES > DADMAC > GLTES > MLTES, indicative of a higher degree of adsorption for the cationic species relative to the neutral compounds. Hence, the ionic binding of small cationic molecules seems to provide a more efficient silica surface coverage. In the case of the covalent modifiers, the fractional surface coverage was assessed using both steady-state and time-resolved anisotropy data. For steadystate anisotropy, the fractional coverage of the surface can be estimated by comparing the minimum and maximum (limiting) steady-state anisotropy values as 1 - {r^JrQ), where r^Jr^ is the fraction of anisotropy remaining due to bound probe after maximum coverage of the surface with the modifier, and thus 1 -(^minM)) is the fraction of sites that are inaccessible to dye, and thus assumed to be bound to the silane coupling agent. Based on this simple equation, the coverage values are thus estimated to be on the order of 45% for the sugarsilane coupling agents at the flocculation limits of 2.5 wt% (GLTES) or 2 wt% (MLTES), 82% for DADMAC (0.3 wt%) and 94% for APTES (0.1 wt%). Using the TRFA data and assessing surface coverage based no changes in (l-/i), significantly different surface coverage values are obtained: 16% for MLTES, 22% for GLTES, 55% for DADMAC and 80% for APTES. In all cases, the estimated surface coverage from steady-state anisotropy measurements of silica modified surfaces is higher than those obtained by timeresolved anisotropy. The difference in these values lies in the presence of the nanosecond component, which corresponds to bound dye that has relatively rapid rotational motion. In general, the fractional components for the nanosecond component and residual anisotropy are consistent with a redistribution of dye between large and small particles. This contributes to a decrease in R6G steady-state anisotropy, which can be mistakenly interpreted as a higher fraction of free dye, and thus a higher surface coverage of silica, when

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only average anisotropy values are measured. This clearly demonstrates that steady-state anisotropy values overestimate the amount of "free" probe, and hence the extent of surface modification. Using TRFA, it is possible to distinguish between free dye and dye that is bound to rapidly rotating particles, providing a more accurate measurement of the true amount of adsorption. This highlights the importance of using time-resolved anisotropy decays to accurately assess the distribution of dye in Ludox systems. 13.4.3.4 Adsorption of Tripeptides More recent studies have extended our previous work with the R6G:Ludox system to the area of peptide:silica interactions. Four peptides were chosen for this study: the cationic tripeptides Lys-Trp-Lys (KWK) and N-acetyl-Lys-TrpLys (Ac-KWK) and the anionic peptides Glu-Trp-Glu (EWE) and N-acetylGlu-Trp-Glu (Ac-EWE). The number of positively charged ammonium groups in these tripeptides vary from 0 (Ac-EWE) to 3 (KWK) and in the case of EWE and Ac-EWE there are also carboxylate groups of the Glu side chains, which should be repelled from the anionic surface of Ludox. The relatively small size of the tripeptides provides a higher flocculation limit than larger cationic peptides such as polylysine,^^ and thus is likely to lead to greater coverage of the silica surface, while the tryptophan residue provides a spectroscopic handle to allow direct observation of peptide adsorption via Trp fluorescence anisotropy. Tripeptide adsorption was monitored both directly, via Trp steady-state anisotropy, and indirectly, using both steady-state and time-resolved fluorescence anisotropy of R6G, by monitoring the competition of the peptide with R6G for binding to the silica surface. In the case of Trp anisotropy, increases in the silica:peptide molar ratio generally led to increases in anisotropy, with the extend of the anisotropy increase correlating with the number of cationic groups present in the peptide. However, the free rotation of the indole ring relative to the peptide backbone led to relatively low limiting anisotropy values, and the Trp anisotropy appeared to depend on both the degree of adsorption and the binding geometry of the peptide, making it difficult to use Trp anisotropy for quantitative assessment of the extent of peptide binding. On the other hand, the competitive binding assay using TRFA of the R6G/Ludox system provided a usefiil method to probe peptide:silica interactions, with adsorption depending on the number of cationic sites present in the peptide. Referring to Table 13.6, it is apparent that the differences in adsorption of the four peptides fall into two categories. The anionic peptides show essentially no adsorption, based on the minimal changes in both/i and g. The cationic peptides, on the other hand, show both increased/i and decreased g values relative to unmodified Ludox, with the tricationic KWK showing larger variations in these parameters than was observed for the dicationic Ac-KWK at similar peptide concentrations. More importantly, it was possible to use the TRFA decay parameters to assess the extent of surface modification for each peptide. At the flocculation limit, both KWK (5 mM) and Ac-KWK (6 mM)

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Table 13.6. TRFA decay parameters for R6G in peptide-modified Ludox suspensions.^^

Anionic: Cationic:

Target plain Ludox 0.7 wt% Si02 Ac-EWE, 10 mM EWE, lOmM KWK, 0.6 mM KWK, 1 mM KWK, 2 mM KWK, 5 mM Ac-KWK, 5 mM

^h^S

0.1810.01 0.1710.01 0.23 ± 0.07 0.1710.04 0.1810.03 0.1610.02 0.1910.02 0.1910.03

(l>2.ns

3.910.5 3.910.3 1417 3.910.3 4.110.4 3.010.4 3.410.5 3.310.9

//

g

0.05 0.05 0.05 0.20 0.24 0.33 0.44 0.35

0.67 0.66 0.72 0.42 0.37 0.26 0.02 0.40

showed surface coverages of-40%, while EWE and Ac-EWE (both at 10 mM) showed essentially no binding to the surface. Overall, the surface modification studies above show that monitoring the R6G anisotropy during the incremental addition of various modifiers or peptides to aqueous Ludox sols resulted in the ability to measure the degree of surface modification of silica particles. Potential advantages of the indirect TRFA method for probing interactions of compounds with surfaces include: (1) no need for labeling the competing compound, which may be difficult, or may introduce unwanted changes; (2) the ability to use the method for many different species (polymers, peptides, proteins) and (3) the ability to change the nature of the probe to assess different types of interactions (electrostatic, hydrogen bonding, hydrophobic), providing a more versatile method for studying interactions of compounds with surfaces. On the other hand, the indirect method has difficulties detecting the adsorption of weakly binding species, such as EWE, the adsorption of which might be observed by the direct measurement of Trp anisotropy. This suggests that weakly binding molecules cannot effectively compete with R6G for binding to the surface and thus a weakly binding fluorescent probe may be more suitable. It should also be noted that at this point TRFA cannot provide absolute quantitation of the surface coverage, but rather provides a relative assessment of the adsorption as a fianction of the charge and concentration of the modifier. Even so, the TRA measurement method still has significant utility, particularly for evaluating potential surface modification methods that may be used to reduce adsorption. Thus, TRFA measurements should be useful for characterization of biomaterials, antifouling surfaces and new chromatographic stationary phases.

13.5. CONCLUSIONS AND OUTLOOK With the implementation of nanoparticle metrology by Geddes and Birch, the TRFA method has moved beyond the doors of the analytical laboratory and is becoming a promising technique for the analysis of problems related to colloid and interface science. The feasibility of this methodology has been

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proven using aqueous DGS sols. The nanoparticle metrology approach has impacted the interpretation of TRPA decays allowing a more realistic understanding of the sol-gel interior in terms of microviscosity. The nanoparticle metrology also revealed the potential of TRFA decays to monitor the adsorption equilibrium between the soluble and silica-bound fractions of a fluorescent probe dispersed in a silica sol. This finding has been exploited in monitoring the surface modification of colloidal silica suspensions by watersoluble polymers, amines, silica precursors and small peptides. While this review has highlighted the potential of TRPA for studies of silica growth and modification, much remains to be explored. First, methods must be developed to allow assessment of absolute surface coverage, in molecules per nm^ surface area, to allow comparison of TRFA modification data to that obtained by other methods. Second, TRFA methods must be developed to assess the potential of modified surfaces to resist or promote protein adsorption. Using our current method based on R6G equilibrium between bound and free states, there are only minor amounts of probe remaining on highly modified surfaces such as APTES coated Ludox. Thus, adsorption of secondary species such as proteins, particularly to APTES-coated areas, will not lead to redistribution of the dye. Covalent attachment of dyes to Ludox NPs, or examination of changes in the correlation times themselves rather than the fractions of the decay components, may allow for observation of the adsorption of large species, such as proteins. Another area where TRFA needs to be further explored in terms of the surface modification and protein adsorption is the sol-gel monolithic matrices. This can avoid issues with flocculation and thus provide a higher range of modifier concentrations to be studied. Studies are now underway to address these issues, and will be reported in due course. 13.6. ACKNOWLEDGEMENTS The authors thank the Natural Sciences and Engineering Research Council of Canada, MDS-Sciex, the Canadian Foundation for Innovation, and the Ontario Innovation Trust for financial support of this work. John D. Brennan holds the Canada Research Chair in Bioanalytical Chemistry. 13.7. REFERENCES 1. W. Jin and J.D. Brennan, Properties and applications of proteins encapsulated within sol-gel derived materials, Anal. Chim. Acta 461, 1-36 (2002). 2. I. Gill, Bio-doped nanocomposite polymers: sol-gel bioencapsulates, Chem. Mater. 13, 34043421 (2001). 3. W. Tan, K. Wang, X. He, X. J. Zhao, T. Drake, L. Wang and R. P. Bagwe, Bionanotechnology based on silica nanoparticles, Med Res Rev. 24, 621-638 (2004). 4. A. A. Vertegel, R. W. Siegel and J. S. Dordick, Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme, Langmuir 20, 6800-6807 (2004). 5. Du Pont. Ludox Colloidal Silica, Properties, Uses, Storage and Handling (Data sheet, 1987). 6. R. K. Her, The Colloidal Chemistry of Silica (ACS, Washington, 1994). 7. H. Eckert and M. Ward, Controlling the length scale through "soft" chemistry: from organicinorganic nanocomposites to functional materials, Chem. Mater. 13, 3059-3060 (2001).

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56. U. Narang, R. Wang, P. N. Prasad and F. V. Bright, Effects of aging on the dynamics of rhodamine 6G in tetramethyl orthosiHcate-derived sol-gels, J. Phys. Chem. 98, 17-22 (1994). 57. K. E. Wheeler, N.S. Lees, R. J. Gurbiel, S. L. Hatch, J. M. Nocek and B. M. Hoffman, Electrostatic influence on rotational mobilities of sol-gel-encapsulated solutes by NMR and EPR spectroscopies, J. Am. Chem. Soc. 126, 13459 - 13463 (2004). 58. C. D. Geddes, J. Karolin and D. J. S. Birch, Fluorescence anisotropy in sol-gels: microviscosities or growing silica nanoparticles offering a new approach to sol-gel structure elucidation,/. Fluoresc. 12, 135-137 (2002). 59. D. Tleugabulova, W. Czardybon and J. D. Brennan, Time-resolved fluorescence anisotropy in assessing side-chain and segmental motions in polyamines entrapped in sol-gel derived silica, J. Phys. Chem. B. 108, 10692-10699 (2004). 60. H. Shirota and H. Segawa, Time-resolved fluorescence study on liquid oligo(ethylene oxide)s: coumarin 153 in poly(ethylene glycol)s and crown ethers, J. Phys. Chem. A 107, 3719 - 3727 (2003). 61. S. Sen, D. Sukul, P. Dutta and K. Bhattacharya, Solvation dynamics in aqueous polymer solution and in polymer-surfactant aggregate, J. Phys. Chem. B 106, 3763 -3769 (2002). 62. S. Zorrilla, G. Rivas and M. P. Lillo, Fluorescence anisotropy as a probe to study tracer proteins in crowded solutions, J. MolRecognit. 17, 408-416 (2004). 63. M. Polverari and T. G. M. van de Ven, Dilute aqueous poly(ethylene oxide) solutions: clusters and single molecules in thermodynamic equilibrium, J. Phys. Chem. 100, 13687 - 13695 (1996). 64. I. Isenberg, On the theory of fluorescence decay experiments. II. Statistics, J. Chem. Phys. 59, 5708-5713 (1973). 65. K. Kinosita Jr., A. Ikegami and S. Kawato, On the wobbling-in-cone analysis of fluorescence anisotropy decay, Biophys. J. 37, 461-464 (1982). 66. P. Damberg, J. Jarvet, P. Allard, U. Mets, R. Rigler and A. Graslund, 13C-1H NMR relaxation and fluorescence anisotropy decay study of tyrosine dynamics in motilin, Biophys. J. 83, 28122825 (2002). 67. G. R. Ziegler and E. A. Foegeding, The gelation of proteins. Adv. Food Nutr. Res. 34, 203-298 (1990). 68. H. Boukari, J. S. Lin and M. T. Harris, Small-angle X-ray scattering study of the formation of colloidal silica particles from alkoxides: primary particles or not? J. Colloid Interface Sci. 194, 311-318(1997). 69. C. H. Byers, M. T. Harris and D. F. Williams, Controlled microcrystalline growth studies by dynamic laser-light-scattering methods, Ind. Eng. Chem. Res. 26, 1916-1923 (1987). 70. T. Matsoukas, E. Gulari, Monomer-addition growth with a slow initiation step: A growth model for silica particles from alkoxides, J. Colloid Interface Sci. 132, 13-21 (1989). 71. V. K. LaMer, Kinetics in phase transitions, Ind. Eng. Chem. 44, 1270-1277 (1952). 72. A. van Blaaderen, J. van Geest and A. Vrij, Monodisperse colloidal silica spheres from tetraalkoxy si lanes: particle formation and growth mechanism, J. Colloid Interface Sci 154, 481501 (1992). 73. G. H.Bogush, C. F. Zukoski, Studies of the kinetics of the precipitation of uniform silica particles through the hydrolysis and condensation of silicon alkoxides, J. Colloid Interface Sci. 142,1-18(1991). 74. G. H.Bogush, C. F. Zukoski, Uniform silica particle precipitation: An aggregative growth model, J. Colloid Interface Sci. 142, 19-34(1991). 75. K. Lee, J. L. Look, M. T. Harris and A. V. McCormick, Assessing extreme models of the Stober synthesis using transients under a range of initial composition, J. Colloid Interface Sci. 194, 7888(1997). 76. R. B. Bhatia, C. J. Brinker, A. K. Gupta and A. K. Singh, Aqueous sol-gel process for protein encapsulation, Chem. Mater. 12, 2434 - 2441 (2000). 77. K. Ishibashi, O. Sato, R. Baba, D. A. Tryk, K. Hashimoto and A. Fujishima, Characterization of the chromophore orientation of rhodamine B amphiphiles in Langmuir Blodgett monolayers. J. Colloid Interface Sci 233, 361-363 (2001). 78. T. Kikteva, D. Star, Z. Zhao, T. L. Baisley and G. W. Leach, Molecular orientation, aggregation, and order in rhodamine films at the fused silica/air interface, J. Phys. Chem. B. 103, 1124-1133 (1999). 79. R. Sasai, T. Fujita, N. lyi, H. Itoh and K. Takagi, Aggregated structures of rhodamine 6G intercalated in a fluor-taeniolite thin film, Langmuir 18, 6578-6583 (2002).

