Bioluminescence and Chemiluminescence: Light Emission: Biology and Scientific Applications, Proceedings of the 15th International Symposium

Proceedings of the 15th International Symposium on BIOLUMINESCENCE AND CHEMILUMINESCENCE Light Emission: Biology and S...

Author: Xua Shen | Xiao-lin Yang | Xin-rong Zhang | Zong Jie Cui | Larry J. Kricka

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Proceedings of the 15th International Symposium on

BIOLUMINESCENCE AND CHEMILUMINESCENCE Light Emission: Biology and Scientific Applications

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Shanghai, P. R. China 13 – 17May 2008

Proceedings of the 15th International Symposium on

BIOLUMINESCENCE AND CHEMILUMINESCENCE Light Emission: Biology and Scientific Applications edited by

Xun Shen Chinese Academy of Sciences, P. R. China

Xiao-Lin Yang People's Hospital of Peking University, P. R. China

Xin-Rong Zhang Tsinghua University, P. R. China

Zong Jie Cui Beijing Normal University, P. R. China

Larry J Kricka University of Pennsylvania, USA

Philip E Stanley Cambridge Research & Technology Transfer Ltd, UK

World Scientific NEW JERSEY * ~ O ~ O O* NSINGAPORE

BElJlNG * SHANGHAI * HONG KONG * TAIPEI * CHENNAI

Published by

World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

BIOLUMINESCENCE AND CHEMILUMINESCENCE Light Emission: Biology and Scientific Applications Copyright © 2009 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN-13 978-981-283-957-2 ISBN-I0 981-283-957-7

Printed in Singapore by World Scientific Printers

PREFACE These are the Proceedings of the 15 th Symposium on Bioluminescence and Chemiluminescence held at the Shanghai Galaxy Hotel on 13-17 May, 2008. This series of symposia started in Brussels in 1978, and a list of the other Proceedings volumes appears at the end of this Preface. As in previous symposia, participants came from far and wide and in all 19 countries were represen ted. The Organizing Secretariat was fortunate to have the continued association with the International Society for Bioluminescence & Chemiluminescence. The organizers are thankful for the kind support of the society. We also thank John Wiley & Sons for publishing the regular abstracts in the journal Luminescence Vol. 23(2) 2008. Editorial Note This volume was compiled without peer review from camera-ready manuscripts of lectures and posters presented at the Symposium. The Editors have, in the interest of rapid publication, made only minor stylistic changes. They take no responsibility for scientific or priority matters. The Editors: Xun Shen, Xiao-Lin Yang, Xin-Rong Zhang, Zong Jie Cui, Larry J Kricka, Philip E Stanley.

THE MARLENE DELUCA PRIZE The Marlene DeLuca prizes were again generously given by Dr Fritz Berthold, together with Berthold Technologies. Dr. Berthold has provided these prizes at each symposium since the 1988 Symposium in Florence. The prize can be awarded to symposium participants under the age of 35 on the day before the starting date of the symposium. The prize is given in memory of Dr. Marlene DeLuca who made major contributions to the science of bioluminescence (see Stanley PE. Dedication to Marlene DeLuca: Journal oj Bioluminescence and Chemiluminesceence 1989;4:7-11 (includes list of her papers). Similarly to previous years' selections, the President of the International Society, Professor Xun Shen (Institute of Biophysics, Chinese Academy of Sciences, China), assembled a selection committee from the society to choose the four winners based on their presentations. The 2008 prize winners were: Zhijuan Cao, School of Pharmacy, Fudan University Shanghai. G-rich sequence-functionalized polystyrene microsphere-based instananeous derivatization for the chemiluminescence-amplified detection of DNA. Julien Claes, Laboratory of Marine Biology, Catholic University of Louvain. Bioluminescence of sharks, a case study: Etmopterus spinax. v

vi

Preface

Elena Eremeeva, Photobiology Laboratory, Institute of Biophysics Krasnoyarsk and Laboratory of Biochemistry, Wageningen Univers ity. The kinetics of coelenterazine binding with apo-obelin and apo-aequorin. Michael Koksharov, Department of Chemistry, Lomonosov State University Moscow. pH-tolerant mutants of Luciola mingrelica luciferase created by random mutagenesis.

INTERNATIONAL SOCIETY FOR BIOLUMINESCENCE AND CHEMILUMINESCENCE 2006-2008 ISBC COUNCIL Council Members: B. Branchini (President), A. A. Szalay (Past President), M. Aizawa (President Elect), Y. Ohmiya (Secretary), P. Pasini (Past Secretary), E. Hawkins (Treasurer & Membership Secretary), L. J. Kricka (Publications Officer). Councilors: H. Akhavan-Tafti, L. Brovko, R. Hart, P. Hill, O. Nozaki, A. Roda, E. Widder, K. Wood 2008-2010 ISBC COUNCIL Council Members: M. Aizawa (President), B. Branchini (Past President), Larry J Kricka (President Elect), Y. Ohmiya (Secretary), P. Pasini (Past Secretary), E. Hawkins (Treasurer & Membership Secretary), L. J. Kricka (Publications Officer). Councilors: H. Akhavan-Tafti, L. Brovko, R. Hart, P. Hill, O. Nozaki, A. Roda, E. Widder, K. Wood

LOCAL ORGANIZING AND PROGRAM COMMITTEE CHAIRMAN: Xun Shen VICE CHAIRMEN: Zong Jie Cui, Xin-Rong Zhang MEMBERS: Guo-Nan Chen, Hua Cui, Zong-Jie Cui, Wei-Jun Jin, Xiang-Gui kong, Jin-Miong Lin, Ya-Ning Liu, Xun Shen, Da Xing, Xiao-Lin Yang, Guo-Qiang Yang, Xin-Rong Zhang, Zhu-Jun Zhang, Hui-Sheng Zhuang SECRETARIAT: Xiao-Lin Yang (Secretary), Ya-Ning Liu (Co-Secretary), JinLing Min (Co-Secretary) MANUSCRIPT EDITORS: Larry J Kricka and P E Stanley

ACKNOWLEDGEMENTS We wish to express our sincere appreciation to the following for their generous support of this symposium.

Preface

vii

HOSTED BY: The Commission for Photobiology, Biophysical Society of China. CO-HOSTED BY: The Commission for Analytical Chemistry, The Chinese Chemical Society, The Commission for Luminescence, The Chinese Physical Society. LOCAL SPONSORS: China Association for Science and Technology, National Natural Science Foundation of China. The Institute of Biophysics, The Chinese Academy of Sciences. SPONSORS: John Wiley & Sons, Ltd, China Medical Technologies, Prom ega Corporation. EXHIBITORS: Chemclin Biotech Co, Ltd. (Beijing); Hamamatsu Photonics K.K. (Beijing); Berthold Technologies GmbH& Co. KG; Berthold Detection Systems GmbH; Prom ega Corporation; Perkin Elmer Instruments (Shanghai) Co., Ltd.; Nature Gene Life Sciences Company Ltd. (Hong Kong); Longmed Bio-Tech. Ltd. (Beijing); Thermo Fisher Scientific (Shanghai) Co., Ltd.; Olympus (Beijing) Sales and Service Co., Ltd.; China Medical Technologies; Nikyang Enterprise Ltd (Hong Kong).