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80. J. Bujdak, N. lyi, Y. Kaneko, A. Czimerova and R. Sasai, Molecular arrangement of rhodamine 6G cations in the films of layered silicates: the effect of the layer charge, Phys. Chem. Chem. Phys. 5, 4680-4685 (2003). 81. R. J. Hodgson, Y. Chen, Z. Zhang, D. Tleugabulova, H. Long, X. Zhao, M. Organ, M. A. Brook and J. D. Brennan, Protein-doped monolithic silica columns for capillary liquid chromatography prepared by the sol-gel method: applications to frontal affinity chromatography. Anal Chem.. 76, 2780-2790 (2004). 82. S. R. Raghavan, H. J. Walls and S. A. Khan, Rheology of silica dispersions in organic liquids: new evidence for solvation forces dictated by hydrogen bonding, Langmuir 16, 7920 - 7930 (2000). 83. J. N. Cha, G. D. Stucky, D. E. Morse, T. J. Deming, Biomimetic synthesis of ordered silica structures mediated by block copolypeptides, Nature 403, 289-92 (2000). 84. P. van der Meeren, H. Saveyn, S. Bogale Kassa, W. Doyen, R. Leysen, Colloid-membrane interactions effects on flux decline during cross-flow ultrafiltration of colloidal silica on semiceramic membranes, Phys. Chem. Chem. Phys. 6, 1408-1412 (2004). 85. M. Kosmulski, Electrokinetic study of specific adsorption of cations on synthetic goethite. Colloid Surf. A 111, 113-118 (2003). 86. J. Laven and H. N. Stein, The electroviscous behavior of aqueous dispersions of amorphous silica (Ludox), J. Colloid Interface Sci. 238, 8-15 (2001). 87. H. Wang, E. C. Y. Yan, Y.Liu, K. B. Eisenthal, Energetics and population of molecules at microscopic liquid and solid surfaces, J. Phys. Chem. B. 102, 4446-4450 (1998). 88. H. Wang, E. Borguet, E. C. Y. Yan, D. Zhang, J. Gutow and K. B. Eisenthal, Molecules at liquid and solid surfaces, Langmuir 14, 1472-1477 (1998). 89. H. M. Eckenrode and H.-L. Dai, Nonlinear optical probe of biopolymer adsorption on colloidal particle surface: poly-L-lysine on polystyrene sulfate microspheres, Langmuir 20, 9202-9209 (2004). 90. M. A. Polizzi, R. M. Plocinik and G. J. Simpson, EUipsometric approach for the real-time detection of label-free protein adsorption by second harmonic generation, J. Am. Chem. Soc. 126,5001-5007(2004). 91. M. Y. Lin, H. M. Lindsay, D. A. Weitz, R. Klein, R. C. Ball and P. Meakin, Universal diffusion-limited colloid aggregation, J. Phys. Condens. Matter 2, 3093-3113 (1990). 92. J. Hester, P. Banerjee and A. Mayes, Preparation of protein-resistant surfaces on poly(vinylidene fluoride) membranes via surface segregation, Macromolecules 32, 1643-1650 (1999). 93. A. K. Gupta, A. S. Curtis, Surface modified superparamagnetic nanoparticles for drug delivery: interaction studies with human fibroblasts in culture, J. Mater. Sci. Mater. Med. 15, 493-496 (2004). 94. Q. Chen, G. L. Kenausis and A. Heller, Stability of oxidases immobilized in silica gels, J. Am. Chem. Soc.llQ, 4582-4585 (1998). 95. J. Heller, A. Heller, Loss of activity or gain in stability of oxidases upon their immobilization in hydrated silica: significance of the electrostatic interactions of surface arginine residues at the entrances of the reaction channels, J. Am. Chem. Soc. 120, 4586-4590 (1998). 96. T. Keeling-Tucker, M. Rakic, C. Spong, J. D. Brennan, Controlling the material properties and biological activity of lipase within sol-gel derived bioglasses via organosilane and polymer doping, Chem. Mater. 12, 3695-3704, (2000). 97. G. A. Baker, S. Pandey, E. P. Maziarz III and F. V. Bright, J. Sol-Gel Sci. Technol. 1999, 75, 37. 98. A. Bronshtein, N. Aharonson, D. Avnir, A. Tumiansky and M. Alstein, Sol-gel matrixes doped with atrazine antibodies: atrazine binding properties, Chem. Mater. 9, 2632-2639 (1997).. 99. T. Coradin, O. Durupty and J. Livage, Interactions of amino-containing peptides with sodium silicate and colloidal silica: a biomimetic approach of silicification, Langmuir, 18, 2331-2336 (2002).

DYNAMICS OF DNA AND PROTEIN-DNA COMPLEXES VIEWED THROUGH TIMEDOMAIN FLUORESCENCE Nabanita Nag\ T. Ramreddy^ Mamata Kombrabail\ P.M. Krishna Mohan^, Jacinta D'souza^, B.J. Rao^, Guy Duportail^, Yves Mely , and G. Krishnamoorthy 14.1. INTRODUCTION In recent times, there has been increasing level of realization that activity of biomolecular systems arises from a combination of structure and dynamics^''^. While this concept has gained substantial experimental support in proteins^""^, the role of dynamics is comparatively less appreciated in DNA where the activity is assumed to be largely controlled by its sequence. This expectation might have arisen due to the fact that DNA is more rigid and structured than proteins and other bio-macromolecules. However, a variety of intramolecular as well as intermolecular interactions often influence the structure and dynamics of DNA. Double-stranded (ds) DNA is generally modeled as a string of rods (or disk) which follows Hookean twisting and bending much like a spring where the counter interacting torsional and flexural deformation play an important role in maintaining its rigidity^. The rigidity of DNA is often characterized by its persistence length which is -- 50 nm for dsWatson-Crick B-form^ and ~ 1.0 nm when present as single stranded^. Furthermore, the structure of DNA double helix is not monotonous but instead exhibits marked sequence dependent variations^. Ds-DNA bends easily at one 1

2

Department of Chemical Sciences, Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India. ^Laboratoire de Pharmacologic et Physicochimie des interactions cellulaires et moleculaires, UMR 7034 du CNRS, Faculte' de Pharmacie, Universite' Louis Pasteur de Strasbourg, 74 Route du Rhin, 67401 lUkirch, France. 311

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plane when compared to the other thus giving rise to 'anisotropic flexibiHty^"^\ This in turn helps ds-DNA to form transient as well as stable complexes with proteins and other co-factors and drugs. Sequence-specific bends in DNA has been recognized as one of the mechanisms for specific binding of proteins^^. Additionally, sequence-specific dynamics along the backbone could be thought of as another interesting mechanism for specific recognition by proteins (see section 4.2 and 4.3). The hierarchy of dynamics could be classified in timescale and in molecular structure. Fluorescence studies have shown that the interior of DNA has the diffusive and viscous dynamics characteristics of fluid, rather than the purely vibrational dynamics of crystals^^. These local motions in DNA could occur in sub-nanosecond timescales. This would include propeller twist and wobbling motion of base pairs involving relatively small segments of DNA. In contrast, movement of DNA backbone giving rise to twisting of double helix (torsion) and in tum long range bending and global tumbling of the DNA involve larger segments and would occur in the timescales of nanoseconds and beyond. Spontaneous and transient opening/ disruptions of double helix have been shown to be segmental rather than continuous^'^. Hydrogen exchange studies by NMR have revealed the occurrence of local fluctuations as well as melting and unstacking of the duplex*^' ^^. Such local and small perturbations could finally determine long range torsional and flexural rigidity and Brownian dynamics of DNA. Capturing high resolution snapshots of the various molecular motions under equilibrium conditions poses technical challenges. A variety of physical techniques such as NMR, X-ray crystallography, electron microscopy, atomic force microscopy (AFM), electrophoretic mobility on gels and fluorescence spectroscopy are being used to study the structure and dynamics of DNA systems. By virtue of its selectivity, sensitivity and large temporal range, fluorescence spectroscopy has become an important and widely used technique for studying structure and dynamics of small as well as large biological macromolecules in solution^^"^^. The exquisiteness of this technique lies on the selectivity, where the small change in the environment around the fluorophore is reflected in the measurable change in the emission properties without interference from the rest of the system. The observation can be made with small amount of sample which gives it sensitivity. The most important feature of fluorescence spectroscopy is the characteristic time scale of the emission process over a time period ranging from picoseconds to nanoseconds which is comparable to the temporal range of biological macromolecular dynamics. The dynamic nature of DNA structure, which is essential for its ftinction, gives its structural heterogeneity. The apparent level of heterogeneity depends upon the time window used for observation. Large time windows result in averaging of the structural parameters, whereas shorter windows produce an instantaneous snapshot of the structural variants populating a distribution. The time window set by fluorescence based methods is linked to the excited state life time of the fluorophore, which lies generally in the range of 10 ps to 10 ns. The rapid timescale of the emission process allows us to take snapshots of

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various structural forms present due to various types of molecular movements including internal motion.

14.2. FLUORESCENCE PROBES FOR DNA DYNAMICS The fluorescent quantum yield of native DNA which is due to its bases is too small (Of- 4x10'Y^'^^ and its fluorescent lifetimes too short (10-60 ps)^^. However, there have been several attempts to use the intrinsic fluorescence of DNA to gain information on its dynamics^^. Furthermore, due to its non-specific nature, its use is limited to study only overall dynamics of DNA such as internal motion of DNA bases^"*. Several extrinsic probes (Figure 14.1 A) have been developed over the years to make DNA fluorescently visible. They fall into two categories, namely specific and non-specific. Non-specific probes such as ethidium bromide^^, DAPI^^ propydium iodide^^, YOYO^^' ^^ and PicoGreen^^ (PG) and their large range of analogs were originally developed as DNA stains to visualize DNA in fluorescence microscopic images. However, probes such as ethidium^^' ^^ and YOYO-1^^' ^^ (see section 3) have found use in studying complex dynamic modes of DNA and as condensation indicators (see section 3.3 and 3.4). Most of these probes are nearly non-fluorescent in their free form and become highly fluorescent when bound to DNA (see section 3.3 and 3.4). Although many of these probes are either mono or bis intercalators, but DAPI^^ work as groove binders. Probe such as PicoGreen^^ has been used to assess the relative population of single-stranded regions and single-strand nicks in ds-DNA^^' ^^. This capability arises presumably due to the modulation of fluorescence quantum yield by the various dynamic modes of DNA (see section 3.5). Owing to its non-specific probing properties, the probes mentioned above are not useful in studying environmental change in a specific location of DNA. Hence site-specific fluorescent probes (Figure 14.IB) which are mainly nucleotide base analogues are most appropriate in studying the dynamics in a specific region of DNA. Selection of these nucleotide analogs is generally based on the criteria that the fluorescent analog mimics the parent nucleotide in its base-pair hydrogen bonding and other interaction potentials. 2-aminopurine (2AP) which is being widely used as an analog of adenine forms hydrogen bonds with thymine very similar to those of adenine (Figure 14.2A). However, 2AP:T base-pair itself is treated as a mismatched base-pair by the mismatch recognition protein, Mut-S^^' ^^ indicating subtle differences in the base-pair structure and dynamics. Although the quantum yield of 2-AP gets substantially reduced (Of ~ 0.1) on incorporation into DNA, it is still sufficient for monitoring subtle changes in the structure and dynamics"^^"^^. Very recently, 8-vinyl-deoxyadanine (8-VA) has been proposed as an improved substitute of 2-AP'^^. The quantum yield of this new adenine analog is significantly higher than that of 2-AP, when inserted in DNA. In addition, 8-VA

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Figure 14.1A: Chemical structures of some of the non-specific fluorescence probes used for monitoring DNA dynamics, (i) Ethidium bromide, (ii) DAPI, (iii) PicoGreen (PG), (iv) Propidium iodide, (v) YOYO-1.

is able to adopt an anti conformation that preserves the Watson-Crick hydrogen bonding. Another fluorescent analog pyrrolo-dC (PdC) was introduced few years back which can pair with Guanine'*'^ (Figure 14.2B). The quantum yield sensitivity of pyrrolo-dC is quite similar to that of 2-AP'^'^' ^^. All the three probes 2-AP, 8-VA and PdC show significant reduction in their quantum yield on base-pair formation and thus serve as indicators of local hybridization. However, PdC is likely to have greater potential since its excitation wavelength (347 nm) overlaps less with that of proteins. Thus PdC is better suited for studies on DNA-protein complexes. Furthermore, PdC has a very unique characteristic when it is mismatched in a ds-DNA. Its quantum yield in this condition is higher than that in the single-stranded species when the mismatched base is adenosine. This unusual behavior has been used to probe dynamics at the mismatched site"^^.

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Several other fluorescent base analogs such as Etheno-A and Etheno-C (Figure 14.IB) developed earlier have limited use due their poor hybridizing nature'*^. Other analogs of adenosine such as 6-MAP (4-amino-6-methyl-8(2-deoxy-beta-d-riboftiranosyl)-7(8H)-pteridone)(Figure 14.IB) and derivatives of guanosine and other nucleotides have also been investigated^^' ^^ One of the commonly used fluorescent labeling of DNA, especially at the ends of DNA, rely upon nucleoside bases derivatized with probes such as fluorescein, rhodamine, Cy3, Cy5 and Alexa derivatives with an aliphatic linker. The linkers are attached to C-5 or C-8 position of pyrimidine or purine bases respectively so that the linker arms are projected into the major groove of the DNA and thus interfere less with base-pairing interactions. These probes, when placed far from the ends, give useful information about the microenvironment inside the groove"^^' ^^ apart from being useful in foot printing studies^^' ^^ involving DNA binding proteins. These fluorescent dyes along with the linker are also tethered to the 5' or 3' terminus of the DNA and extensively used in applications such as estimation of DNA bends^^, interaction with proteins from the change in its tumbling motion^^, formation of hairpin bends^^ and DNA hybridization^^' ^'^.

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Figure 14.IB: Chemical structures of some of the site-specific fluorescence probes used for monitoring DNA dynamics, (i) Etheno-A, (ii) Etheno-C, (iii) 6-MAP, (iv) 2-Aminopurine (2-AP), (v) 8-Vinyladenine (8-VA), (vi) Pyrrolo-dC (PdC).

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14. 3. PROBING DNA DYNAMICS WITH NON-SPECIFIC PROBES 14.3.1. DNA condensation Biological interest in DNA is generally confined to its sequence. However, higher order structures and structural changes in DNA, which are sequence-independent, have profound control on several processes that involve DNA. Condensation of DNA, whereby DNA transforms from an open and

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fluctuating architecture into a compact and relatively static form is encountered in a variety of situations both natural and artificial. Chromatinized DNA in the nucleus of mammalian cells^^ and DNA packaged into phage heads^^ exemplify the phenomenon of natural condensation. A major part of artificial condensation of DNA is related to the development of gene delivery (therapy) systems in which DNA is condensed and compacted by a variety of agents such as cationic lipids^^' ^^, polycations^^' ^^, cationic polymers, and cationic detergents^'* before being taken up into cells by endocytic pathway^^' ^^. The process of DNA condensation has to achieve a fine balance between efficient compaction to economize the size while preserving the function such as gene expression in chromosomes. This makes studies on the structure and dynamics of condensed forms of DNA quite rewarding. Condensation of DNA has been studied by a variety of physical techniques mainly based on light and X-ray scattering^^' ^^, spectroscopy^"^, imaging^^"^^ and viscometry . Atomic force microscopy ' and electron microscopy have revealed that condensed form of DNA has either toroidal or rod-like structures having dimensions in the range of 50-100 nm. Despite the use of a large variety of experimental techniques to DNA condensates, atomic level high-resolution structures are lacking. For example, even information such as relative localization of the condensing agent with respect to the DNA backbone is not known with certainty although it has been shown that condensation results in protection of DNA from nucleases^^. Similarly, questions such as how the therapeutic DNA which is condensed by various agents is integrated into the genome and does transcription of this DNA require decondensation, are largely unanswered. In this scenario it becomes quite relevant and interesting to look for detailed information on the comparative dynamics of DNA backbone and base in condensed and extended forms of DNA. 14.3.2. YOYO-1 as an indicator of DNA condensation We had shown recently that YOYO-1, a DNA bis-intercalator, is a convenient and robust marker of DNA condensation^'*. The fluorescence quantum yield of YOYO-1 gets enhanced by several orders of magnitude on intercalation presumably due to suppression of internal rotation-induced nonradiative decay when bound to DNA^"^. This model for the remarkable level of enhancement in the fluorescence gets support from the observation that the quantum yield of YOYO-1 gets significantly enhanced in a viscous solvent such as glycerol. Bis-intercalation of analogs of YOYO-1 has been confirmed from NMR structures'^ When used at relatively high levels (YOYO-1: DNA phosphate, D:P > 1/50), the quantum yield of DNA-bound YOYO-1 shows a remarkable decrease following the condensation of DNA. Detailed spectroscopic investigations^^ showed that the formation of H-dimers of YOYO-1 (Figure 14.3A) as the cause of condensation-induced decrease in quantum yield. The major evidence for the formation of H-dimers comes from the observation of excitonic blue shift ' ' in the absorption spectrum of DNA-bound YOYO-1^^ Furthermore, the closeness of DNA-bound YOYO-1 in the presence of condensing agents was demonstrated by time-resolved

318

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Figure 16.7. Fluorescence spectra of fluorescein (2.5%) in aqueous solution (intrinsic) and in a tissue phantom (distorted) composed of fluorescein (2.5%), Direct Blue absorber (|aa=ll cm'^ at 500 nm) and polystyrene particles (|as'=20 cm"', g=0,82 at 500 nm). The corrected spectrum is shown in the same figure. The measurements were made using a single-fiber probe. The squared estimation error between the corrected fluorescence spectrum and the intrinsic fluorescence spectrum was 2.8x10 ^.