NEXT SYMPOSIUM The next Symposium will be held in Lyon, France in 2010. Details of the 16th BL&CL Symposium will be posted on, http://www.isbc.unibo.it. PROCEEDINGS OF PREVIOUS SYMPOSIA 14th 2006 San Diego, CA, USA Bioluminescence & Chemiluminescence: Chemistry, Biology and Applications. Editors: Szalay AA, Hill PJ, Kricka LJ, Stanley PE. Singapore: World Scientific 2007. pp. 283. ISBN 981-270-816-2. 13 th 2004 Yokohama, Japan Bioluminescence & Chemiluminescence: Progress and Perspectives. Editors: Tsuji A, Matsumoto M, Maeda M, Kricka LJ, Stanley PE. Singapore: World Scientific 2004. pp. 520. ISBN 981-238-156-2. 12th 2002 Cambridge, UK Bioluminescence & Chemiluminescence: Progress & Current Applications. Editors: Stanley PE, Kricka LJ. Singapore: World Scientific 2002. pp. 520. ISBN 981-238-156-2. 11 th 2000 Monterey, CA, USA Proceedings of the 11th International Symposium on Bioluminescence & Chemiluminescence. Editors: Case JF, Herring PJ, Robison BH, Haddock SHD, Kricka LJ, Stanley PE. Singapore: World Scientific 2001. pp. 517. ISBN 98102-4679-X.

viii Preface 10 th 1998 Bologna, Italy Bioluminescence and Chemiluminescence: Perspectives for the 21 51 Century. Editors: Roda A, Pazzagli M, Kricka LJ, Stanley PE. Chichester: Wiley 1999. pp. 628. ISBN: 0-471-98733-6. 9 th 1996 Woods Hole, MA, USA Bioluminescence and Chemiluminescence: Molecular Reporting with Photons. Editors: Hastings JW, Kricka LJ, Stanley PE. Chichester: Wiley 1997. pp. 568. ISBN: 0-471-97502-8. 8 th 1994 Cambridge, UK Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. Editors: Campbell AK, Kricka LJ, Stanley PE. Chichester: Wiley 1994. pp. 672. ISBN: 0-471-95548-5. 7th 1993 Banff, Canada Bioluminescence and Chemiluminescence: Status Report. Editors: Szalay AA, Kricka LJ, Stanley PE. Chichester: Wiley. 1993, pp. 548. ISBN: 0-471-94164-6. 6th 1990 Cambridge, UK Bioluminescence and Chemiluminescence: Current Status. Editors: Stanley PE, Kricka LJ. Chichester: Wiley 1991. pp. 570. ISBN: 0-471-92993-X. 5th 1988 Florence, Italy Bioluminescence and Chemiluminescence: Studies and Applications in Biology and Medicine. Editors: Pazzagli M, Cadenas E, Kricka LJ, Roda A, Stanley PE. Chichester: Wiley 1989. pp. 646. (published as volume 4, issue 1 of the Journal a/Bioluminescence and Chemiluminescence, 1989). ISBN: 0-471-92264-1. 4th 1986 Freiburg, Germany Bioluminescence and Chemiluminescence: New Perspectives. Editors: Sch61merich J, Andreesen R, Kapp A, Ernst M, Woods WG. Chichester: Wiley 1987. pp. 600. ISBN: 0-471-91470-3. 3 rd 1984 Birmingham, UK Analytical Applications of Bioluminescence and Chemiluminescence. Editors: Kricka LJ, Stanley PE, Thorpe GHG, Whitehead TP. London: Academic Press 1984. pp. 602. ISBN: 0-12-426290-2. 2 nd 1980 San Diego, CA, USA Bioluminescence and Chemiluminescence: Basic Chemistry and Analytical Applications. Editors: DeLuca MA, McElroy WD. New York: Academic Press 1981. pp.782. ISBN: 0-12-208820-4. 1st 1978 Brussels, Belgium International Symposium on Analytical Applications of Bioluminescence and Chemiluminescence. Proceedings 1978. Editors: Schram E, Stanley PE. Westlake Village, CA: State Printing & Publishing, Inc., 1979, pp. 696.

INTRODUCTION On behalf of the Organizing Committee of 15th International Symposium on Bioluminescence & Chemiluminescence, held May 13-17, 2008, I would like to thank the International Society of Bioluminescence and Chemiluminescence (ISBC) for their trust and support to host this exciting meeting. The symposium brought scientists from different parts of the world to Shanghai, China's most comprehensive industrial and commercial city. Since the first symposium was held in 1978 in Brussels, Belgium, the symposium has subsequently been held every two years in Europe, America and Japan. This is the first time that this symposium has been held in China. Thus, it gave Chinese scientists, interested in bioluminescence and chemiluminescence, an opportunity, to interact closely with the international bioluminescence and chemiluminescence community. It also gave the scientists from Europe, America and other parts of Asia an opportunity to learn that Chinese scientists are catching up the world in all aspects of science, including research and application of bioluminescence and chemiluminescence. In the last decade, great advances have been made in fundamental research and in the applications of bioluminescence and chemiluminescence. Bioluminescence imaging has emerged as a powerful new optical imaging technique. It offers realtime monitoring of spatial and temporal progression of biological processes in living animals. The bioluminescence resonance energy transfer (BRET) methodology has also emerged as a powerful technique for the study of protein-protein interactions. Luciferase reporter gene technology represents one of the major recent achievements of molecular biology. Luciferase genes can be artificially introduced into a cell to monitor gene expression and used to explore molecular mechanisms in the regulation of gene expression. Furthermore, chemiluminescence detection and analysis have been more and more applied to life science research. For example, chemiluminescent labels and substrates have been widely used to replace radioisotope-labeling and have become the most efficient and sensitive method for detecting proteins in various immunoassays. In this symposium, five outstanding experts delivered keynote lectures describing recent advances in molecular imaging using bioluminescence, chemical mechanisms involved in squid bioluminescence, novel applications of electrochemiluminescence, luminescence-based point-of-care testing devices in biomedical diagnostics, and molecular imprinted chemiluminescence imaging sensors. In the final plenary session, Professor J. Woodland Hastings, the world renowned pioneer in understanding bioluminescence, reviewed the history of the discoveries in bioluminescence and its applications. We were fortunate to have oral and poster presentations given by scientists from 19 countries, as well as active participation from industrial exhibitors. The sessions included luciferase-based bioluminescence, photoprotein-based bioluminescence, fundamental aspects and applications of chemiluminescence, luminescence imaging, fluorescence quantum dots and other inorganic fluorescent materials, phosphorescence and ultraweak luminescence, instrumentation and new methods. ix

x

Introduction

On May 12, 2008, just one day before the symposium, a major earthquake measuring 8.0 on the Richter scale hit Wenchuan County in southwest China's Sichuan province. It is the biggest disaster in Chinese history. As many as 70,000 people died, 20,000 people were missing and millions of people became homeless. To express our sympathy and help the people in the earthquake area, the symposium participants benevolently donated more than 1200 US dollars during the symposium. On behalf of the Organizing Committee, I would like to thank all of the donors for their kind support to the people in earthquake area. The organizers and I are grateful to all the generous sponsors for their financial support of the symposium. Special thanks are owed to the China Association for Science and Technology and the National Natural Science Foundation of China for their sponsorship, and Promega Corporation and China Medical Technologies for their financial support. I would like to thank my co-organizers, Drs. Xiaoping Yang, Zong Jie Cui, Xinrong Zhang, Yaning Liu and all my competent and friendly staff, Shunyi Wei, Yue Wang and Wenli Xu, who aided the participants of the 15th International Symposium. In particular, I would like to thank Dr. Larry J. Kricka for his great effort in editing the manuscripts. Without them, this symposium would not be so successful.

Cordially,

Xun Shen President The 15th International Symposium on Bioluminescence and Chemiluminescence

CONTENTS

Preface

v th

Introduction to the 15 Symposium

ix

PART 1. BASIC BIOLUMINESCENCE Plenary lecture - Progress, perspectives and problems in basic aspects of bioluminescence HastingsJW Bioluminescence of sharks, a case study: Etmopterus spinax Claes JM and Mallefet J Chemiexcitation mechanism for Cypridina (Vargula) and Aequorea bioluminescence Hirano T, Ohba H, Takahashi Y, Maki S, Kojima S, Ikeda H andNiwaH

3

15

19

Site-directed mutagenesis of Lampyris turkestanicus luciferase: The effect of conserved residue(s) in bioluminescence emission spectra among firefly luciferases Hosseinkhani S, Tafreshi N Kh, Sadeghizadeh M, Emamzadeh R, Ranjbar Band Naderi-Manesh H

23

Chemiluminescent and bioluminescent analysis of plant cell responses to reactive oxygen species produced by a new water conditioning apparatus equipped with titania-coated photo-catalytic fibers Kagenishi T, Yokawa K, Lin C, Tanaka K, Tanaka R and KawanoT

27

pH-tolerant mutants of Luciola mingrelica luciferase created by random mutagenesis Koksharov MI and Ugarova NN

31

xi

xii Contents Bacterial bioluminescence with flavinmononuc1eotide activated by N-methylimidazole Krasnova 01, Tyulkova NA and Doroshenko 10 New method of measuring bacterial bioluminescence Krasnova 01, Tyulkova NA and Doroshenko 10

35

39

Enhancement of thermostability of Luciola mingrelica firefly luciferase by mutagenesis of non-conservative residues CYS62 and CYS146 Lomakina GY, Modestova YA and Ugarova NN

43

Web-resource: "Bioluminescence and luminous organisms" of the IBSO culture collection Medvedeva SE, Kotov DA and Rodicheva EK

47

Chemistry of symplectin bioluminescence with fluorodehydrocoelenterazine Nakashima Y, Kongjinda V, Tani N, Kuse M and Isobe M

51

Mechanisms of heavy atom effect in bioluminescent reactions Nemtseva EV, Kirillova TN, Brukhovskih TV and Kudryasheva NS

55

Theoretical analysis on the absorption spectra of intermediates of firefly luciferin in deoxygenated dimethyl sulfoxide Sakai Hand Wada N

59

Biophoton emission of biological systems in terms of odd and even coherent states Kun SI, Liu C and Jia H- Y

63

Study on ATP-dependent luminescence reaction of the arm light organs of the luminous squid Watasenia scintillans Teranishi K and Shimomura 0

67

Mechanism of bacterialluciferase: Energetic and quantum yield Considerations TuS-C

71

Contents

Mechanism responsible for the spectral differences in firefly bioluminescence UgarovaNN Luminous mushrooms Vydryakova GA, Psurtseva NV, Belova NV, Gusev AA, Pashenova NV, Medvedeva SE, Rodicheva EK and Gitelson JI Use of Cypridina luciferin analog for assessing the monoamine oxidase-like superoxide-generating activities of two peptide sequences corresponding to the helical copper-binding motif in human prion protein and its model analog Yokawa K, Kagenishi T and Kawano T

xiii

75

79

83

PART 2. APPLIED BIOLUMINESCENCE Bioluminescent assay of antibiotic susceptibility of clinical samples Frundzhyan VG and Ugarova NN

89

BART: Smart biochemistry, bright bioluminescence, low-cost hardware Gandelman GA, KiddIe G, McElgunn CJ, Rizzoli M, Murray JAH and Tisi LC

93

BART applications in medical and food diagnostics Gandelman GA, KiddIe G, Rizzoli M, Murray JAH and Tisi LC

97

Change of expression efficiency of natural and cloned lux-operon in conditions of famine GusevAA

. 101

Construction of recombinant luminescence bacteria vector to evaluate genetoxic environmental pollutants Huang X-X; He M, Shi H-C and Cai Q

105

Development ofa novel bioluminescent assay for nitric oxide by using soluble guanylate cyclase Sano Y, Seki M, Suzuki S, Abe S, Ito K and Arakawa H

109

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PART 3. BASIC CHEMILUMINESCENCE Mass spectrometric approach to elucidation of chemiexcitation of dioxetanes Ijuin HK, Ohashi M, Tanimura M, Watanabe Nand MatsumotoM Theoretical considerations on the roles of hydrogen bonding in thermal decomposition of peroxides lsobe H, Yamanaka S, Okumura M and Yamaguchi K A new bright chemiluminescent reaction: Interaction of acetone with solid-phase potassium monoperoxysulfate in the complex of europium nitrate Kazakov DV, Safarov FE, Schmidt Rand Kazakov VP Study of novel aryloxalate chemiluminescence reaction without addition of hydrogen peroxide Kishikawa N, Ohyama K, Nakashima K and Kuroda N

115

119

123

127

Nucleophilic acylation catalysts effect on luminol chemiluminescence Marzocchi E, Grilli S, Della Ciana L, Mirasoli M, Simoni P, Prodi Land Roda A

131

Effect of surfactants on peroxyoxalate chemiluminescence reaction Nakashima K, Abe K, Nakamura S, Wada M, Harada S andKurodaN

135

Solvent-promoted chemiluminescent decomposition of bicyclic dioxetanes bearing a 4-(benzothiazol-2-yl)-3-hydroxyphenyl Tanimura M, Watanabe N, ljuin HK and Matsumoto M Synthesis and characterization of near-infrared chemiluminescent probes Teranishi K

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Generation of high-energy chemiluminophores in ambient light Tsaplev Yu B, Vasil' ev RF and Trofimov A V Alkaline metal ion enhanced chemiluminescence of bicyclic dioxetanes bearing a 3-hydroxynaphthalen-2-yl group Watanabe N, Kakuno F, Hoshiya N, Ijuin HK and Matsumoto M

xv 147

151

PART 4. APPLIED CHEMILUMINESCENCE Plenary lecture - Analytical challenges for luminescence-based point-of-care testing devices in biomedical diagnostics Roda A, Guardigli M, Mirasoli M, Michelini E, Dolci LS, and Musiani M