Figure 16.7 shows a normalized intrinsic fluorescence spectrum Ft of fluorescein in an aqueous solution, a normalized distorted fluorescence spectrum for the same fluorophore in tissue phantom Fm and the normalized corrected fluorescence spectrum F^. The model permitted to correct the distortion of fluorescence spectra due to the turbidity of the medium. Such an approach can be implemented in fluorescence spectral and imaging techniques to account for the spatial variation due to tissue optical properties in order to enhance the detection features that are related to the tissue malignancies. 16.6. CONCLUSIONS We have reviewed several optical approaches being studied for assessing melanoma by imaging, light-induced fluorescence spectroscopy and elastic scattering spectroscopy. Current fluorescence spectroscopy and imaging techniques are convenient for noninvasive diagnosis of certain cancers and noncancer diseases. Although the skin is easily accessible by optical techniques, there are challenges and limitations to the application of fluorescence spectroscopy for distinguishing malignant from benign nevi. The development of new optical reporters for melanoma, with higher selectivity and reduced skin photosensitivity and toxicity may lead to reestablishing larger investigations for assessing pigmented skin lesions. It is important to acquire fluorescence signatures that are independent of optical properties of tissue for the elucidation of fluorescence diagnostic signatures.

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Elastic scattering spectroscopy is a sensitive technique for detecting changes in sub-cellar morphology that accompany the transformation of normal cells to cancerous cells . Preliminary measurements have shown promise for the differentiation of benign, dysplastic nevi and malignant melanocytic lesions. The ultimate utility of ESS will require larger studies to benchmark the diagnostic accuracy histology, and then compare the accuracy with the clinical assessment of dermatologists and general practitioners, in order to establish the clinical benefits of ESS in the management of melanoma in humans. Larger scale studies are in progress. Finally, the combination of ESS and LIFS might provide a better solution for diagnosing melanoma With a slight modification, one instrumentation can be used to acquire both ESS and LIFS measurements. Such technique will provides wider information about tissue morphology and biochemistry for enhancing the differentiation between benign nevi and malignant melanoma.

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105. J. Mourant, J Freyer, A Hielscher, A Eick, D Shen, T Johnson, Mechanisms of light scattering from biological cells relevant to noninvasive optical-tissue diagnostics, Applied Optics 37, 3586-3593(1998). 106. R. Marchesini, M. Brambilla, C. Clemente, M. Maniezzo, A. E. Sichirollo, A. Testori, D. R. Venturoli and N. Cascinelli, In vivo spectrophotometric evaluation of neoplastic and nonneoplastic skin pigmented lesions~I. Reflectance measurements. Photochemistry and Photobiology 53(1), 77-84 (1991). 107. D. Rallan, C. C. Harland, Skin imaging: is it clinically useful?, Clinical and Experimental Dermatology, 29(5):453-9 (2004). 108. J. Boyer, J. R. Mourant and I. J. Bigio, Monte Carlo investigations of elastic scattering spectroscopy applied to latex spheres used as tissue phantoms, Proc. SPIE 2389, 103-12 (1995). 109. I. J. Bigio, J. R. Mourant, J. Boyer, T. Johnson and T. Shimada, Noninvasive identification of bladder-cancer with sub-surface backscattered light, Proc. SPIE 2135 26-35 (1994). 110. L. G. Henyey and Greenstein JL, Astrophysics Journal 93, 70 (1941). 111.1. J. Bigio, S. G. Bown, G. Briggs, C. Kelley, S. Lakhani, D. Pickard, P. M. Ripley, I. Rose and C. Saunders, Diagnosis of Breast Cancer using Elastic-Scattering Spectroscopy, Preliminary Clinical Results, Journal of Biomedical Optics 5, 221-228 (2000). 112. J. R. Mourant, I. J. Bigio, J. Boyer, T. M. Johnson, J. A. Lacey, A. G. Bohorfoush, and M. Mellow, Elastic-scattering spectroscopy as a diagnostic tool for differentiating pathologies in the gastrointestinal tract, preliminary testing. Journal of Biomedical Optics 1(2), 192-199 (1996). 113. D. C. Pickard, L. B. Lovat, M. Novelli, P. M. Ripley, C. Kelly, I. J. Bigio and S. G. Bown, Diagnosis of dysplasia in Barrett's oesophagus with in-situ elastic-scattering spectroscopy. Optical Biopsy and Tissue Optics, Proc. SPIE 4161, 122-130 (2000). 114. J. R. Mourant, I. J. Bigio, J. Boyer, R. L. Conn, T. M. Johnson and T. Shimada, Spectroscopic diagnosis of bladder cancer with elastic light scattering. Lasers in Surgery and Medicine 17, 350-357(1995). 115. M. L. Wolbarsht, A. W. Walsh and G. George, Melanin, a unique biological absorber, Applied Optics 20, 2184-2186 (1981). 116. R. Marchesini, C. Clemente, E. Pignoli and M. Brambilla, Optical properties of in vitro epidermis and their possible relationship with optical properties of in vivo skin. Journal of Photochemistry and Photobiology B, Biology 16(2), 127-140 (1992). 117. G. Zonios, J. Bykowski and N. Kollias. Skin melanin, hemoglobin and Hght scattering properties can be quantitatively assessed in vivo using diffuse reflectance spectroscopy, Journal of Investigative Dermatology 117(6), 1452-1457 (2001). 118 M. Canpolat and J. R. Mourant, Particle size analysis of turbid media with a single optical fiber in contact with the medium to deliver and collect white light, Applied Optics 40, 37923799(2001). 119 A. Amelink, M. P. L. Bard, S. A. Burgers, H. J. C. M. Sterenborg, Single-scattering spectroscopy for the endoscopic analysis of particle size in superficial layers of turbid media, Applied Optics 42, 4095-4101 (2003). 120 V. Backman, R. Gurjar, K. Badizadegan, I. Itzkan, R. RO Dasari and L. T. Perelman, MS Feld, Polarized light scattering spectroscopy for quantitative measurement of epithelial cellular structure in situ, IEEE Selected Topics in Quantum Electrons 5, 1019-1026 (1999). 121 K. Sokolov, R. Drezek, K. Gossage and R. Richards-Kortum, Reflectance spectroscopy with polarized light: is it sensitive to cellular and nuclear morphology. Optics Express 5, 302-317 (1999). 122 J. R. Mourant, T. M. Johnson, S. Carpenter, A. Guerra, T. Alda and J. P. Freyer, Polarized angular dependent spectroscopy of epithelial cells and epithelial cell nuclei to determine the size scale of scattering structures. Journal of Biomedical Optics 7, 378-387 (2002). 123. J. R. Mourant, J. Boyer, A. H. Hielscher and I. J. Bigio Influence of the scattering phase function on light transport measurements in turbid media performed with small sourcedetector separations. Optics Letters 21(7), 546-548 (1996).

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124. D. F. Kusewitt, L. A. Applegate, C. D. Bucana and R. D. Ley, Naturally occurring malignant melanoma in the South American opossum (Monodelphis domestica), Veterinary Pathology 27, 66-68 (1990). 125. R. Marchesini, N. Cascinelli, M. Brambilla, C. Clemente, L. Mascheroni, E. Pignoli, A. Testori and D. R. Ventroli, In vivo spectrophotometric evalulation of neoplastic and non-neoplastic skin pigmented lesions. II: discriminant analysis between nevus and melanoma, Photochemistry and Photobiology 55(4), 515-522 (1992). 126. R. Marchesini, S. Tomatis, C. BartoH, A. Bono, C. Clemente, C. Cupeta, I. Del Prato, E. Pignoli, SichiroUo AE and Cascinelli N., In vivo spectrophotometric evaluation of neoplastic and non-neoplastic skin pigmented lesions. III. CCD camera-based reflectance imaging, Photochemistry and Photobiology 62(1),151-154 (1995). 127. M. Moncrieff, S. Cotton, E. Claridge, P. Hall, Spectrophotometric intracutaneous analysis: a new technique for imaging pigmented skin lesions, British Journal of Dermatology 146(3):448-457 (2002). 128. L. M. Mcintosh, R. Summers, M. Jackson, H. H. Mantsch, J. R. Mansfield, M. Howlett, A. N. Crowson and J. W. P. Toole. Towards non-invasive screening of skin lesions by near-infrared spQctroscopy, Journal of Investigative Dermatology 116, 175-181 (2001). 129. V. P. Wallace, D. C. Crawford, P. S. Mortimer, R. J. Ott and J. C. Bamber, Spectrophotometric assessment of pigmented skin lesions, methods and feature selection for evaluation of diagnostic performance, Physics in Medicine and Biology 45(3), 735-51 (2000). 130. V.P Wallace, JC Bamber, D. C. Crawford, R. J. Ott and P. S. Mortimer, Classification of reflectance spectra from pigmented skin lesions, a comparison of multivariate discriminant analysis and artificial neural networks. Physics in Medicine and Biology 45(10), 2859-2871 (2000). 131. J. J. Scarisbrick, C. D. O. Pickard, A. C. Lee, G. M. Briggs, K. Johnson, S. G. Bown, M. Novelli, M. R. S. Keshtgar and I. J. Bigio, R. Yu, Elastic scattering spectroscopy in the diagnosis of pigmented lesions: Comparison with clinical and histopathological diagnosis, Diagnostic Optical Spectroscopy II, Proc. SPIE 5U\ (2003). 132. N. N. Zhadin and R. R. Alfano, Correction of the internal absorption effect in fluorescence emission and excitation spectra from absorbing and highly scattering media: theory and experiment. Journal of biomedical optics, 3 (2), 171 -186 (1998). 133. Q. Zhang, M. G. MuUere, J. Wu and M. S. Feld, Turbidity-free fluorescence spectroscopy of biological tissue. Optics letters 25 (19), 1451-1453 (2000). 134. J. Y. Qu and J. Hua, Calibrated fluorescence imaging of tissue in vivo, Applied Optics 78 (25), 4040-4042(2001). 135. O. M. A'Amar and I. J. Bigio, Correction of fluorescence spectra using data from elastic scattering spectroscopy and a modified Beer's law. Optical Society of America Topical Meetings, Miami (2002).

QUANTITATIVE FLUORESCENCE HYBRIDIZATION USING AUTOMATED IMAGE CYTOMETRY ON INTERPHASE NUCLEI Applications on chromosome imbalances Khuong Truong, Anne Gibaud, Nicolas Vogt, and Bernard Malfoy^

17.1. INTRODUCTION Chromosome imbalances are the hallmark of malignancies in several human pathologies. In contrast to normal cells, alterations of the amount of chromosomes or chromosome arms (chromosome aneuploidy) are consistently observed in virtually all cancers [1]. There is much controversy about the cause and effect with regard to malignancy [ 2 - 4 ] . However, evidence such as specific gains or losses of chromosomes segments within specific tumour types, presence of aneuploidy in various pre-neoplastic conditions and increased frequency of genetic instability in aneuploid cell lines compared to diploid cells suggest that this phenomenon possibly plays an active role in carcinogenesis. Whatever their function may be, the presence of chromosome imbalances in somatic tissue, is a strong diagnostic indication for malignant transformation. Dramatic consequences of chromosome imbalances are also well established for several constitutional diseases. For example, trisomy of chromosome 21 (Down's syndrome) is the most frequently observed aberration in newborns [5]. Thus, the detection of numerical chromosome aberrations remains the main purpose of prenatal diagnosis. Several methods have been developed to detect chromosome number abnormalities in situ using fluorescence-based approaches. Data acquisition is currently performed by human observers using

Khuong Truong, IMSTAR. Paris France. Anne Gibaud, Nicolas Vogt, Bernard Malfoy. Institut Curie-CNRS-UPMC UMR7147. Institut Curie, 26 Rue d'Ulm 75248 Paris Cedex 5 France. 387

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digital cameras for image recording and visual counting of the signals. In order to improve the reliability of these molecular cytogenetic approaches, we have developed a quantitative two-colour fluorescence in situ hybridization (FISH) method based on differential chromosome or chromosome arm paintings for detection of imbalances on interphase nuclei using automated image cytometry. 17.2. CHROMOSOME IMBALANCES IN HUMAN DISEASES. 17.2.1. Cancers. Considerable efforts have been devoted to characterize biological indicators of human cancers with relevance to clinical outcome. Cancer cells can be characterized by their genetic alterations having initiated the evolution from normal to malignant cells. Such alterations can be reflected either at the molecular or chromosomal level. In the latter case, the analysis of karyotypes reveales chromosome aberrations with gains and losses of chromosome segments leading to chromosome aneuploidy. Variations in the number of gene copies following an aneuploidy may induce overexpression (oncogenes, receptors,) or underexpression (suppressor genes) of genes at the origin of the tumour development. Thus, specific chromosome gains and losses may indicate how to establish tumour classifications for specific therapies. In human solid tumours, specific chromosome aberrations were particularly well characterized in breast and lung cancers. The most recurrent clonal alterations within breast cancers affect chromosome 1, with principally gain of the long arm (Iq) and loss of the short arm (Ip) leading to Iq/lp imbalance [6,7]. These aberrations are regarded as early events in tumorigenesis resulting from various rearrangements between chromosomes 1 and/or 16 [8,9]. Bronchic cancers can be divided into two categories: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) [10]. The short arm of chromosome 3 is partially or totally lost in more than 90% of SCLC and 50-80% of NSCLC [11,12]. This loss is considered as an early event in the tumour progression since it is also observed in pre-neoplastic lesions [13,14]. In addition, recurrent gains of the long arm of chromosome 3 are also observed. In particular, in about 50% of NSCLC cases, gains in the whole 3q arm are found [15,16]. Thus, loss of 3p associated with gain of 3q leads to an imbalance between the long and short arms within many lung tumours. 17.2.2. Constitutional diseases Children affected by chromosome trisomy usually have a range of birth defects, including delayed development and intellectual disabilities. Apart from Down's syndrome (trisomy 21), trisomy 13 and trisomy 18 allow live bom children [17 - 20]. However, their incidence remain lower, about one in every 5000 or 3000 births for trisomy 13 and 18, respectively, and one in 1000 births for trisomy 21. The presence of an extra copy of genes localized on the third chromosome leads to overexpression of some genes involved in the child's

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development. It is estimated that only a small percentage of these presently unidentified genes, may be involved in producing the symptoms of Down syndrome. The addition of an extra chromosome usually occurs spontaneously during conception. The cause of this is unknown and therapy remains difficult [5]. Prenatal diagnosis is the only way to provide informed consent to pregnant women.