157

Plenary lecture - Molecular imprinted polymer-based chemiluminescence sensors Zhang Z

161

Flow injection chemiluminescence determination of hydroxylamine hydrochloride Baezzat MR and Izadpanah M

173

Study on gold-sensitised chemiluminescence for the determination of norfloxacin Bao J-F, Jiang Z-H and Yu X-J

177

Conjugates of (acridinium)x-BSA-anti-HCV core to enhance the detection of HCV core antigen Chang CD, Chang KY, Jiang L, Sablilla VA and Shah DO

181

Chemiluminescence determination of rutin based on a micelle-sensitizing N-bromosuccinimide-H20 2 reaction Du JX, Hao Land Lu JR

185

Luminol-dependent chemiluminescence increases with formation of phenothiazine cation radicals by horseradish peroxidase Hadjimitova VA, Traykov T and Bakalova R

189

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Contents

Variety of chemiluminescent methods for antioxidant activity: Investigation of Crataegus oxicantha extract Hadjimitova VA, Traykov Tand Bakalova R

193

Simultaneous mUltiplex bio- and chemiluminescent enzyme immunoassay for PCR products derived from genetically modified Papaya Ito K, Tanaka Y, Maeda M, Gomi K, Inouye S, Akiyama H and Arakawa H

197

Effect of sugars on aluminum-induced oxidative burst and cell death in suspensions of tomato cells Kadono T, Kawano T, Yuasa T and Iwaya-Inoue M

201

Chemiluminescence determination of sparfloxacin using Ru(bipY)32+-Ce(IV) system Karim MM, Choi JH, Alam SM and Lee SH

205

Flow injection analysis with chemiluminescence detection: Determination of gatifloxacin using the KMn04-formaldehyde system Khan MA, Alam SM and Lee SH

209

Determination of ciprofloxacin in pharmaceutical formulation by chemiluminescence method Khan MA, Lee SH, Alam SM, Wabaidur SM and Chung HY

213

Chemiluminescence flow-through biosensor for hydrogen peroxide based on enhanced HRP activity by gold nanoparticles Lan D and Li B

217

Flow injection chemiluminescence determination of thiamine by the enhancement of luminol- K3Fe(CN)6 system Li YH, Yang Y and Lu JR

221

Chemiluminescent and electron spin resonance spectroscopic measurements of reactive oxygen species generated in water treated with Titania-coated photocatalytic fibers Lin C, Tanaka K, Tanaka L and Kawano T

225

Contents

A sensitive micellar-enhanced chemiluminescence method for the determination of ofloxacin by flow injection analysis Ma H, Zhang Y, Miao L and Sun X Excessive extracellular chemiluminescence and necrosis of neutrophils in bovine neonates and potentially supportive role of vitamin C Mehrzad J, Mohri M and Burvenich C Chemiluminescence of 9-benzylidene-l O-methylacridans with electron-donating groups by chemically generated singlet oxygen - Application to metal ion sensing using azacrowned compound Motoyoshiya J, Tanaka T, Kuroe M and Nishii Y Effects of l,4-butanediol dimethacrylate on HL-60 cells metabolism Nocca G, De Sole P, De Palma F, Martorana GE, Rossi C, Corsale P, Antenucci M, Giardina Band Lupi A Determination of pyrogallol by imidazole chemiluminescence enhanced with hydrogen peroxide Nozaki 0, Munesue M, Momoi H, Shizuma M, Kawamoto H and Ikeda T

xvii

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233

237

241

245

Chemiluminescence study on the regulation of NADPH oxidase activity by thioredoxin reductase in vascular endothelial cells Shen X and Liu Z-B

249

Quantitative detection of singlet oxygen with a chemiluminescence probe during photodynamic reactions Wei Y, Xing D, Luo S, Xu Wand Chen Q

253

Flow-injection chemiluminescence determination of human serum albumin based on fluoresceinyl Cypridina luciferin analog-'02 reaction Xu W, Wei Y, Xing DA, Luo S and Chen Q

257

Charge-transfer-induced luminescence (CTIL) mechanisms of chemi- and bioluminescence reactions Yamaguchi K, Isobe H, Yamanaka S and Okumura M

261

xviii Contents A novel synergistic enhancer for HRP-Luminol-H 20 2 based chemiluminescence and its application in immunoassay Yang X and Sun X

265

Separation and detection of amino acids with a novel capillary electrophoresis chemiluminescence system Yin DG, Xie CJ, Liu BH and Wu MH

269

A novel chemiluminescent immunoassay of total thyroxine using the acridinium ester 2' ,6' -dimethyl-4' -(N-succinimidyloxycarbonyl) phenyl-1O-methyl-acridinium-9-carboxylate methosulfate as label Yin DG, He YF, Liu YB, Shen DC, Han SQ, Luo ZF, Xie CJ, Zhang L, Liu BH and Wu MH

273

Determination of ascorbic acid by a flow injection chemiluminescence method with a novel rhodanine Yu J, Zhang C, Tan Y, Ge S, Dai P and Zhu Y

277

Study of superweak luminescence in plants and application to salt tolerance in alfalfa Zhou H, Yang Q and Liu Y

281

Development and optimization of a quantitative western blot and dot blot procedure for the determination of residual host cell proteins present in inactivated polio vaccine using a GZll based signal reagent Zomer G, Hamzink M, De Haan A, Kersten G and Reubsaet K Development and optimization of a fast and sensitive ELISA for polio D-antigen using a GZll based signal reagent Zomer G and Hamzink M

287

291

PART 5. APPLIED ELECTROLUMINESCENCE Detection of Xanthomonas oryzae pv. Oryzicola by electrochemiluminescence polymerase chain reaction method Wei J and Zhang L

297

Contents

xix

A novel electrochemiluminescent sensor based on cationic polymer/chitosan for ultrasensitive detection of hydrogen peroxide Wu X, Wang Y, Dai H and Chen G

301

Capillary electrophoresis - electrochemiluminescence detection of ciprofloxacin in biological fluids Zhou X and Jia L

305

PART 6. BIOMEDICAL APPLICATION OF FLUORESCENT PROTEINS A novel multicolor fluorescent protein from the soft coral Scleronephthya gracillima Kuekenthal Kato Y, Jimbo M, Sato C, Takahashi T, lmahara Yand Kamiya H

311

Fluorescence from STlevel of complexes of tryptophan with europium (III) in water-ethanol solution Osina 10, Ostahov Sand Kazakov V

315

Identification of developmental enhancers using targeted regional electroporation (TREP) of evolutionarily conserved regions Pira CU, Caltharp SA, Kanaya K, Manu SK, Greer LF and Oberg KC

319

PART 7. DEVELOPMENT AND BIOMEDICAL APPLICATIONS OF QUANTUM DOTS AND OTHER INORGANIC FLUORESCENT MATERIALS Quantum dots as fluorescent resonance energy transfer donors in antibody-antigen systems Hu S, Yang H, Cai R, Zhang Q and Yang X