17.3. EXPERIMENTAL APPROACHES FOR THE IN SITU DETERMINATION OF CHROMOSOME IMBALANCES. 17.3.L Metaphase chromosomes. The 23 human chromosome pairs within metaphase are formed during the cell division by condensation of interphase chromatin. For experimental purposes, they are generally obtained by growing in vitro cells coming from in vivo material (tumour fragments or embryonic tissues). Preparations are spread out on slides and treated in classical cytogenetics to develop chromosome specific banding patterns (non fluorescent method) or processed using FISH (Fluorescence in situ hybridization, fluorescent method). Classical cytogenetic analysis of G or R-banded metaphase chromosomes has extensively been used for analysis of tumour aneuploidy [21,22] and remains the standard test for identification of trisomies in prenatal diagnosis [23,24]. However, its level of resolution is low and the use requires intervention of highly trained experts. Molecular cytogenetics has opened a new way for analysis of chromosome abnormalities, improving sensitivity of conventional banding analysis. In FISH, a DNA probe is labelled with chemically modified nucleotides. The modified probe hybridizes specifically to the homologous sequence on the spread chromosomes. Probes labelled with fluorescent adducts may be directly detected. Altematively, probes are localized using fluorescent labelled antibodies specific of adducts. Using adducts with different emission or excitation wavelengths allow simultaneous detection and localization of several probes on the same preparation. Several probe families of various sizes can be used. They range from a DNA fragment of a few thousand of base pairs specific of a single gene, to complex mixtures (painting) covering a whole chromosome or chromosome arm (review in [25 - 27]). FISH on metaphase chromosomes is a very efficient method to determine chromosome aneuploidy. However, the method has several limitations. The main difficulty is to obtain metaphase chromosomes in sufficient quality and quantity for analysis. In fact, it is not always possible to obtain such preparations from a patient's sample. For example in breast cancers, metaphase chromosome preparations are obtained in no more than 50% of the cases, whereas in some bronchic cancers, this value does not exceed 10% [15,28]. Another bias is introduced using the in vitro culture. Only cells able to divide in the given conditions will be observed and a false evaluation of the actual clonal composition of the sample is possible [29]. In addition, metaphase

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chromosome preparation requires a minimum of 3 - 5 days inducing a long waiting time before the diagnostic result is known. 17.3.2. Interphase chromosomes. In interphase nuclei, chromosomes adopt a more extended structure compared to metaphase chromosomes. FISH with DNA probes may be performed directly on interphase nuclei from various tissue samples (fineneedle samplings, pleural effusions, biopsies, tissue slides...). This approach solves numerous problems associated with metaphase chromosome preparations with loss in resolution. In contrast to metaphase chromosome where probes may be precisely localized on an identifiable chromosome, hybridization on interphase nuclei show fluorescent spots which may be enumerated without possibilities of precise location (review in [30 - 33]). Whole chromosome and chromosome arm specific paintings generate a diffuse signal when hybridized on nuclei, not allowing a precise localization of the spots particularly in cases of complex chromosome rearrangements. Specific probes of centromeric repeated DNA may also be used. However, these probes do not detect chromosome rearrangements without involvement of a variation in the number of centromeres, such as formation of isochromosomes. Probes of several hundred of kilobase pairs such as Bacterial Artificial Chromosomes (BAC or PAC) specific of single sequences may be used. A commercial probe of this type is available for trisomy detection in prenatal diagnosis [34]. Whatever the used probe may be, a bias is remaining in tumour studies due to the ignorance of the cell ploidy. In fact, the copy number of a chromosome segment has not the same significance in a diploid or an aneuploid cell. The use of a reference probe located on another chromosome does not solve the problem as the number of copies of each chromosome may vary in tumour cells. In addition, enumeration of FISH spots either by human observers or by automated image cytometry reveals major difficulties concerning objective criteria for signal discrimination or segmentation. Hybridization efficiency and variable signal intensity within nuclei may hamper correct and objective spot enumeration. In order to go beyond these limitations, we developed an automated image cytometry approach [35 - 39]. 17.4. QUANTITATIVE FISH BY AUTOMATED IMAGE CYTOMETRY ON INTERPHASE NUCLEI. Image cytometers are automated imaging systems for cellular analysis on glass slides in fluorescence and bright field microscopy using a wide range of dedicated analysis modules. The platform that has been chosen for in our studies was Pathfinder^^ from IMSTAR (France). The basic unit is a microscope-based unit combined with a motorized stage, a high-resolution CCD camera, and fast auto-focus mechanism (Figure 17.1). The Pathfinder^^ instrument has been designed for easy set up and use, which is achieved through an intuitive graphical user interface fiilly adjustable for each user type, defined parameters ensuring optimal, reproducible analysies. A powerful combination

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of multi-wavelength fluorescence imaging, including flexible analysis algorithms and full system automation enable quality information for the analysis. Results can be interactively verified due to permanent link between extracted data and the images. Thus, the system provides tools for rapid and objective quantitation of multiple cell parameters allowing human control. For chromosome or chromosome arms imbalance measurements with image cytometers, slides were processed using standard methods for FISH. DNA in the nuclei was stained by 4,6-diamino-2-phenylindole (DAPI). The two chromosomes or chromosome arms specific paintings were labelled using digoxigenin or biotin modified nucleotides and detected using fluorescein isothiocyanate (FITC) or Texas red labelled adduct specific antibodies. Reference slides with normal lymphocytes or fibroblasts were simultaneously hybridized to the samples. Quantitation of hybridization signals was performed as followed: images were acquired at different excitation wavelengths corresponding to DAPI (330 nm), FITC (green fluorescence, 485 nm) and Texas Red (red fluorescence, 577 nm). No bleeding of any red or green signal was observed when changing the filters. Detection of the nuclei was performed on the basis of DAPI counter-staining. After acquisition of 300 fields with a 40x objective, which contained altogether 1000-1500 nuclei, object segmentation and fluorescence measurements for each nucleus were performed automatically by the PATHFINDERS^ system. For each experiment, optimal integration time was determined with the reference slide and kept constant for all slides. Artefacts, falsely segmented nuclei and nuclei containing no green or red fluorescence were excluded interactively. The amount of excluded cells could reach up to 20% depending on the hybridization efficiency. A minimum of 800 cells with analyzable data was obtained. The histogram representing the chromosome or chromosome arm ratio of the whole cell population and its mean value were automatically generated by the software after background subtraction. Two examples with different applications are presented: chromosome 3 arms imbalance in bronchic cancer and prenatal diagnosis of trisomy 21. 17.4.1. Chromosome 3 arms imbalances in bronchic cancers. A first series of analyses was performed on short-time-cultured lung tumour samples [36]. It was shown that high quality double-colour FISH could be obtained with on these samples (Figure 17.2A) and that the imbalance could be determined with reliability. To investigate the importance of our method in clinical work, we further analyzed a series of tissue sections from snap-frozen NSCLC and SCLC specimens [39] (Figure 17.2B) Integrated fluorescent signals from 3q and 3p arm-specific painting were separately quantified in each nucleus and their ratio measured. A histogram representing the 3q/3p ratio of the whole cell population was automatically generated by the software after background subtraction. Detection of peaks representing each cell population was performed automatically based on curve fitting of the Gaussian mode. The standard deviation of the mean fluorescence was taken as an indicator for dispersion of the values. The automated calculation based on statistical analysis of the histogram allowed the exclusion of nuclei containing no green or red

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Figure 17.1. Image cytometer Pathfinder (IMSTAR).

fluorescence as well as artefacts with abnormally high fluorescence values. The proportion of excluded cells could reach up to 10% depending on the hybridization efficiency. An example of the quantified signals within nuclei is shown in a biopsy and in a control (Figure 17.3). Green/red (long arm painting/short arm painting) ratios were determined for each nucleus of a slide. Analysis of the resulting histograms, using specifically developed software allowed a determination of mean values for the whole preparation. Imbalances were obtained by dividing the mean value of the 3q/3p ratio of tumour samples by the mean value of the 3q/3p ratio from normal lymphocytes. When no imbalance was present, the expected ratio was 1, in case of excess of long arm, the ratio would be higher than 1; it would be below 1 if short arms were in excess. In the studied biopsies, calculated imbalances varied from 1.0 to 2.6 with a mean of 1.4 (median: 1.5) indicating excess of 3q [39]. No excess of 3p was observed. Variability of calculated imbalances were verified between successive experiments and adjacent sections. Data showed variations of about 10 %. 48 % of the samples showed an imbalance. In one sample, two populations were observed on the same biopsy, one revealing an imbalance, one without an imbalance in accordance with histological heterogeneity of this tumour (Figure 17.4). The distribution of individual nuclei carrying imbalances on the whole tissue section allowed the establishment of two populations with specific distributions (Figure 17.2C). However, the overlap between the experimentally established value of the imbalance in cell nuclei for normal and abnormal 1 populations limits the possibilities to detect the presence of cellular clone represented by only a few cells. Experiments performed on artificially composed samples of normal and aneuploid cells have shown that the current detection limit corresponds to the ratios of 1 to 20.

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Figure 17.4. Automated curve fitting. The curve enveloping of the histogram, can be decomposed in 2 Gaussian components. In the box, the characteristics of the gaussian components are presented: amplitude, position (Xo) and standard deviation (sigma) determined automatically. In comparaison to the control (not shown), the first gaussian (Xo=1.165) corresponds to an imbalance of 1. The position of the second gaussian gives the value of the imbalance of the second population: 1.6.

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17.4.2. Prenatal Diagnosis of Trisomy 21. For pre-natal diagnosis of trisomy 21, the ratio between the number of chromosome 21 and 22 was measured [38] (Figure 17.2D). The chromosome 22 was chosen as a reference because of its similar size with chromosome 21. Cultured blood of patients with Down's syndrome and of healthy donors were first used to establish the sensitivity of the technique. Then, samples from amniotic fluids from pregnant women at risk chosen after classical diagnostic tests were analyzed in parallel with standard metaphase analysis. Integrated fluorescent signals from chromosome 21 and 22 specific paintings were separately quantified and their ratio measured. The ratio of the signals of chromosomes 21 and 22 normalized with data from normal cells (normal lymphocytes or fibroblasts), which were processed simultaneously allowing determination of the copy amount of chromosome 21. When two copies of both chromosomes 21 and 22 were present in the cells, the ratio was expected to be 1, in case of trisomy, the ratio would be 1.5. An example of the quantified signals within interphase nuclei is shown in figure (Figure 17.5). Green/Red ratios were determined on each nucleus in every field and the resulting histogram allowed determination of the mean value for the whole sample. The ratio calculated by comparison of blood from various donors, ranged from 0.95-1.05 for normal blood (not shown). For patients with trisomy, ratios ranged from 1.5 to 1.6, showing good correlation with the expected value of 1.5 (not shown). The low dispersion of the data allows clear discrimination between normal and trisomic blood specimens. For amniotic fluids, normal fibroblasts were used as a reference because results were less dispersed (smaller standard deviations) than using normal lymphocytes. The reason for this difference between the two cell types may lie in the size difference of the nuclei and the cytoplasm. The cytoplasm of the fibroblasts seems to have less permeability for probes and antibodies. Cells from amniotic fluids reacted in a similar way. Therefore, fibroblasts are recommended as a reference. On 20 analyzed cases, 7 were positive (imbalance range 1.45-1.6) and 13 were normal (imbalance range 0.95-1.1). Results showed 100 % concordance with conventional cytogenetics. 17.5. CONCLUSIONS AND PERSPECTIVES. The data obtained indicated that chromosome or chromosome arm imbalances could be detected efficiently on interphase nuclei using a quantitative comparison between the integrated fluorescent signals from specific paintings. The possible bias during cell culture necessary for metaphase analysis may be eliminated. Our method presents several advantages compared to the currently used interphase FISH approaches. Enumeration of FISH spots by human observers showed difficulties related to objective and correct spot discrimination requiring presence of experienced observers. Our automated technique allows objective analysis of FISH signals on a great number of cells (500-1000 or more). It also avoids segmentation problems due to variable signals within one nucleus. Moreover, inter-experimental variations of the

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Ratio Figure 17.5. Histograms representing the distribution of the fluorescence ratios of chromosome 21 and 22 paintings in an amniotic fluid and in normal control fibroblasts. The mean ratio were 1.3 for the ammiotic fluid and 0.8 for the control. Thus, the measured imbalance was 1.6, indicating a trisomy 21.

signal do not interfere with the measurement because the imbalance is calculated with regard to a reference slide that is added at each experiment. Finally, the variability of the signal within the same nucleus does not affect the measurement either, since the entire integrated fluorescence is analyzed. The efficiency of the approach was proved for detection of chromosome 21 trisomy. Aneuploidy of other chromosomes of interest as chromosomes 13, 18, X or Y could also be studied in a similar way using appropriate chromosome painting. For cancers, the data obtained indicate that chromosome imbalance can be detected efficiently on the nuclei of clinical samples. The method allows the detection of cancer cells directly on the histological heterogeneous tumour sample. The presence of sub-populations with different chromosome imbalances can be precisely established. The possibility to localize each nucleus including the chromosome imbalance value allows reconstitution of the multi-clonal panel of the apparently homogeneous section. This reconstitution could be a powerful approach for the understanding of tumour progression.

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The described quantitative FISH method gathers the advantages of common interphase analysis with a faster and expert-independent way of analyzing chromosome aneuploidies for cancer or prenatal diagnosis. This approach could be a response to the challenge of performing reliable, fast and large-scale analysis of patients. In particular, this approach may be used for techniques of detection of rare cells such as micrometastasis or ciriculating fetal cells within maternal blood for non-invasive prenatal diagnosis. 17.6. REFERENCES 1. 2. 3. 4. 5.

6. 7.

8. 9.

10. 11.

12. 13.

14.

15.

16.

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H. Rajagopalan and C. Lengauer (2004). Aneuploidy and cancer Nature 432(7015), 338-341. G. Pihan and S. J. Doxsey (2003). Mutations and aneuploidy: co-conspirators in cancer? Cancer Cell 4(2), 89-94. Z. Storchova and D. Pellman (2004). From polyploidy to aneuploidy, genome instability and cancer Nat Rev Mol Cell Biol 5(1), 45-54. V. M. Draviam, S. Xie, and P. K. Sorger (2004). Chromosome segregation and genomic stability Curr Opin Genet Dev 14(2), 120-125. S. E. Antonarakis, R. Lyle, E. T. Dermitzakis, A. Reymond, and S. Deutsch (2004). Chromosome 21 and down syndrome: from genomics to pathophysiology Nat Rev Genet 5(10), 725-738. B. Dutrillaux (1995). Pathways of chromosome alteration in human epithelial cancers Adv Cancer Res ^159-^2. F. Farabegoli, M. A. Hermsen, C. Ceccarelli, D. Santini, M. M. Weiss, G. A. Meijer, and P. J. van Diest (2004). Simultaneous chromosome Iq gain and 16q loss is associated with steroid receptor presence and low proliferation in breast carcinoma Mod Pathol 17(4), 449-455. I. Bieche, M. H. Champeme, and R. Lidereau (1995). Loss and gain of distinct regions of chromosome Iq in primary breast cancer Clin Cancer Res 1(1), 123-127. N. Kokalj-Vokac, A. Alemeida, M. Gerbault-Seureau, B. Malfoy, and B. Dutrillaux (1993). Two-color FISH characterization of i(lq) and der(l;16) in human breast cancer cells Genes Chromosomes Cancer 7(1), 8-14. D. P. Carbone (1997). The biology of lung cancer Semin Oncol 24(4), 388-401. Wistuba, II, C. Behrens, S. Milchgrub, D. Bryant, J. Hung, J. D. Minna, and A. F. Gazdar (1999). Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma Oncogene 18(3), 643-650. K. L. Braithwaite and P. H. Rabbitts (1999). Multi-step evolution of lung cancer Semin Cancer Biol 9(4), 255-265. C. A. Powell, S. Klares, G. O'Connor, and J. S. Brody (1999). Loss of heterozygosity in epithelial cells obtained by bronchial brushing: clinical utility in lung cancer Clin Cancer Res 5(8), 2025-2034. J. Sanz-Ortega, M. C. Saez, E. Sierra, A. Torres, J. L. Balibrea, F. Hernando, J. SanzEsponera, and M. J. Merino (2001). 3p21, 5q21, and 9p21 allelic deletions are frequently found in normal bronchial cells adjacent to non-small-cell lung cancer, while they are unusual in patients with no evidence of malignancy J Pathol 195(4), 429-434. J. R. Testa, Z. Liu, M. Feder, D. W. Bell, B. Balsara, J. Q. Cheng, and T. Taguchi (1997). Advances in the analysis of chromosome alterations in human lung carcinomas Cancer Genet Cytogenet 95(1), 20-32. J. Pei, B. R. Balsara, W. Li, S. Litwin, E. Gabrielson, M. Feder, J. Jen, and J. R. Testa (2001). Genomic imbalances in human lung adenocarcinomas and squamous cell carcinomas Genes Chromosomes Cancer 31(3), 282-287. M. J. Parker, J. L. Budd, E. S. Draper, and I. D. Young (2003). Trisomy 13 and trisomy 18 in a defined population: epidemiological, genetic and prenatal observations Prenat Diagn 23(10), 856-860. I. Witters, K. Devriendt, E. Legius, G. Matthijs, D. Van Schoubroeck, F. A. Van Assche, and J. P. Fryns (2002). Rapid prenatal diagnosis of trisomy 21 in 5049 consecutive uncultured amniotic fluid samples by fluorescence in situ hybridisation (FISH) Prenat Diagn 22(1), 2933.

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S. A. Rasmussen, L. Y. Wong, Q. Yang, K. M. May, and J. M. Friedman (2003). Populationbased analyses of mortality in trisomy 13 and trisomy 18 Pediatrics 111(4 Pt 1), 111-l^A. R. Wapner, E. Thom, J. L. Simpson, E. Pergament, R. Silver, K. Filkins, L. Piatt, M. Mahoney, A. Johnson, W. A. Hogge, R. D. Wilson, P. Mohide, D. Hershey, D. Krantz, J. Zachary, R. Snijders, N. Greene, R. Sabbagha, S. MacGregor, L. Hill, A. Gagnon, T. Hallahan, and L. Jackson (2003). First-trimester screening for trisomies 21 and 18 N Engl J Me^/349(15), 1405-1413. B. Dutrillaux (2000). Chromosome and gene alterations in human cancers in relation to aging Chromosomes Today 13207-223. F. J. Mitelman, B. Mertens, F.E., Mitelman database of chromosomes aberrations in cancers, 2001. L. Verma, F. Macdonald, P. Leedham, M. McConachie, S. Dhanjal, and M. Hulten (1998). Rapid and simple prenatal DNA diagnosis of Down's syndrome Lancet 352(9121), 9-12. B. Eiben, W. Trawicki, W. Hammans, R. Goebel, and J. T. Epplen (1998). A prospective comparative study on fluorescence in situ hybridization (FISH) of uncultured amniocytes and standard karyotype analysis Prenat Diagn 18(9), 901-906. M. Muhlmann (2002). Molecular cytogenetics in metaphase and interphase cells for cancer and genetic research, diagnosis and prognosis. Application in tissue sections and cell suspensions Genet Mol Res 1(2), 117-127. S. D. Mundle and I. Sokolova (2004). Clinical implications of advanced molecular cytogenetics in cancer Expert Rev Mol Diagn 4( 1), 71 -81. T. Schwarzacher (2003). DNA, chromosomes, and in situ hybridization Genome 46(6), 953962. A. Flury-Herard, E. Viegas-Pequignot, H. De Cremoux, C. Chlecq, J. Bignon, and B. Dutrillaux (1992). Cytogenetic study of five cases of lung adenosquamous carcinomas Cancer Genet Cytogenet 59(1), 1-8. K. Truong, M. N. Guilly, M. Gerbault-Seureau, B. Malfoy, P. Vielh, and B. Dutrillaux (1999). Evidence for in vitro selection during cell culturing of breast cancer: detection by flow and image cytometry Cancer Genet Cytogenet 114(2), 154-155. J. M. Bartlett (2004). Fluorescence in situ hybridization: technical overview Methods Mol Med 9111-Sl. T. Liehr, H. Starke, A. Weise, H. Lehrer, and U. Claussen (2004). Multicolor FISH probe sets and their applications Histol Histopathol 19(1), 229-237. S. Langer, J. Kraus, I. Jentsch, and M. R. Speicher (2004). Multicolor chromosome painting in diagnostic and research applications Chromosome Res 12(1), 15-23. K. K. Jain (2004). Current status of fluorescent in-situ hybridisation Med Device Technol 15(4), 14-17. M. A. Hulten, S. Dhanjal, and B. Pertl (2003). Rapid and simple prenatal diagnosis of common chromosome disorders: advantages and disadvantages of the molecular methods FISH and Q¥-PCR Reproduction 126(3), 279-297. K. Truong, M. N. Guilly, M. Gerbault-Seureau, B. Malfoy, P. Vielh, C. A. Bourgeois, and B. Dutrillaux (1998). Quantitative FISH by image cytometry for the detection of chromosome 1 imbalances in breast cancer: a novel approach analyzing chromosome rearrangements within interphase nuclei Lab Invest 78( 12), 1607-1613. K. Truong, M. Gerbault-Seureau, M. N. Guilly, P. Vielh, G. Zalcman, A. Livartowski, A. Chapelier, M. F. Poupon, B. Dutrillaux, and B. Malfoy (1999). Quantitative fluorescence in situ hybridization in lung cancer as a diagnostic marker J Mol Diagn 1(1), 33-37. K. Truong, P. Vielh, M. N. Guilly, J. Klijanienko, X. Sastre-Garau, F. Soussaline, B. Dutrillaux, and B. Malfoy (2002). Quantitative FISH analysis on interphase nuclei may improve diagnosis of DNA diploid breast cancers Diagn Cytopathol 26(4), 213-216. K. Truong, A. Gibaud, J. M. Dupont, M. N. Guilly, F. Soussaline, B. Dutrillaux, and B. Malfoy (2003). Rapid prenatal diagnosis of Down syndrome using quantitative fluorescence in situ hybridization on interphase nuclei Prenat Diagn 23(2), 146-151. K. Truong, A. Gibaud, G. Zalcman, M. N. Guilly, M. Antoine, F. Commo, C. Fouquet, J. Cadranel, T. Soussi, B. Dutrillaux, and B. Malfoy (2004). Quantitative fish determination of chromosome 3 arm imbalances in lung tumors by automated image cytometry Med Sci Monit 10(ll),BR426-432.

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IMPORTANCE OF MEASURING FREE ZINC IN CELLS Rebecca A. Bozym, Richard B. Thompson, and Carol A. Fierke

18.1. INTRODUCTION Zinc is the second most abundant trace element in the body and essential for eukaryotic and prokaryotic organisms. Zinc is required as a cofactor or structural component for more than 300 metalloenzymes in all six classes of enzymes ^"^. Zinc is a vital cofactor for zinc fingers of transcription factors '^j the immune system ^, and the reproductive system ^. More than 1% of human gene products are zinc finger proteins ^. A deficiency in zinc leads to impairment of growth, immune activity, and brain functions ^' ^^. Interest in zinc is growing among researchers in medicine and neuroscience. In the brain, synaptically released zinc has both physiological and pathological relevance ^"^^ where the level of free zinc after release from vesicles may reach a range of 10100 fiM in the synaptic cleft ^^. High amounts of vesicular zinc are seen in the hippocampus,fu cerebral cortex, and amygdala ^'^. Zinc released from synaptic vesicles has been suggested to inhibit N-methyl-D-aspartate (NMDA) and yaminobutyric acid ( G A B A A ) receptors, as well as potentiate a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and act as an antagonist of voltage-gated calcium channels ^^' ^^. Although zinc is essential for proper brain function, excessive zinc may also act as a neurotoxin ^^; Frederickson and later Koh, et al., showed that neurons were filled with fi*ee zinc following prolonged seizure ^ or an ischemic insult ^^' ^^. Zinc has also been increasingly linked to apoptosis, although how it influences this process is still unclear. Several groups have reported that zinc at various levels (up to * Rebecca A. Bozym and Richard B. Thompson, University of Maryland School of Medicine, Baltimore, MD, 21201. Carol A. Fierke, University of Michigan, Ann Arbor, MI, 48109. 399

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hundreds of micromolar) induces apoptosis ^^. Canzoneiro, et aL, ^^ have shown that chelation of extracellular zinc inhibits zinc influx and eventual neuronal death. However, they also found that addition of the membranepermeant chelator TPEN caused neuronal death to occur, presumably due to excessive chelation of zinc, as also as shown by Truong-Tran et al ^^. Other investigators have shown that oxidative insults, including administration of nitric oxide, lead to the release of free zinc from intracellular sources ^^"^^. Maret, along with Bossy-Wetzel et al, have also shown that metallothionein can release bound zinc under oxidizing conditions ^^' ^^. Though zinc plays important biological roles, little is known about the process(es) of distribution of this metal in the body, or amongst metalloproteins which require it for function. Eukaryotic cells generally are rich in zinc with a total concentration in the range of 100 ^M \ The abundance of zinc ligands in cells, including, glutathione, histidine, and cysteine, as well as metallothionein (MT) and a myriad of proteins ^^' ^^, assures that the vast majority of cellular zinc is bound and not free. Thus it has been suggested that there is effectively very little (fM) or no free zinc in bacterial cells ^^. However, femtomolar free zinc equates to less than one atom of free zinc per bacterial cell and only a handful in eukaryotic cells, which seems improbable in view of the high total concentration and abundant ligands with medium affinity (nM to ^M). An alternative hypothesis proposes that there is a "pool" of free and rapidly exchangeable zinc ions complexed with kinetically labile ligands which are available for inclusion into metalloproteins and for other purposes. Rapidly exchangeable zinc is likely the biologically active form of zinc, whose level is closely regulated. Metallothionein is thought to act as a buffer of cytosolic zinc ^^. Zinc is enriched in certain compartments within the cell such as endosomes/lysosomes which aid in storage or detoxification ^^. Similar types of organelles are also found in synaptic vesicles and secretory granules ^^'^^. Significant amounts of chelatable zinc are seen in certain cell types: pancreatic P cells ^^, hippocampal neurons, and sperm cells ^^''^\ Two families of zinc transporters also aid in regulating zinc homeostasis. The ZIP (ZRT, IRT-like protein) family facilitates influx of zinc into the cytosol ^^'^^. The CDF (cation diffusion facilitator) family acts in the efflux of zinc from the cytosol ^\ Different subtypes of these families are seen in different cells and different areas of the cell such as the CDF family member ZnT-3 which is highly expressed in the hippocampus, cerebral cortex, and amygdala ^"^ It seems likely there should be a process for rapidly delivering zinc to upregulated transcription factors and the 300+ metalloenzymes that utilize zinc. In the case of copper, specific chaperones exist for each copper enzyme even though it fixnctions in only -12 enzymes. Presumably due to its reactivity and potential toxicity, copper is tightly regulated and there is very little, if any, free copper in the cell ^'^. Copper chaperones deliver copper from transporters to particular cytosolic proteins and to organelles. To date, no similar chaperone for zinc has been identified. Yet if free/exchangeable levels are truly picomolar or lower, the kinetics of binding to newly synthesized zinc proteins by diffusion alone would be very slow: for instance, the measured association rate

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constant for zinc binding in vitro to apocarbonic anhydrase II (kon ) is 1.1 x 10^ |iM"^s"^ ^^ , such that even at 0.1 nM zinc concentrations would take more than an hour to saturate the apoenzyme, which seems unacceptably slow in comparison to the rest of the gene expression apparatus. If the intracellular free/rapidly exchangeable level is typically picomolar ^^ even proteins with diffusion-controlled zinc association rate constants would take hours to equilibrate. However, if intracellular free zinc levels are rapidly maintained at constant concentration (i.e., pM) by transporters and chelators then the kinetics of zinc incorporation will depend on the concentration of the apoprotein in question. In this case, the half time for zinc incorporation by diffusioncontrolled binding is more rapid, varying from 70 seconds for nanomolar apoprotein to 70 msec for an apoprotein at micromolar levels. O'Halloran and his colleagues have suggested that zinc addition to apoproteins is under kinetic rather than thermodynamic control '^^. One way to significantly increase the rate of zinc incorporation is for chelated zinc, which is at much higher concentration than free zinc, to serve as donor. It has long been known that 2,6-dipicolinate ^^ is able to catalyze the binding to and the dissociation from carbonic anhydrase of zinc. It remains to be shown whether other small molecules or proteins are also capable of catalyzing the association/dissociation of zinc. Also, apoproteins are thermodynamically less stable in their metal-free state, adding to the need for rapid zinc insertion either by small molecules or chaperones. However, the hundreds of proteins requiring zinc would seem to require a large number of specific protein chaperones, which makes small molecules perhaps more appealing as chaperones. The determination of free zinc concentration in cells is important for a number of reasons: 1) the level of free (or rapidly exchangeable) zinc essentially reflects the occupancy of all other binding sites, and is most closely related to the zinc activity; 2) free levels (as opposed to total levels) establish thresholds of zinc toxicity, both high and low, with intracellular and extracellular concentrations maintained at different levels; 3) to establish if free zinc is involved in signaling in the brain or elsewhere then it must be quantitated, preferably with an imaging method; 4) free zinc concentrations may provide an overall measure of zinc nutriture, ultimately useful in assessing deficiency; 5) understanding the roles of zinc in pathological states and processes, such as amyloid plaque formation, seizures, apoptosis, ischemia and oxidative stress. The free zinc level in cells has been observed under various conditions ^^""^^j but many of these studies were performed in cells known to be rich in zinc (e.g., mossy fiber neurons, pancreatic islet cells). By comparison, the majority of resting cultured cells studied exhibit little cytoplasmic staining ^^, except for perinuclear punctuate staining ^^ ^^, suggesting that cytoplasmic resting free zinc levels are lower than the detection limits of these indicators, generally not much under one nanomolar. Some difficulties in measuring free zinc at nanomolar levels and below are widely appreciated, such as potential interference by Ca^^ and Mg^^ which are present at much higher concentrations. Less well accepted are the influences of substantial (and variable) concentrations of medium strength ligands such as serum albumin as well as zinc itself in culture media.

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18.2. TSQ DERIVATIVES The first biologically useful fluorescent zinc indicator, TSQ (ptoluenesulfonamide quinoline) was used to observe free zinc in brain tissue sections ^^. TSQ shows an increase in fluorescence upon formation of the Zn:TSQ complex and is able to cross the cell membrane. This was the first indicator that was selective enough to measure zinc in the presence of high concentrations of calcium and magnesium ^'^. However TSQ may also form a water insoluble complex and is found to partition in the cell membrane as well. The fluorescence of TSQ is also pH dependent, and the stoichiometry of zinc binding is not unequivocal ^^, complicating the ability to quantitatively measure free zinc in cells. Due to this nature derivatives of TSQ were made for use in cell systems, such as zinquin ^^. Zinquin coordinates zinc by two nitrogen atoms with a large increase in fluorescence upon addition of zinc. Ca^^ and Mg^^ have little effect on the fluorescence of zinquin whereas Fe^^ and Cu^^ quench any fluorescence. The ethyl ester of zinquin is excited at 368 and emits at 490 nm ^^. Studies have found that zinquin A likely forms a ternary complex with zinc and the protein or biological molecule it is attached to ^^. This suggests that it is a semiquantitative indicator for measuring intracellular free zinc. Another TSQ derivative used in imaging free zinc in mossy fiber boutons is TFLZn, N-(6-methoxy-8-quinolyl)-p-carboxybenzoylsulfonamide ^'^. This indicator is water soluble and exhibits an increase in fluorescence emission by 100-fold upon excitation at 360 nm in the presence of zinc '^^. TFLZn has a IQ of approximately 20 ^M with no apparent interference by Ca and Mg at ImM '^^. Due to the extremely low levels of available zinc, the affinity of this indicator renders it generally less useful for application in cells.