325

Synthesis and photoluminescence of green-emitting X2-(Y,GdhSiOs:Tb3+ phosphor under VUV excitation Zhang ZH, Wang YH and Li XX

329

Luminescent properties of Na2CaMg2Si401s:Tb3+ nano-sized phosphor Zhou L-Y, Yi L-H, Huang J-L, Wei J-S and Gong F-Z

333

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Contents

PART 8. BIOLUMINESCENCE, CHEMILUMINESCENCE AND FLUORESCENCE IMAGING The measurement of cytosolic ATP during apoptosis: Bioluminescence imaging at the single cell level Akiyoshi R and Suzuki H Bioluminescence imaging of bacteria-host interplay: Interaction of E. coli with epithelial cells Brovko LY, Wang H, Elliot J, Dadarwal R, Minikh 0 and Griffiths MW Ultrasensitive chemiluminescent immunochemicallocalisation of protein components in painting cross-sections Dolci LS, Sciutto G, Rizzoli M, Guardigli M, Mazzeo R, Prati S and RodaA Development of a new device for ultrasensitive electrochemiluminescence microscope imaging Dolci LS, Rizzoli M, Marzocchi E, Zanarini S, Della Ciana L and RodaA

339

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347

351

Visualization of sequential response in intra cellular signal transduction cascade by fluorescence and luminescence imaging in the same living cell Hatta-Ohashi Y, Takahashi T and Suzuki H

355

Bioluminescence imaging of intracellular calcium dynamics by the photoprotein obelin The! MM, Sugiyama T and Suzuki H

359

Applications of delayed fluorescence and laser confocal scanning microscope techniques in monitoring artificial acid rain stress on plants

363

Zhang H, Wen F and Zhou X Delayed fluorescence and optical molecule imaging techniques for detecting the stress response of plants to high temperature Zhang Land Wen F

367

Contents

xxi

PART 9. ASPECTS OF FLUORESCENCE AND PHOSPHORESCENCE

The interaction of Tb 3+-protocatechuic acid complex with nucleic acids and its application in determination of nucleic acids based on fluorescence quenching Chen Y, Yang Yand Yang J

373

Fluorescence enhancement of KI for the morin-fsDNA system and its analytical application 377 Ding H, Wu X, Yang J and Wang F Microemulsion sensitized determination of BSA with 3-(4'-methylphenyl)-5-(2'-sulfophenylazo) rhodanine by resonance Rayleigh scattering method Ge S, Dai p, Yu J, Li B and Tan Y Fluorimetric determination of rutin using rutin-Fe(IlI) system Karim MM, Jean CW, Lee SH and Wabaidur SM Micelle enhanced fluorimetric determination of benserazide in pharmaceutical formulations Lee SH, Kim WH, Meea K and Khan MA Improvement in carbaryl assay by fluorescence in a micellar medium Lee SH, Jean CW, Kim WH, Chung HY, Wabaidur SM, Park HW, Suh YS and Khan MA

381

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389

393

Study of the interaction between human serum albumin and 7-ethyl-1Ohydroxycamptothecin Li G and Liu Y

397

Resonance Rayleigh scattering method for determination of alginic sodium diester with methylene blue Liu Yand Li G

401

Effects of metal ions on peroxynitrite nitrifying protein Luo Y, Cui S, Zhang L and Zhong R

405

xxii Contents Mechanism and properties of bio-photon emission and absorption of protein molecules in living systems Pang X-F The mechanism of photon emission of bio-tissues and its properties Pang X-F and Cao X-Y Synthesis of a novel fluorescence probe of P-CD and cuprous iodide pyridine and its application Qiao J, Dong R, Li D, Dong C and Shuang S Phosphorescence properties of 2-bromoquinoline-3-boronic acid in sodium deoxycholate and its potential application in recognition of carbohydrates Shen QJ, Zou WS, Jin WJ and Wang Y

409

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421

425

Study on the interaction between methyl blue and HSA in the presence of P-CDIHP-P-CD by molecular spectroscopy Song S, Hou X, Shuang S and Dong C

429

Study on the interaction of kaempferol with human serum albumin by spectroscopy and molecular modeling Tian J, Liu J, Hu Z and Chen X

433

Selection of salt-tolerant rice variety using light-induced delayed fluorescence Wang J, Xu W, Xing D and Zhang L

437

Effects of LMWOA on biodegradation of phenanthrene studied by fluorimetry Wei XY, Sang LZ, Zhu YX and Zhang Y

441

Alleviation effects of salicylic acid and lanthanum on ultra weak bioluminescence in maize leaves under cadmium stress Wei ZL, Jiao CZ, Su YN and Tian ZH

445

Rhodamine B-quinoline-8-amide as a fluorescent "ON" probe for Fe3+ in acetonitrile Xiang Y, Li ZF and Tong AJ

449

Contents

xxiii

Studies on determination of deoxyribonucleic acid by second order scattering with a novel rhodanine Yu J, Li B, Zhu Y, Cheng X and Zhang L

453

Fluorescence characteristics of novel chlorophenyl-arsenoxylphenylazo rhodanines and application in the determination of thallium (I) Yu J, Cheng X, Ge S, Tan Y and Li B

457

Molecular recognition of amino acids by hematoporphyrin and metallohematoporphyrin receptors Zhang Y, Lei Y-C and Liu D-S

461

Determination of BSA by its enhancement effect on second order scattering of 3-(4'-methyl phenyl)-5-( 4'-methyl-2'-sulfophenylazo) rhodanine Zhu Y, Yu J, Dai P, Zhang C and Li B

Index

465

469


PARTl BASIC BIOLUMINESCENCE


PROGRESS, PERSPECTIVES AND PROBLEMS IN BASIC ASPECTS OF BIOLUMINESCENCE JWHASTINGS Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA

INTRODUCTION th It is a great pleasure to participate in this 15 Symposium on Bioluminescence and Chemiluminescence, thirty years after the first, brilliantly conceived and organized in Brussels by Eric Schram and Philip Stanley, later to be joined by Larry Kricka, and to express my gratitude to the organizing committee for inviting me. It is also an overwhelming experience to see the greatly transformed Shanghai. There has 'also been a profound transformation in the field of bioluminescence over these thirty years, progressing from the vision in Brussels that luciferase systems could be used for analytical purposes in biochemistry and medicine) to the now widespread use of genes of luciferases and GFP as reporters to track expression of other genes in time and location. 2 In parallel, there have been many important advances is basic aspects. 3 Color mutants of both luciferases and green fluorescent protein have been put to great advantage in studies where they are used as reporters and, along with other mutants, contribute to our understanding of reaction mechanisms. Crystal structures have been obtained for luciferases from four systems- bacterial, firefly, coelenterate and dinoflagellate, and much has been elucidated concerning the structures of emitters and reaction intermediates. Here I will discuss specific aspects of each of the four systems for which luciferase structures are available, starting with the coelenterate system and the use of the term photoprotein. Coelenterates: Aequorin & photoproteins are luciferase intermediates. For many years the biochemistry of the brilliantly luminescent jellyfish Aequorea was a real enigma. Cold-water extracts gave bright and long-lived emission, but the luciferin-Iuciferase test was frustratingly negative. Shimomura made the seminal discovery that the reaction requires calcium, and found that cold-water extracts made in the presence of EDTA yielded a protein that gave light upon the addition of excess caIcium. 4 He named the protein aequorin, and later dubbed it a photoprotein, the precise nature of which was not well appreciated at first. It was later shown to be a luciferase intermediate, effectively the "substrate" in the assay because turnover is slow, and is destroyed in hot water extracts of the luciferin-Iuciferase test. 5 Sessions at this symposium are divided into luciferase-based bioluminescence and photoprotein-based bioluminescence. But both use luciferases; the photoprotein aequorin is simply a stable luciferase-peroxy-Iuciferin intermediate in which a 67 subsequent reactant has been withheld, as confirmed by its crystal structure. • Such intermediates in this or other systems, when accumulated, can provide the substrate 3