18.3. FLUORESCENT INDICATORS BASED ON FLUORESCEIN 18.3.1. The Zinpyr family Fluorescein and its derivatives are widely used in biological applications such as flow cytometry, confocal scanning microscopy, DNA sequencing, and immunoreagents ^^. The high quantum yield, long wavelength excitation maxima, high extinction coefficient, and water solubility of fluorescein in comparison with the quinolines make it a good candidate for the development of zinc indicators. The many members of the Zinpyr (ZP) family (Figure 18.1) utilize the spectral properties of fluorescein while adding the selectivity of the zinc chelator di(2-picolyl)amine (DPA) forming a photoinduced electron transfer (PET) zinc indicator ^^' ^'^' ^^. The first-generation indicators ZPl and ZP2 contain two DPA moieties at the 4' and 5' positions of the xanthenone ring, forming a symmetrical indicator that exhibits enhanced fluorescence upon addition of zinc or cadmium ^^. In the absence of zinc the fluorophore is switched "off, likely due to electrons on the nitrogen donor atom that transfer to the fluorophore, subsequently quenching emission. Zinc coordinates to the nitrogen donor, reducing the quenching so that fluorescence

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increases 3- to 6-fold ^^. However, ZPl and 2 demonstrate high background fluorescence and can bind two zinc atoms. Nolan et al. ^^ describe three new ZP indicators, ZP5, 6, and 7, based on the structure of ZP4, which contains an unsymmetrical fluorescein platform. ZP5-7 are electronic variations of ZP4 at the aniline nitrogen ^^. These indicators exhibit fluorescence enhancement upon the addition of zinc along with a slight blue shift of 10 nm. They also exhibit a lower quantum yield in the unbound state due to the reduced pKa of the nitrogen atom. For ZP5 and ZP6 the pKa's are 9.6 and 6.3 respectively, making ZP6 a good candidate because PET is not inhibited at physiological pH. Zinc binding affinities were measured using a dual-metal buffering system in which both Ca and EDTA are kept at a constant concentration while varying the zinc concentration ^\ The K^'s for both ZP5 and 6 are 0.50 ± 0.10 nM; however, the fluorescence intensity only increases approximately 2-fold for ZP5 and 6, and changes negligibly for ZP7. For both ZP5 and 6, Cd^^ and Zn^^ exhibit similar fluorescence increases, while there is no fluorescence enhancement in the presence of Ca^^, Mg^^, and Mn^^ (no Zn^^). In the presence of Fe^^, Co^^, Ni^^, and Cu^^, Zn^^ does not successfully compete to bind with the fluorophore, so there is no fluorescence enhancement. These indicators (ZP4-6) are not cell permeable. By comparison ZP3 seems to be the most promising indicator as it is cell permeable and exhibits a six-fold increase in fluorescence in the presence of zinc ^\ However ZP3 is still an intensity-based indicator which means that it is dependent on the amount of indicator present, the excitation intensity, and sample conditions. ZP3 has been used to image endogenous pools of zinc in hippocampal neurons and slices using confocal microscopy ^^. More recently ZP3 has been shown to undergo two-photon excitation for microscopy with excitation at 800 nm ^^. Overall, ZPl-3 (Figure 18.1) exhibit a high quantum yield in the presence of zinc. ZP4-7 have a lower quantum yield in the presence of zinc. ZP8 has also been developed on the monosubstituted difluorescein platform and is asymmetrical, as are ZP4-7 *l Upon addition of zinc ZP8 exhibits an eleven-fold increase in fluorescence intensity with a IQ of 0.6 ± 0.1 nM ^^. ZP8 has the same response to other metals as ZP4-7 and is also not cell permeable. Improvements to these indicators have been made in order to minimize background fluorescence due to protonation of the tertiary nitrogen atom of the PET switch ^\ In order to achieve this, ZP indicators were substituted with electron withdrawing groups on fluorescein ^\ ZPFl, ZPCU, ZPBrl, and ZPF3 all display pKa values below physiological pH with a low quantum yield ^^ Other fluorescent indicators for zinc which have recently been described are also based on a fluorescein platform ^^' ^^. The indicator Zinnaphthopyr 1 (ZNPl), described by Chang et al. ^^, is based on seminaphthofluorescein where it switches between tautomeric forms of fluorescein and naphthofluorescein. This indicator utilizes a single excitation source with dual emission at 624 nm and 528 nm. Upon addition of zinc the emission intensity increases at 624 nm with very minimal changes at 528 nm. The reported apparent K for zinc was 0.55 nM, with

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ZPI:X = CI,Y = H ZP2:X»H,Y»H ZP3: X « F, Y = H ZPF1:X = CI»Y»F ZPCI1:X = CI,Y«CI ZPBrl:X = CI,Y = Br ZPF3: X = F, Y = F

ZP4:X = H,Y = H,Z = CI ZP5:X = F,Y = H,Z«CI ZP6:X«CI,Y = H,Z = a ZP7:X = 0Me,Y = H,Z = CI ZP8:X«H,Y = F,Z»F

Figure 18.1. Structures of the Zinpyr family of zinc indicators. ZPl-3 along with the electronically substituted ZP's are based on a symmetrical fluorescein platform with substitutions on both the xanthene ring and benzoic acid moiety. ZP4-8 are based on an asymmetrical fluorescein platform with substitutions on the xanthene ring. Redrawn from

an 18-fold increase in emission intensity ratio after zinc binding. When ZNPl is added to COS-7 cells as shown by Chang et al. ^^, minimal fluorescence of the cells is observed. Upon addition of 50 |iM Zn(pyrithione)2 an increase in cellular fluorescence.is observed 18.3.2. The Zinspy Family The Lippard group is also developing zinc indicators based on a fluorescein reporting group and a pyridyl-amine-thioether ligand ^^ This new set of zinc indicators are water soluble and increase their fluorescence upon addition of zinc. The Zinspy (ZS) family of indicators undergo PET as well. ZSl contains dichlorofluorescein, ZS2 contains difluorofluorescein, ZS3 contains benzylbromide, and ZS4 contains carboxaldehyde (Figure 18.2). The change from Cl-1 to F-1 in ZSl to ZS2 results in a decrease of the quantum yield in the apo form, but does not lower the pKa of the tertiary amine nitrogen, unlike the ZP indicators 1 and 3. The increase in fluorescence intensity upon zinc binding is only 2 fold for ZS2 and 1.4 fold for ZSl. ZS3 has a pKa of 9.3 suggesting that protonation will interfere with PET quenching in the apo form. Upon addition of zinc there is no fluorescence increase rendering this indicator unsuitable for zinc sensing at neutral pH. ZS4 exhibits a quantum yield of 0.12 in the apo form and 0.5 upon zinc addition with a 4.5-fold increase in fluorescence intensity immediately after adding zinc. Unfortunately, several minutes after mixing there is approximately a 25% decrease in fluorescence

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Figure 18.2. Structures of the Zinspy family; redrawn from^

and precipitation occurs after 15 minutes. In summary, ZSl and ZS2 exhibit relatively high quantum yields in the absence of zinc, with modest fluorescence emission enhancement upon zinc binding. ZS3 has a fairly high pKa for the tertiary amine responsible for PET and thus displays a high quantum yield at neutrality in the absence of zinc with no enhancement when zinc is added. ZP4 demonstrates a 4.5-fold increase in fluorescence emission upon addition of zinc; however, this signal also decreases over time with the formation of a precipitate. 18.3.3. The ZnAFs The fluorescent indicators in the ZnAF family also detect zinc through the PET mechanism while employing fluorescein as the fluorophore with an attached N,N-Bis(2-pyridylmethyl)ethylenediamine (a TPEN derivative) (Figure 18.3) ^'*. ZnAF-1 and ZnAF-2 show little fluorescence in the absence of zinc with fluorescence enhancement of 17-fold and 51-fold, respectively, in the presence of zinc ^^. Below pH 7.0 the fluorescence of both ZnAF-1 and 2 decreases due to protonation of a hydroxyl group on fluorescein which has a pKa of 6.43 ^\ Therefore ZnAF-IF and ZnAF-2F were designed with 2 fluorine

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atoms on fluorescein in order to lower the pKg value for use at physiological pH. The quantum yield in the absence of zinc is greatly reduced with ZnAF-lF and 2F. Calibrations of binding affinities for each ZnAF were performed by buffering zinc in HEPES with 10 mM NT A. Studies were performed with other cations such as Ca^^, Mg^^, Mn^^, Co^^, Ni^^, and Cu^^ to see if they interfere with the fluorescence intensity. At up to 5 mM, Ca^^, Mg^^, and Mn^^ showed no enhancement of fluorescence; Fe^^, Co^^, and Ni^^ indicated a slight enhancement of fluorescence; and Cu^^ quenched fluorescence ^^. In order to achieve intracellular measurements of free zinc the diacetyl derivative of ZnAF2F was made so that it can permeate the cell membrane ^^. Cell staining was demonstrated in mouse macrophage cells and rat hippocampal slices ^'^. This series of indicators is useful in determining the free zinc level in the range of 0.1-10 nM zinc however there are a few drawbacks in using these indicators to detect free zinc inside of cells. The ZnAFs are intensity-based indicators which mean that their emission is dependent on excitation intensity, indicator concentration, the environment around the indicator, and the thickness of the cells. These indicators also utilize UV excitation which might limit their use in fluorescence microscopy. ZnAF-Rl and ZnAF-R2 have also been developed using benzofuran derivatives as fluorophores and TPEN as the chelator ^^. These indicators are excitation ratiometric and operate through the internal charge transfer (ITC) mechanism. They are designed with an electron donating group (amino) conjugated to an electron withdrawing group (carboxylate) that undergoes ITC from donor to the acceptor ^^. There is a cation-induced blue shift in absorbance which is used to ratio the excitation intensities at an emission wavelength of 495 nm. ZnAF-R2 has a better quantum yield in the presence of zinc than ZnAF-Rl; however, both have relatively low quantum yields. ZnAFR2 was shown to be selective for zinc over other divalent cations such as 5 mM Mg^^, Ca^^, Na^^, and 5^M Fe^^, Ni^^, Mn^^, and Cu^^ with a large response to Cd^^^^ Metal ion selectivity was determined by adding the indicated amount of each cation with 5|LIM ZnAF-R2 in HEPES buffer and the fluorescence ratio (335/365 nm) was calculated for each. ZnAF-R2 was made cell permeable by formation of the ethyl ester derivative and was used to stain mouse macrophage cells producing ratiometric images (340/380 nm). Although ZnAF-R2 appears to be a useful tool for measuring intracellular zinc it is ratiometric in the UV range which may increase the number of artifacts due to cellular autorluorescence. 18.4. ZINC INDICATORS BY MOLECULAR PROBES (NOW NVITROGEN) A number of the indicators which are used for Ca+2 detection actually bind zinc with higher affinity ^^. These BAPTA-based (l,2-bis(oaminophenoxy)ethane-N,N,N',N'-tetraacetic acid) Ca^^ indicators, such as Fura-2, have been used to measure zinc in the presence of low [Ca^^]. Fura-2 has an apparent IQ for zinc of 3 nM in the absence of Ca^^, and remains sensitive to zinc in the presence of 25-100 nM free Ca+2 68. Other related

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ZnAF-lF

407

l:ZiAF-Rl 2: ZiAF-iU

Figure 18.3. Selected structures of the ZnAF family; redrawn from ^'^^.

indicatorsfor Mg^^ , such as MagFura-2 and MagFura-5 (Kd for zinc of 20 nM), have also been used for measuring zinc in neurons ^^. Another series of indicators has been developed to measure free zinc in the 0.1-100 \xM range for use in monitoring synaptically released zinc. FuraZin-1 and lndoZin-1 exhibit excitation and emission shifts, respectively, upon zinc addition. These indicators have apparent Kd's for zinc of 8 iiM and 3 ^M. FluoZin-2 and RhodZin-1 show an increase in fluorescence emission upon zinc binding with Kd's of 2 jiM and 23 ^M, respectively. FluoZin-3 exhibits a Kd for zinc of 15 nM and has been shown to respond to calcium at concentrations above 1 ^M '^^ ^^, making it of questionable utility for quantitating extracellular zinc ^\ Moreover, this indicator is intensity-based with an increase in fluorescence with increasing concentrations of zinc. The AM ester of RhodZin-3 localizes in the mitochondria, exhibiting a 75-fold increase in fluorescence with saturating zinc, having a Kd of 65 nM ^^. While this may be a useful tool in determining the role of zinc in apoptosis, it is a straight intensity indicator which is subject to the usual artifacts; it may be, however, that it exhibits a useful lifetime change. The Newport Green indicators exhibit ^M affinity for zinc (1 ^iM and 30 ^M) ^^ with no interference by mM calcium or magnesium ^^. These indicators use DPA as the chelator and undergo fluorescence enhancement upon zinc binding; Newport Green DCF also exhibits a lifetime shift which was useftjl in quantifying intracellular zinc ^^. Overall the zinc indicators available through Molecular Probes are useful in measuring higher zinc concentrations, particularly where calcium is at a minimum. Substantial data indicate that much of the biology of zinc occurs at < 10'^ M [Zn^^jfree where these probes are unable to accurately measurefi*eezinc ion. The majority of these indicators are intensity-based, making it difficult to use them for actual quantitation of free zinc in a biological system.

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18.5. A ZINC INDICATOR BASED ON BENZOXAZOLE As mentioned before ratiometric indicators for zinc provide a more accurate measurement of free zinc in biological systems. Using this approach the measurement is largely independent of indicator concentration, excitation intensity, or differences in the sample. Taki et al. '^^ have recently described an emission ratiometric indicator using benzoxazole as the fluorescent component and an aminomethyl pyridine moiety as the zinc ligand named Zinbo-5 ^^. Upon addition of zinc to Zinbo-5 there was a decrease in the absorbance band at 337 nm which is seen in the apo form, and an increase of a new band at 376 nm forming an isosbestic point ^'^. When excited at the isosbestic point at 356 nm there is a band at 407 nm that shifts to 443 nm with increasing concentrations of zinc. In determining the apparent K^ of this indicator a zinc/EGTA buffer system was used to achieve free zinc values ranging from 0 to 11 nM. The ratio of 443/395 nm was plotted versus zinc concentration giving rise to a IQ of 2.2 ± 0.1 nM ^'*. Two photon excitation microscopy was used to determine if this indicator is usefril in biological applications. When Zinbo-5 is added to mouse fibroblast cells, there is very minimal fluorescence seen (excitation 710 nm) even after the brightness of the image had been increased '^^. Zinbo-5 is responsive to changes in zinc concentration as addition of zinc with the ionophore pyrithione leads to an increase in the ratio image; whereas addition of TPEN caused the fluorescence to decrease and thus match the original ratio image. However all of this was performed in fixed cells (4% formaldehyde on ice for 20 min) and the staining process was also done on ice which thus may not reflect the actual resting level of free zinc inside these cells. 18.6. LANTHANIDE CHEMOSENSORS FOR ZINC Lanthanides exhibit good spectral properties for sensing such as long luminescence lifetimes (up to msec), Stokes' shift larger than 200 nm, narrowband emission, and high water solubility ^^. Lanthanides are also suitable for time-resolved fluorescence (TRF) measurements, which have the ability to suppress background resulting in a better signal-to-noise ratio. The europium (Eu^^) complex in particular is useful in this area. The lanthanide requires a chromophore for high luminescence which will absorb the light and undergo ISC (intersystem crossing) to populate the triplet state. Then an intramolecular energy transfer occurs to the lanthanide which then emits at a particular wavelength. However it should also be mentioned that this type of fluorophore is also susceptible to water quenching, which would be difficult to overcome in a biological system. In the study by Hanaoka, et al., the chromophore is a quinoline-containing TPEN ligand which also acts as a sensitizer. This is combined with the Eu^^ - diethylenetriaminepentaacetic acid (DTPA) complex forming [Eu-7] ^^. This complex was characterized using time-delayed luminescence with a delay of 0.05 ms in aqueous solution. Upon addition of zinc, the luminescence emission increased by 8.5-fold. For IQ determinations the zinc concentration was controlled by using a NTA system

MEASURING FREE ZINCIN CELLS

J

409

L^

coo

Eu*^

(Eu-71

Figure 18.4. Structure of the lanthanide chemosensor [Eu-7],using a quinolyl ligand as the chromophore; redrawn from ^^.