4

Hastings JW

for a rapid flash in living cells if the lacking reactant is rapidly added, thus calcium for aequorin. The flash decay will thus be first order and attributable to the rate constant for the decay of the intermediate formed after calcium addition (Fig. I), and the total light emitted in the flash will be proportional to the amount of intermediate. Also, it should be noted that for the flash to decay to baseline, the prior enzymatic reaction step(s) must be very slow so that little if any more intermediate will be reformed during the course of the flash, during which time the triggering substance can be withdrawn so that new intermediate can be accumulated.

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Fig. 1. Kinetics of the reaction of aequorin with calcium mixed in a stopped-flow apparatus at 23 0 C. Firefly: the regulation of the flash. Although the luciferin-Iuciferase reaction appeared to "work" in firefly extracts, it turned out that the components were not those specified in the long-established protocol. McElroy discovered 8 that ATP is the component exhausted in cold water extracts of fireflies, while both luciferin and luciferase remain (Fig. 2), while the hot-water extract contains ATP. In McElroy's lab, we established that the reaction of ATP and lucifer in with purified luciferase involves two steps;9 the first forms an active intermediate, later determined to be the adenylate, and the second is the reaction with oxygen, leading to an excited state and light emission. The prompt decline of luminescence over the first minutes was shown to be due to luciferase inhibition, not substrate exhaustion. All evidence indicates that the flash of the firefly is initiated by the introduction of oxygen into the photocytes, triggered by a nerve impulse, which actually does not end on the photocytes, but on adjacent cells. IO- 12 More recently, nitric oxide (NO)

Progress, Perspectives and Problems in Basic Aspects of Bioluminescence

5

has been proposed to be a humoral agent involved in transmission of the signal from the nerve ending to the photocyte to initiate a flash. 13 •14 The evidence for this is not strong, and I believe the proposed mechanism to be incorrect.

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lime (min.) Fig. 2. Depiction of the steps and conditions for a luciferin-Iuciferase reaction in which an exhausted cold-water extract is mixed with a hot-water extract to give light emission. How it differs in firefly extracts is also noted.

Briefly, the NO mechanism postulates that mitochondrial oxygen consumption maintains photocytes anaerobic in spite of a continuous input of oxygen from tracheoles. A flash is initiated through a cascade of transduction steps from the nerve ending that result in NO production in the photocytes, where it inhibits this respiration, allowing oxygen to reach luciferase and initiate the reaction. As NO production ceases, along with some other possible factors, the mitochondrial utilization of oxygen resumes and the luciferase reaction declines. The kinetics of the rise phase of the flash, which in many species is less than 100 msec, seems difficult to attribute to a cascade of signal transduction events. But the extinction of the flash is most certainly not caused by the withdrawal of a reactant. Instead, it has kinetics attributable to the reaction of a luciferase intermediate whose

6

Hastings JW

precursor is accumulated in the absence of oxygen, comparable to the case of the jellyfish flash. Some years ago I demonstrated that such a "biochemical" flash can be produced in the test tube. 9,15 If oxygen is excluded from a firefly luciferase reaction mixture and then added rapidly back, a bright flash occurs, some 100 to 200 times brighter than the baseline intensity (Fig. 3). This comes from the reaction of the luciferyl adenylate "active" intermediate accumulated in the absence of oxygen. Note that the decay of the flash is not due to the removal of oxygen, but to the utilization of the luciferase-peroxide intermediate, so the baseline returns to a low level (Fig. 4), defined by the slow rate of reaction of ATP with lucifer in. It is well known that the kinetics of firefly flashes are species specific and of functional importance in courtship communication, fixed by the rate constant for the first order decay of the peroxide intermediate formed from the adenylate.

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Fig. 3. Flashes in response to the rapid addition of oxygen to firefly luciferase reactions initiated in the complete absence of oxygen. 9 A: Time course of normal reaction in air. B,C,D: started under strict anaerobic conditions; oxygen added later at times indicated.

Fig. 4. Kinetics of a flash obtained by addition of oxygen, as described in Figure 3. 9

Bacteria: A peroxide intermediate, quorum sensing and milky seas. Although the luciferin-Iuciferase test in bacterial extracts was negative, Strehler 16 discovered that light emission in extracts could be obtained by adding reduced pyridine nucleotide, underlining the fact that bioluminescence is not a phenomenon separate


7

from all other cell biochemistry, but linked to it in different ways in different systems. Light emission in bacteria is continuous, deriving electrons for the reduction of flavin, the luciferin in this system, from the respiratory pathway, as indicated in Fig 5. Reports that it occurs as pulses have not been confirmed. 17 This luciferase reaction also forms a semi-stable peroxide intermediate, which we demonstrated some years ago 1S and later isolated. 19 It is reasonably stable in the absence of aldehyde and might, in principle, be accumulated in the cell and triggered to emit a flash by aldehyde addition. Indeed, bioluminescence in tunicates, which utilizes a bacterial luciferase system 20 derived from endosymbionts,21 emits light as flashes, the biochemical basis for which has not been investigated. An important phenomenon, now called quorum sensing, was discovered from studies of bacterial bioluminescence, in which it was found that growth and luminescence are controlled separately.22 After inoculating a culture into fresh medium, growth is exponential with no lag, but the amount of luciferase remains constant for the first three hours, after which its synthesis and light emission increase very, very rapidly (Fig. 6). This was shown to be due to the production and release into the medium of a substance that we named auto inducer; upon reaching a critical concentration, it induces the synthesis of luciferase and other proteins involved in the bioluminescence. Eberhard and colleagues determined the structure to be a homoserine lactone and synthesized it. 23 SUBSTRATE ~ NAOH ~ CYTOCHROMES ~ O2

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8 Hastings JW 100 t:..O.D.- 660 NM IN VIVO LUM. IN VITRO LUM .