below 398 nM but was unbuffered above 200 jiM zinc ^^. The apparent IQ for [Eu-7] was calculated to be 59 nM, which is less tight than expected ^^. The luminescence of [Eu-7] is stable at physiological pH. Various metals were added to [Eu-7] (50 |iM) in HEPES buffer in order to determine the selectivity. The presence of 5 mM Ca^^ and Mg^^ did not result in any luminescence emission enhancement. The same result was seen with the addition of 50 jiM Cu^^ Ni'^^ Co'^^ Fe^^ and Mn^l In the presence of zinc the luminescence of Eu-7 was quenched upon the addition of Ni , Cu , and Co . A large enhancement of luminescence was seen upon addition of Cd^^ to [Eu-7] ^^. While it is advantageous to use this indicator due to its long emission wavelength and lifetime, unless a lifetime change occurs it remains an intensitybased indicator with the attendant shortcomings. 18.7. EXCITED-STATE INTRAMOLECULAR PROTON TRANSFER Ratiometric indicators offer a better solution for analyte detection than simple intensity indicators due to their dependence on the cation concentration, but not on indicator concentration, spectral properties of the instrument, etc. Henary et ah ^^ describe a new emission ratiometric indicator that is based on inhibition of excited-state intramolecular proton transfer (ESIPT). ESIPT occurs when a proton is transferred either to a base or another atom on the molecule in the excited state which results in a large shift in emission. In the process described by Henary, et ah, they use a benzimidazole derivative which undergoes ESIPT ^^. When the fluorophore is in the excited state, the proton becomes highly acidic, thus the coordination of a metal cation will displace the proton and thus inhibit ESIPT ^^. Ligands 6b, 7b, 8b, and 9b are water soluble and the pKa of the sulfonamide nitrogen atom is above 8.0 for each one. The absorption spectra for 6b-9b are very similar in that they all have a peak around 300 nm in the protonated form and a red shifted band around 330 nm in the deprotonated state in aqueous solution. Upon excitation at the peak wavelength, 6b-9b exhibit a single band at 460 nm '^^. In the presence of zinc the absorbance spectra of 6b, 7b, and 9b resemble the spectra seen in the

410

R.A. BOZYM ETAL

c / \

Figure 18.5. Structures of ligands 6b and 9b; redrawn from ''''.

deprotonated state, indicating loss of the nitrogen proton and zinc binding. Interestingly 8b does not contain a red shifted peak in the absorbance spectrum indicating that the nitrogen atom is protonated even in the presence of zinc and does not coordinate to zinc. The emission spectra in the presence of zinc resulted in a large blue shift of the peak emission around 460 nm to a peak around 400 nm with an isosbestic point at 428 nm ^^. In calculating the zinc binding affinities of 6b and 7b, metal ion buffers were evidently not used which calls into question the accuracy of the measured affinities. For 8b Zn/EGTA buffers were used to calculate the affinity for zinc and for 9b Zn/HEDTA (N-(2hydroxyethyl)ethylenediaminetriacetic acid) buffers were used. The binding affinities reported for 6b-9b are fxM, mM, nM, and pM, respectively ^^. Calibration curves for 6b-9b were performed by taking the ratio of emission at 400 and 500 nm, indicating 9b and 8b can be used to measure zinc between 0.2 - 20 pM and 1 - 1 0 nM respectively. 7b has very low affinity for zinc thus it is not useful for biological applications. 6b is suitable for zinc concentrations between 30 |xM - 3 mM ^^. 18.8. PEPTIDES AS ZINC INDICATORS Other zinc indicators have been developed which utilize the properties of zinc finger domains ^^"^^. Although these indicators exhibited high selectivity for zinc the Imperiali group expanded their development to smaller peptides for easier use in biological systems. The second group of indicators developed by this group were only seven amino acids long with the reporting group being 8methoxy-quinoline ^\ These indicators underwent chelation enhanced fluorescence (CHEF) upon zinc binding. More recently the Imperiali group has developed peptides with tunable affinity for zinc in the nanomolar to micromolar range ^^. The indicators make use of three components: a Zn ligand amino acid, a p-tum sequence, and the fluorophore containing zinc ligand. The fluorophore is incorporated into the peptide via a novel amino acid Sox, which contains 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline ^^. Each

MEASURING FREE ZINCIN CELLS

411

of the peptides synthesized are only 6-8 amino acids in length, all of which include 2 serine residues for solubility and cysteine or histidine residues for binding zinc ^^. Upon addition of zinc the Sox peptides emit at 500 nm with excitation at 360 nm exhibiting a 30-fold increase in fluorescence through CHEF. Metal competition experiments were performed to determine if these peptides also respond to other divalent cations either in a positive (cation binding results in fluorescence) or negative manner (cation binding compromises the ability of the peptide to bind zinc). Only a few Sox peptides (P7, PI2, and PI3) exhibited fluorescence in the presence of ImM Ca^^ and Mg^^ whereas all the peptides gave a positive response to Cd^^. However, Cu^^ prohibits Zn^^ binding for all peptides, Ni^^ quenches peptides that use imidazole and carboxylate ligands, and Mn^^ and Co^^ compete with the binding of zinc for peptides with weaker affinity for zinc ^^. It is possible to increase the zinc affinities of the Sox peptides by making a more flexible turn or increasing the number of ligands incorporated into the peptide. When combined with one another these indicators may be used to determine a range of zinc concentration when one peptide gives a response and others do not. However it would be difficult to utilize these Sox peptides over a broad range using just one color. 18.9. CARBONIC ANHYDRASE AS A BIOSENSOR FOR ZINC We have been developing a fluorescent indicator system using apocarbonic anhydrase as a sensor transducer ^'^'^^. Human carbonic anhydrase II (CA) exhibits a 4 pM affinity for zinc at pH 7.5 and its response is unaffected 10 mM Ca^^ or 50 mM Mg^^ , levels significantly higher than those encountered in vivo ^^. In using a biological molecule we are uniquely able to alter or adapt the molecule to our needs through site-directed mutagenesis '^^. Single amino acid substitutions in the sequence can improve the selectivity, sensitivity, and response-time of carbonic anhydrase ^^, making it a uniquely flexible and responsive indicator. For instance, a variant CA with a single amino acid mutation (E117A) exhibits a several hundred-fold faster association rate constant than the wild type, with only a 10-fold drop in affinity ^^. When compared with other indicators, CA has excellent selectivity (100fold) over cadmium ^^. Apocarbonic anhydrase has been adapted to an excitation ratiometric fluorescent biosensor based on fluorescence resonance energy transfer (FRET) from a zinc-bound aryl sulfonamide to a fluorescent label on the protein ^^. Zinc levels are measured by taking the ratio of intensities at two different excitation wavelengths ^^. This approach, as depicted in Figure 18.6, is based on FRET from the zinc-dependent binding of Dapoxyl sulfonamide to the fluorescent label, Alexa Fluor 594, covalently attached to the protein. In the absence of zinc, Dapoxyl sulfonamide doesn't bind and there is only very weak emission from free Dapoxyl sulfonamide at 617 nm upon excitation at 365 nm. When zinc is bound to the active site of the enzyme, Dapoxyl sulfonamide binds to zinc replacing water as the fourth ligand, and there is strong emission at 617 nm with UV excitation, due to FRET from bound Dapoxyl exciting

412

R,A,BOZYM ETAL H36C-CA

H36C-CA

TAT

WEAK

/

y

N/

V

• Zn'.

617nm*^ STRONG

e-5000 OAPOXYL

OJ EM.eOOnm QY«0.01

TAT

\

^

^n

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CA KJU

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(BOUNO>

SULFONAMIDE (FREE) EXC.365nm

Figure 18.6. Schematic of ratiometric zinc determination with apoTAT-H36C-Alexa Fluor 594 carbonic anhydrase and Dapoxyl sulfonamide. In the absence of zinc, Dapoxyl sulfonamide does not bind to CA, therefore no FRET occurs to the fluorescent label on the protein and very weak emission at 617 nm is observed. In the presence of zinc, Dapoxyl sulfonamide binds, is excited with UV light, and FRET occurs from Dapoxyl to the Alexa Fluor, thus exciting the fluorescent label causing emission to occur at 617 nm. The emission when excited with 365 nm is normalized to the amount of labeled protein by directly exciting Alexa Fluor 594 with 543 nm and dividing the two intensities.

Alexa Fluor. The change in emission when excited in the UV is normalized to the amount of labeled protein present by exciting the Alexa Fluor 594 directly (543 nm, where Dapoxyl sulfonamide does not absorb) and dividing the two intensities. Additional advantages of this approach (beyond being ratiometric) are that there is no interference from the emission of Dapoxyl sulfonamide bound to adventitious carbonic anhydrase, lipids, or other proteins because it emits in the blue, and the sensitive range and kinetics can be adjusted by small changes in the protein structure. Also the recognition and transduction elements are capable of being in separate moieties of the molecule; the fluorophore need not be the metal ligand ^^. This approach was tailored to intracellular use by attaching a TAT (transactivator of transcription) peptide to carbonic anhydrase which then induces the cells to take up the protein ^^. The TAT peptide is encoded by HIV1 and contains an 11 amino acid protein transduction domain (PTD) allowing the cell to carry the protein across the membrane and into the cytoplasm. Dapoxyl sulfonamide penetrates the cell readily. Thus intracellular free zinc levels are able to be imaged and quantitated using this ratiometric approach. Calibrations for this system were performed using both the steady state fluorometer and the epifluorescence microscope. For calibrations performed on the microscope, wells of a 1536 well-plate were filled with apoH36C-AF594CA, Dapoxyl, and buffers of increasing free zinc concentration. The wells were imaged using the Nikon 4x objective. The mean ratio for each well was calculated and used to plot the calibration curve from the microscope against the one acquired on the fluorometer (Figure 18.7). The curve from the microscope reveals a IQ of 70 ±15 pM whereas the fitted Kj using the fluorometer is 137 ±18 pM. The optical trains of the microscope (using filters) and the fluorometer used for the cuvette measurements (using monochromators) are quite different, such that agreement to within a factor of two appears

413

MEASURING FREE ZINCIN CELLS

H36C-AF596 -

0.8

DAPOXYL

i

EM. 617 nm

c

5 ^

0-7

?

9 10-1*

j(j-iJ

^^-10

jo^«

FREE [ Z n * * ] . N A N O M O L A R

Figure 18.7. Zinc dependent ratio of 366 nm over 548 nm with emission at 618 nm of apo-H36CAF594 and Dapoxyl sulfonamide performed on the microscope in a 1536-well plate (o) and on a steady state fluorometer in a cuvette (n). Kd = 70 ±15 pM (o) and 137±18 pM (D).

Figure 18.8. Microscope calibration with a 1536 well plate. Ratio images of each well (365/543 nm) containing zinc ion buffers, apoH36C-AF594 CA and Dapoxyl sulfonamide; pseudo colored for visual comparison. (See color insert section.)

satisfactory. The ratio increases with increasing zinc concentration by almost 50%, which is slightly less than that achieved with the fluorometer. A representative set of wells was used to create Figure 18.8 in order to visually compare the intensity ratio of different zinc concentrations. The ratio images of the wells were pseudo colored using a rainbow color scheme in IPLab. The calibration color bar was formed using the values in the calibration curve and matching them with the well plate. Measuring and imaging the free zinc level in cells is accomplished by adding apoTAT-H36C-AF594 CA and Dapoxyl sulfonamide. The TAT peptide quickly transports the label into the cells (incubation time of only 15 min) with bright fluorescence seen immediately. Figure 18.9 shows a representative set of cells after staining with apoTAT-H36C-AF594 CA and Dapoxyl sulfonamide. In the excitation 543 and 365 nm images (emission 617 nm), fluorescence is uniformly distributed throughout the cells. Overall the free zinc is approximately 5pM, which is low, but not zero and well above the femtomolar levels proposed for prokaryotic cells ^^. Similar results were obtained using the E117A variant which has a ten-fold lower affinity for zinc and faster zinc equilibration ^^, reinforcing the conclusion that our measurement is near equilibrium conditions and therefore the free zinc in cells is well above femtomolar.

414

R.A. BOZYM ETAL

Figure 18.9. Measuring free zinc inside of PC-12 cells with apoTAT-H36C-AF594 and Dapoxyl sulfonamide. Top left: bright field; top right: ratio image pseudo color rainbow; bottom left: excitation 543 nm, emission 617 nm, exposure time 20 ms, pseudo color red; bottom right: excitation 365 nm, emission 617 nm, exposure time 500 ms, pseudo color red. The calibration bar (at right) indicates the level of cytoplasmic free zinc. (See color insert section.)

18.10. CONCLUSION The number of papers published on developing zinc indicators and the importance of zinc in biochemistry has been steadily increasing. There is substantial incentive to develop selective, sensitive, and quantitative zinc indicators in order to elucidate the many unknown roles of zinc in the body. How zinc is sequestered and bound to the vast number of enzymes is still unknown, although the likelihood of a cfuatalytic delivery mechanism is very high. The roles of zinc in neurodegenerative disorders (Alzheimer's), intracellular signaling, and apoptosis are the subject of substantial investigation which will be elucidated by the use of zinc indicators just as calcium indicators opened the door to calcium signaling. It is important to use the appropriate indicator for experiments. The kinetic parameters are also of importance in choosing an appropriate indicator. Faster association/dissociation rates are needed for measurements where the zinc concentration is going to change rapidly, as in neuronal activity. For a quantitative measurement of the free zinc level a ratiometric probe is most useful as it is much less affected by environmental factors (indicator concentration, excitation intensity, etc.). Intensity-based indicators are susceptible to these issues and it is difficult to obtain reliable quantitation of free zinc using these indicators. The comparable advantages of lifetime- and polarization-based indicators for these applications should not be overlooked. How the indicator is transported into cells is a main focus in many zinc indicator studies. Many indicators are not cell-permeable with some being injected into cells ^^, others use the AM ester form to get through the cell membrane, meanwhile others are developed to be transported into the cell. We have reported the novel use of the TAT peptide in transporting our CA-based indicator into cells. The TAT peptide allows the cell to readily take in the protein in an efficient manner without any apparent adverse affects to the cell, or need for disruptive procedures such as microinjection. Our

MEASURING FREE ZINCIN CELLS

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system is also excitation ratiometric which allows for a more accurate quantitation of the free zinc level in the cell. 18.11. REFERENCES 1. 2. 3. 4. 5. 6. 7.

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Kimura, E.; Aoki, S., Chemistry of zinc(II) fluorophore sensors. Biometals 2001, 14, (3-4), 191-204. Fahmi, C. J.; O'Halloran, T. V., Aqueous coordination chemistry of quinoline-based fluorescence probes for the biological chemistry of zinc. Journal of the American Chemical Society 1999, 121, 11448 - 11458. Zalewski, P. D.; Forbes, I. J.; Betts, W. H., Correlation of apoptosis with change in intracellular labile Zn(II) using Zinquin [(2-methyl-8-p-toluenesulphonamido-6quinolyloxy)acetic acid], a new specific fluorescent probe for Zn(II). Biochemical Journal 1993, 296, 403-408. Hendrickson, K. M.; Geue, J. P.; Wyness, O.; Lincoln, S. F.; Ward, A. D., Coordination and fluorescence of the intracellular Zn2+ probe [2-methyl-8-(4-toluenesulfonamido)-6quinolyloxyjacetic acid (Zinquin A) in ternary Zn2+ complexes. J Am Chem Soc 2003, 125, (13), 3889-95. Haugland, R. P., Handbook of Fluorescent Probes and Research Chemicals. Sixth ed.; Molecular Probes, Inc.: Eugene, Oregon, 1996; p 679. Burdette, S. C ; Frederickson, C. J.; Bu, W.; Lippard, S. J., ZP4, an improved neuronal Zn2+ sensor of the ZinPyr family. Journal of the American Chemical Society 2003, 125, 1778 1787. Nolan, E. M.; Burdette, S. C ; Harvey, J. H.; Hilderbrand, S. A.; Lippard, S. J., Synthesis and characterization of zinc sensors based on a monosubstituted fluorescein platform. Inorg Chem 2004, 43, (8), 2624-35. Chang, C. J.; Nolan, E. M.; Jaworski, J.; Burdette, S. C ; Sheng, M.; Lippard, S. J., Bright fluorescent chemosensor platforms for imaging endogenous pools of neuronal zinc. Chem Biol 2004, 11, (2), 203-10. Chang, C. J.; Nolan, E. M.; Jaworski, J.; Okamoto, K.; Hayashi, Y.; Sheng, M.; Lippard, S. J., ZP8, a neuronal zinc sensor with improved dynamic range; imaging zinc in hippocampal slices with two-photon microscopy. Inorg Chem 2004, 43, (21), 6774-9. Nolan, E. M.; Lippard, S. J., The zinspy family of fluorescent zinc sensors: syntheses and spectroscopic investigations. Inorg Chem 2004, 43, (26), 8310-7. Hirano, T.; Kikuchi, K.; Urano, Y.; Nagano, T., Improvement and biological applications of fluorescent probes for zinc, ZnAFs. J Am Chem Soc 2002, 124, (23), 6555-62. Sun, W.-C; Gee, K. R.; Klaubert, D. H.; Haugland, R. P., Synthesis of Fluorinated Fluoresceins, y Org Chem 1997, 62, (19), 6469-6475. Maruyama, S.; Kikuchi, K.; Hirano, T.; Urano, Y.; Nagano, T., A novel, cell-permeable, fluorescent probe for ratiometric imaging of zinc ion. J Am Chem Soc 2002, 124, (36), 106501. Hirano, T.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T., Novel Zinc Fluorescent Probes Excitable with Visible Light for Biological Applications We thank Prof E. Kimura and Prof T. Koike for advice on the chemistry of macrocyclic polyamines. Angew Chem Int Ed Engl 2000,39,(6), 1052-1054. Atar, D.; Backx, P. H.; Appel, M. M.; Gao, W. D.; Marban, E., Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels. J Biol Chem 1995, 270, (6), 2473-7. Simons, T. J., Measurement of free Zn2+ ion concentration with the fluorescent probe magfura-2 (furaptra). JBiochem Biophys Methods 1993, 27, (1), 25-37. Gee, K. R.; Zhou, Z. L.; Qian, W. J.; Kennedy, R., Detection and imaging of zinc secretion from pancreatic beta-cells using a new fluorescent zinc indicator. J Am Chem Soc 2002, 124, (5), 776-8. Kay, A. R., Evidence for chelatable zinc in the extracellular space of the hippocampus, but little evidence for synaptic release of Zn. Journal of Neuroscience 2003, 23, (17), 6847 - 6855. Sensi, S. L.; Ton-That, D.; Weiss, J. H.; Rothe, A.; Gee, K. R., A new mitochondrial fluorescent zinc sensor. Cell Calcium 2003, 34, (3), 281-4. Thompson, R. B.; Peterson, D.; Mahoney, W.; Cramer, M.; Maliwal, B. P.; Suh, S. W.; Frederickson, C. J., Fluorescent zinc indicators for neurobiology. Journal of Neuroscience Methods 2002, 118,63-75. Taki, M.; Wolford, J. L.; O'Halloran, T. V., Emission ratiometric imaging of intracellular zinc: design of a benzoxazole fluorescent sensor and its application in two-photon microscopy. J Am Chem Soc 2004, 126, (3), 712-3.