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Fig. 6. Time courses showing that the development of luminescence and luciferase (both in vitro activity and by antiluciferase, CRM) lag cell growth. 22 For many years this phenomenon was believed to be simply a special curious feature of luminous bacteria, but when DNA sequences became available, genes homologous to those responsible for auto inducer production were found to occur widely in the bacterial world. Up to then it had been generally believed that bacterial cells are mostly loners, essentially autonomous in their activities. But this discovery demonstrated that bacteria produce substances that control expression of different genes in many other bacteria, both in the same and different species, thus constituting chemical communication. 24,25 A major function of luminescence in bacteria is to provide light when cultured in specialized light organs of a higher organism. There, the production of luciferase and light are delayed until cell numbers are high enough for the light to be visible to other organisms. In some pathogenic bacteria toxin production may be delayed until the invading population is high: a surprise attack can produce massive amounts of toxin and overwhelm before resistance can be mounted. Luminous bacteria can be isolated from sea water almost anywhere in the world, but the number is typically very few, so the autoinducer in the water should and does not reach the concentration needed to induce luciferase in isolated cells. 26 Yet ever since records of ship voyages have been kept, there have been repeated reports of continuous luminous light emission in the ocean, all around the ship as far as the eye

r r(}v r",.s_

Perspectives and Problems in Basic Aspects of Bioluminescence

9

can see.27 This has been called "Milky Sea", for it does indeed look like the ship is on a sea of milk! Although no explanation of the phenomenon had been reported in the literature, a group of scientists wondered if earth-imaging satellite cameras might be able to detect the light emission. Checking the archives, they found a ship log reporting the phenomenon in 1995 when a camera had been overhead. They retrieved the satellite and detected a weak signal on three consecutive nights; with background subtracted it revealed a luminous area of about 14,000 km 2 , its exact structure changing from night to night (Fig. 7).28,29 The reported positions of the ship when it entered and exited the area corresponded exactly to the coordinates obtained from the satellite data. The location off the Horn of Africa is where reports in of Milky Seas have been most frequent. 27 Because the emission is continuous it had been speculated, and many scientists that luminous bacteria might be responsible. But, if so, how might the auto inducer concentrations needed be achieved? The answer to this is not nor is it certain that the light is actually due to lum inous bacteria of the kind cultured. But a clue comes from reports of merchant sailors, who from time to time what they saw in a bucket of water from the milky sea. A was that the water " ... contains thousands of very thin lines of

7. Bioluminescence of milky seas recorded by satellite imaging for 3 consecutive nights. Raw data, A,B,C; with background subtracted, locations of images. 28

10 Hastings JW

approximately 13 mm long ,,27 If bacteria are concentrated on a substrate, perhaps a filamentous of some autoindueer could accumulate. Future studies should give the answer.

be

triggers the flash; two functions in one These unicellular marine plankton, which my laboratory has studied for many years, are for the sparkling oceanic luminescence, earlier called pn,osrmclre,;cence Most of our work has with the photosynthetic species, Gonyaulax polyedra), which emits brief (0.1 s) flashes from small named scintillons. 3,3o They contain two major luciferase and a luciferin binding protein (LBP); the activities of both ""If'''''''''''''' The luciferin is a tetrapyrrole, probably derived from The sequences of the N-terminal -100 residues of the two nr,>tpl!1c identical but the remaining regions have no similarities 31 In the molecule 37kDa) is comprised of three repeat homologous each with a located independent catalytic site, where the sequences are about 95% identical. Each individual domain has luciferase and each has four conserved which have been shown to be involved in the by

8. Structure for Noctiluca luciferase (top) showing that it occurs as tandem of a gene possessing a sequence homologous to a domain luciferase (bottom) together with a sequence homologous to a full luciferin protein. The Noctiluca protein lacks the first N-terminal -100 amino acids found in both Lp proteins. 34


11

A crystal structure of one of the domains reveals a catalytic pocket and residues 33 responsible for regulation by pH. The LBP has four homologous domains, but their sequence similarities are not great. 34 The luciferase genes and proteins are very similar in seven different luminous photosynthetic species. They are about the same length and all have three domains, and occur as tandem repeats but with very different intergenic sequences. 35 ,36. The individual domains of different species are more similar to each other than to either of the other two domains of the same species. But in the heterotroph Noctiluca sc intillans the catalytic and luciferin binding sequences are both found in a single gene, and are expressed as a single protein (Fig. 8). The N-terminal -100 sequences found in L. polyedrum, which might be functional for protein-protein association, are completely absent. There is only a single luciferase domain, and it is truncated on the N-terminal side, with three of the four histidines found the three-domain luciferases absent. Aside from the N-terminal -100 sequences, the luciferin binding sequence is similar in size and homologous to the LBP in L. polyedrum, including the four domain structure. Bioluminescence originated independently many different times in evolution From a biological point of view bioluminescence is truly unusual by virtue of its evolutionary origins. As well illustrated by the four systems described, the genes, proteins and substrates involved are altogether different, as are the regulatory and functional aspects of the systems. This is most readily explained by assuming that the different systems arose independently,37 some being related to genes coding for proteins with completely different functions (coelenterates, fireflies), others with no known affinities (bacteria, dinoflagllates). How could this have been? Why is luminescence different in this respect from many, perhaps most, other genes, which have relationships to genes with similar functions in phylogenetically distant organisms? I propose that this is because the different bioluminescence systems actually have different functions, thus not subject to being carried out by the same proteins. For the systems reviewed, coelenterate flashes may startle predators and deter predation; fireflies communicate in courtship by flash patterns; bacteria provide light for various uses for hosts that culture them in different specialized organs, and dinoflagellates flash in response to mechanical stimulation by their predators, thus revealing their presence to their own predators (the burglar alarm theory). Some years ago I estimated that there may be up to 30 different bioluminescent systems.37 Researchers interested in luciferases, as well as mechanisms and functions of light emitting organisms, will thus still find a diversity of new systems for exploration with the prospect of many new and different applications. I hope that researchers will pursue such studies with vigor in the years to come.

12

Hastings JW

REFERENCES 1.

2. 3. 4.

5. 6. 7.

8. 9.

10.

II. 12. 13.

14. 15. 16. 17. 18.