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LASER-BASED DETECTION OF ATMOSPHERIC HALOCARBONS Jean-Franois Gravel and Denis Boudreau*

19.1. ABSTRACT

Among the various atmospheric trace species that play a role in environmental issues, volatile halogenated alcanes have attracted considerable interest because of their massive industrial use and of their documented effects on global warming and ozone depletion. Whereas the monitoring of halogenated alcanes at or near ground level can be accomplished routinely using a variety of established techniques, their determination at higher altitude poses considerable technological and/or logistic challenges. One class of detection techniques that has been experiencing accelerated development in recent years is the use of laser sources to remotely excite and/or fragment the target molecules, followed by their identification and quantification using their optical emission. The purpose of this review is to describe the current status and future trends in remote detection of atmospheric halocarbons by laser-based spectroscopic techniques, with a special emphasis on the use of luminescence techniques to detect the analytes of interest.

* Department of chemistry and Centre d'optique, photonique et laser (COPL), Universite Laval, Quebec (QC), Canada GIK 7P4; phone (418) 656-3287; fax (418) 656-7916; email: denis.boudreau @ chm.ulaval .ca 421

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19.2. INTRODUCTION The key role of atmospheric trace species on many environmental issues such as global warming, ozone depletion and photochemical smog formation is now well recognized, and the knowledge of the fate and behavior of these species in the troposphere, as well as the ability to measure their concentration, lifetime and reactivity in different atmospheric layers are crucial in order to gain a better understanding of the complex mechanisms that lead to atmospheric or climatic changes and potential adverse health effects.' Among these species, volatile halogenated alcanes are of particular interest. Owing to their enviable physicochemical properties, they were massively used over the last decades as refrigerants, propellants/blowing agents and flame retardants. Since the central role of chlorinated and brominated alcanes in the stratospheric ozone layer depletion phenomenon was recognized,^ many replacement products with similar properties have been proposed, first HCFCs (hydrogen-containing chlorofluorocarbons) and then - when these were also shown to have severe impacts on the stratospheric ozone balance^ and on human health'* ^ - hydrofluorocarbons (HFCs). The latter are very stable with respect to atmospheric degradation and have limited effects on the ozone layer depletion. However, because they tend to accumulate in the atmosphere^ and are relatively strong IR absorbers, they are now recognized as significant anthropogenic greenhouse gases and are included in a list of target compounds to control (Kyoto Protocol, 1997). Another class of halocarbons which are also cause for concern are the perfluorocarbons (PFCs), which are among the most potent greenhouse gases.^ ^ As a result, the development of monitoring methods for halogenated species has received increased attention in recent years. Their monitoring at or near ground level can be done routinely using a variety of sensitive and selective analysis techniques, often using on-site air sampling with cryogenic preconcentration, followed by gas chromatographic analysis.^^^ Despite the high detection sensitivities that these off-line techniques offer, they often involve hour-long sampling times and measurement cycles. As highlighted by Sigrist,' spectroscopic techniques offer many significant advantages for on-line and in situ monitoring of trace atmospheric species; those based on laser excitation, in particular, offer the unique combination of real-time analysis with the high sensitivity and selectivity provided by the directionality, high irradiance and spectral purity of lasers. Moreover, the use of pulsed laser sources with synchronous signal detection allows the temporally and spatially-resolved monitoring of target atmospheric species from a remote location. A number of reviews on the application of laser-based techniques for remote atmospheric sensing have been pubhshed.'^ Halogenated alcanes have been monitored using many different spectroscopic approaches. For example, conventional open-path spectroscopic techniques such as Fourier transform infrared spectroscopy (FTIR), tunable diode laser absorption spectroscopy (TDLAS) as well as differential optical absorption spectroscopy (DOAS), allow for the in situ halocarbon determination

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by propagating a light beam through the space to be monitored. These techniques, based on the rovibrational absorption spectral signature of halogenated alcanes in the IR region from 7 to 12.5 |im,^ ^^^^ have provided detection limits reaching the ppb range and good analytical selectivity for a number of halocarbons (CF4, C2F6, C2H3CI, C2HCI3, C2CI4, C2H5CI, C2H4CI2, C4H5CI, CF2CI2, CFCla).^^'^^ However, they usually require that the probe beam be propagated over long (sometimes km-long) absorption paths to reach the desired sensitivity, either using multiple reflection sampling cells or an openpath configuration which, in the latter case, limits the spatial resolution of the measurements.^^ Among the other spectroscopic approaches that have been applied to atmospheric monitoring and that could in principle be used for halocarbon detection, laser-induced fluorescence (LIP) generally offers in situ, on-line monitoring capability, high sensitivity, and spatially resolved measurements.^^ However, as pointed out by several authors,^ ^^^"^ it is often the case for polyatomic molecules, such as halogenated alcanes, to have poor electronic transition probabilities together with broad, poorly defined spectral bands, which makes LIP unsuitable for ultrasensitive measurements.^^ This can be attributed to the existence of numerous closely spaced electronic energy levels, which aggravate signal losses through non-radiative decay processes, as well as fast intersystem crossing^^ and collisional quenching by major atmospheric constituents.^ Ongoing efforts towards the development of efficient laser-based in situ detection of halogenated alcanes therefore often involve breaking the molecule of interest into smaller, yet characteristic fragments, before probing the latter using various optical detection schemes. Among these, photofragmentation (PF) coupled to laser-induced fluorescence (LIP) has seen the widest application and perhaps the highest degree of success outside the research laboratory to perform real-time, spatially resolved, highly sensitive and selective monitoring of halogenated species in various environments.^^ Another technique, Laser induced breakdown spectroscopy (LIBS), while relying on atomic emission rather than fluorescence to detect the analyte of interest, has also shown a great potential for in situ measurements of halogenated alcanes and is included in the present review. Finally, the unique propagation characteristics of intense femtosecond laser pulses in air make the latter a very promising fragmentation and excitation source for the spatially-resolved, sensitive and selective monitoring of halogenated species and other pollutants ranging from groundlevel, in situ measurements to remote sensing of the upper atmosphere.

19.3. LASER INDUCED BREAKDOWN SPECTROSCOPY Laser induced breakdown spectroscopy (LIBS, also known as laserinduced plasma spectroscopy, LIPS) is a versatile analytical method that is applicable to almost any type of sample matrix including solids, liquids and

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gases.^^"^^ It relies on the generation of a hot plasma obtained by tightly focusing laser pulses (typically Nd:YAG laser light in the NIR) to reach high intensities (typically in the GW/cm^ range). The time-resolved emission spectrum of the plasma plume is then measured with a gated detection system to provide fast, quantitative multielemental analysis. While most of the applications of LIBS are related to the analysis of solid samples,^^ it is also considered as an analytically useful gas sensing technique. This is particularly true for the analysis of gaseous halogenated alcanes bearing chlorine and fluorine atoms, owing to the high plasma temperatures (in the 20,000-25,000 K range^^) which are high enough for efficient collision-induced population of high-lying excited atomic states of chlorine and fluorine (e.g. the first radiatively-coupled excited state of fluorine lies -13 eV above the ground state). Over 20 years ago, Cremers et al.^^ reported the first use of LIBS for the time-resolved detection of chlorine- and fluorine-containing halogenated alcanes in air at atmospheric pressure. They studied different parent molecules (i.e. (CH3)2CHC1, CCI2F2, C2CI3F3, CCI4) and showed that the LIBS technique was insensitive to the molecular structure of the analytes, as long as the plasma was not physically or chemically perturbed by the samples. They also reported that the emission spectrum was mainly composed of the atomic lines of major air constituents, namely oxygen and nitrogen, and that the most intense emission lines for CI (I) and F (I) ( X. = 837.6 nm and 685.6 nm, respectively) were free from interferences with these major constituents. As is generally the case for LIBS experiments, satisfactory analytical performance was reached following the optimization of experimental parameters such as laser pulse energy, detection gate delay and integration time relative to the onset of plasma ignition. This is due to the fact that the emission spectrum is first dominated by an intense, short-lived background continuum, followed by the more persistent atomic lines. Therefore, by using time-resolved detection, the signal-to-noise ratio (SNR) can be maximized. Interestingly, Cremers et al. noted that optimal gate delays and integration times strongly depended on the species to be detected.^^'^^ Most of the work done with different halogenated alcane showed that the CI and F emission intensities were directly proportional to the total number of each atom contained in the parent molecule, an indication of complete dissociation of different species in the plasma.^' ^^ Measurement of line intensity ratio for different elements allowed for the discrimination and prediction of the molecular structure of different halogenated alcanes. As an example, Dudragne et al. ^^ measured the relative CI, F and C intensity ratios to distinguish between a series of six different HCFCs. Haisch et al." used the same approach with CI and H Hnes to discriminate between CCI4, CHCI3 and CH2CI2, while Williamson et al.^'* used F and H lines to distinguish between a series of fluorocarbons and hydrofluorocarbons. However, as pointed out by Cremers et al.,^' this procedure is not applicable to evaluate the molecular concentration or stoechiometry for mixtures of different parent molecules. Therefore, LIBS can only be considered

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as a selective technique to distinguish classes of compounds. Typical limits of detection (LOD) for the analysis of halogenated alcanes by LIBS are in the low ppmw range. For example, reported LODs for CI or Cl-containing compounds ranged from 2 to 90 ppmw for CCl^ CF2CI2 and CF4 parent molecules.^^^"^ It is interesting to note that, for all references that provide information on the time required for halocarbon determination in air using LIBS, the analysis time per sample was on the order of a minute or less. Williamson et al.^"^ even showed the possibility to obtain acceptable data from a single laser pulse. Haisch et al.^^ also showed the possibility to develop a miniaturized system based on a compact Nd:YAG laser (dimensions of 13 x 5 x 3 cm). The rapid data acquisition time combined with the possibility to develop compact, field deployable instrumentation for the in situ determination of halogenated alcanes led to the use of LIBS as a diagnostic tool in various applications such as the on-line monitoring of chlorine emissions from waste management facilities (such as incinerators, landfill sites or semiconductor etching processes^^), the sensitive detection of chlorine and fluorine for the monitoring of chemical weapons such as nerve and blister agents^^ or even the real-time and in situ detection of fluorine concentration for the evaluation of different replacement products for halons in severely hostile fire suppression environments such as military aircraft and ground combat vehicle engine compartments.^"^

19.4. LASER PHOTOFRAGMENTATION - FRAGMENT DETECTION The laser photofragmentation - fragment detection approach (hereafter called PF/FD) shares many characteristics with LIBS in terms of in situ detection capability combined with high temporal and spatial resolution. The most important features of PF-luminescence approaches include their applicability to non fluorescent species as well as the ability to target a class of compound rather than a single molecular species by monitoring commonly shared fragmented functional groups. Compared to LIBS, FD is generally more sensitive (ppm to low ppb detection limits) and can be made more selective (or species-specific), especially when LIF or photoionization detection is considered. It is outside the scope of the present paper to review and compare all the various detection approaches that can be implemented to monitor the photofragmentation products. Detailed and comprehensive reviews have been pubUshed by Simeonsson et al.^^' ^"^ on this topic. Therefore, only the basics of PF/FD will be highlighted herein. PF/FD detection schemes can be divided in two distinct processes, as illustrated in Figure 1. The analyte species R-F is first excited using one laser source, hv^ through linear or multiphoton absorption, which leads to the fragmentation of the parent molecule into the characteristic fragment F (and its counterpart R), which can be atomic or molecular species of typically up to 2-3 atoms. As depicted on the energy level diagram of Figure 1, the fragments of interest will be characterized by their internal energy distribution E(j,v,e),

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denoting the rotational (]), vibrational (v), and electronic excitation (e) contributions. Of course, the photofragments themselves contribute to the selectivity of the method since they are characteristic of the chemical composition of the parent molecule. However, unlike the LIBS approach, the first fragmentation step can contribute to enhance the selectivity of the detection technique since molecular absorption occurs through a transition characteristic of the targeted R-F species. Since high laser intensities are generally required for the photofragmentation step, it should be kept in mind that potential interferences from other species must be considered and the selection of an appropriate detection scheme is of crucial importance to obtain both selective and sensitive measurements. After fragmentation, a second laser source, hv2, can be used to probe the fragments. As shown on the energy level diagram of Figure 1, fragment detection can be accomplished by various means including the luminescence methods of laser-induced fluorescence (LIP), prompt emission (PE, sometimes termed fragment fluorescence), stimulated emission (SE) or laser ionization recombination emission (LIRE). Fragment detection via photoionization approaches (photoionization, PI; prompt photoionization, PPI) coupled to mass spectrometry is also possible and has been extensively studied but will not be covered in the current review. As stated by Simeonsson et al., among the most common optical detection schemes, PF-LIF has seen the widest application and perhaps the highest degree of success outside the research laboratory to perform real-time, spatially resolved, highly sensitive and selective monitoring of halogenated species in various environments.^^ ^"^ Analytical applications as well as more fundamental studies of the use of PF-LIF, PF-PE and PF-SE can be found in the literature for the measurement of F-, C1-, Br- and I- containing halogenated alcanes and involve the detection of halogen-containing molecular fragments as well as halogen atoms. For the purpose of the present review article, we have selected the papers that best highlight the different detection strategies that can be implemented and the fields that have been covered the most in terms of applications. Chlorinated hydrocarbons (CHCs) have certainly been the most targeted species due to their massive use in many industrial and domestic products combined with their negative impact to the environment. An attractive field of application for PF-based applications was the monitoring of incinerator performance during CHC-containing waste disposal, which must be evaluated in situ and in real time to ensure complete CHC destruction and avoid the release of toxic by-products such as tetrachloroethylene (TCE).^"^^^^

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2. Fragment Detection rnrv-f

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