Schram E, Stanley P. eds. International Symposium on Analytical Applications of Bioluminescence and Chemiluminescence. Westlake, CA: State Printing & Publishing, Inc. 1979: 696 pp. Hastings JW, Johnson C. Bioluminescence and chemiluminescence. Meth Enz. 2003;360:75-104. Wilson T, Hastings JW. Bioluminescence. Annu Rev Cell Devel BioI 1998;14:197-230. Shimomura 0, Johnson F, Saiga Y. Extraction, Purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 1962;59:223-39. Shimomura 0, Johnson F. Regeneration of the photoprotein aequorin. Nature 1975;256:236-8. Head J, Inouye S, Teranishi K, Shimomura 0. The crystal structure of the photoprotein aequorin at 2.3 angstrom resolution. Nature 2000;405:372-6. Liu Z-J, Vysotski E, Rose J, Lee J and Wang B. De novo structure determination of the photoprotein obelin at 1.7 angstrom resolution using single wavelength sulfur anomalous scattering data. Protein Sci 2000;9:2085-93. McElroy WD. The energy source for bioluminescence in an isolated system. Proc Natl Acad Sci 1947;342-5. Hastings JW, McElroy WD, Coulombre J. The effect of oxygen upon the immobilization reaction in firefly luminescence. J Cell Comp Physiol 1953;42:137-50. Case J, Strause L. Neurally controlled luminescent systems. In: Herring P. Ed Bioluminescence in Action. London: Academic Press, 1978:331-45. Timmins G, Robb F, Wilmot C, Jackson S, Swartz H. Firefly flashing is controlled by gating oxygen to light-emitting cells. J Exp BioI 2001 :2795-2801. Ghiradella H, Schmidt J. Fireflies at 100: A new look at flash control. Integrat Comp BioI 2004;44:202-12. Trimmer B, Aprille D, Dudzinski D, Lagace C, Lewis C, Michel T, Qazi S, Zayas R. Nitric oxide and the control of firefly flashing. Science 2001 ;292:2486-8. Aprille J, Lagace C, Modica-Napolitano J, Trimmer B. Role of nitric oxide and mitochondria in control of firefly flash. Integrat Comp BioI 2004;44:213-19. McElroy WD, Hastings JW. Initiation and control of firefly luminescence. In: Prosser C. Ed. Physiological Triggers. New York, NY:Ronald Press, 1956:80-4. Strehler B. Luminescence in cell-free extracts of luminous bacteria and its activation by DPN. J Am Chern Soc 1953;75:1264. Haas E. Bioluminescence from single bacterial cells exhibits no oscillation. Biophys J 1980; 31: 301-12. Hastings JW, Gibson Q. Intermediates in the bioluminescent oxidation of reduced flavin mononucleotide. J BioI Chern 1963;238:2537-54.


13

19. Hastings JW, Balny C, Le Peuch, C, Douzou P. Spectral properties of an oxygenated luciferase-tlavin intermediate isolated by low-temperature chromatography. Proc Natl Acad Sci 1973 ;70:3468-72. 20. Nealson K, Hastings JW. Luminescent bacterial endosymbionts in bioluminescent tunicates. In: Schwemmler W, Schenk J, eds. Endocytobiology, Berlin: Walter de Gruyter & Co, 1980: 461-6. 21. Mackie G, Bone Q. Luminescence and associated effector activity in Pyrosoma (Tunicata pyrosomida). Proc Roy Soc London Ser B 1978;202:483-95. 22. Nealson K, Platt T, Hastings JW. The cellular control of the synthesis and activity of the bacterial luminescent system. J Bact 1970;104:313-22. 23. Eberhard A, Burlingame A, Eberhard C, Kenyon G, Nealson K, Oppenheimer N. Structural identification of autoinducer of Photobacterium jischeri luciferase. Biochemistry 1981 ;20:2444-9. 24. Fuqua C, Winans S, Greenberg EP. Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu Rev MicrobioI1996;50:591-624. 25. Bassler B, Losick R. Bacterially speaking. Cell 2006;125:237-46. 26. Booth C, Nealson K Luminous bacteria from the ocean emit no light. Biophys J 1975;15:56a. 27. Herring P, Watson M. Milky seas: a bioluminescent puzzle. Marine Observer 1993;63:22-30. 28. Miller S, Haddock S, Elvidge C, Lee T. Detection of a bioluminescent milky sea from space. Proc Natl Acad Sci 2005;102:14181-4. 29. Nealson K, Hastings JW. Quorum sensing on a global scale: massive numbers of bioluminescent bacteria make milky seas. Appl Environ Microbiol 2006;72:2295-7. 30. Hastings JW. Bioluminescence, microbial. Encyl Microbiol 2000; 1:520-9. 31. Li L, Hong R. Hastings JW. Three functional luciferase domains in a single polypeptide chain. Proc Natl Acad Sci 1997;94:8954-8. 32. Li L, Liu L, Hong R, Robertson D, Hastings JW. N-terminal intramolecularly conserved histidines of three domains in Gonylaulax luciferase are responsible for loss of activity in the alkaline region. Biochemistry 2001 ;40: 1844-9. 33. Schultz W, Liu L, Cegielski M, Hastings JW. Crystal structure of a pHregulated luciferase catalyzing the bioluminescent oxidation of open tetrapyrrole. Proc Natl Acad Sci 2005;102:1378-83. 34. Liu L, Hastings JW. Two different domains of the luciferase gene in the heterotrophic dinotlagellate Noctiluca miliaris occur as two separate genes in photosynthetic species. Proc Nat! Acad Sci 2007; 104:696-70 1. 35. Liu L, Wilson T, Hastings JW. Molecular evolution of dinotlagellate luciferases, enzymes with three catalytic domains in a single polypeptide. Proc Natl Acad Sci 2004;101:16555-60.

14

36.

37.

Hastings JW

Liu L, Hastings JW. Novel and rapidly diverging intergenic sequences between tandem repeats of the luciferase genes in seven dinoflagellate species. J Phycol 2006; 42:96-103. Hastings JW. Biological diversity, chemical mechanisms and evolutionary origins of bioluminescent systems. J Mol Evol 1983; 19:309-21.

BIOLUMINESCENCE OF SHARKS, A CASE STUDY: ETMOPTERUS SPINAX 1M CLAES,I,2 1 MALLEFET 1,2 1Laboratory of Marine Biology, Catholic University of Louvain, 3 Place Croix du Sud, Kellner Building, B-1348 Louvain-la-Neuve, Belgium 2 Biodiversity Research Centre Email: [email protected]

INTRODUCTION Bioluminescence arose independently in a wide range of species, from bacteria to fishes, which are the only luminous vertebrates. Consequently, luminescent species demonstrate a great diversity in the structure, in the control, as well as in the function of their photogenic system.! Among luminous organisms, cartilaginous fishes are probably the least investigated group and incredibly few information is available concerning their bioluminescence.' Even if it has been once suggested for some sharks of the genius Somniosus and Megaschasma,J·4 symbiotic luminescence, common in teleosts, seems unlikely in chondrichtyes, This group contains however numerous self-luminous species, with at least one species of ray (Benthobatis moresbyi), and probably more than 50 different sharks (-13% of current shark species).,,6 Luminescent sharks belong to 2 squalid families, the Etmopteridae (lantern sharks) and the Dalatiidae (dwarf mesopelagic sharks), which evolved separately 90 million years ago, it is therefore possible that the bioluminescence arose 2 times independently in sharks: Until now, only information regarding the photogenic structures of these sharks is available in the literature. Dalatiidae have photophores constituted of a single photocyte (=photogenic cell) placed in a pigmented cup and covered by a lens formed by a group of small cells, while photogenic organs of Etmopteridae are more elaborated, composed of a pigmented sheath containing several photocytes, one of several lens cells, and an iris-like structure which has been suggested to allow a control of light emission:· 7 In both groups photocytes have granules supposed to contain the luminescent materia!.",8" Luminous sharks have also a specialized squamation allowing photophore accommodation in the skin.' The physiological control, the biochemistry, and the function of bioluminescence in these fishes remain totally unknown due to a lack of experimental data. Based on simple observation of the luminous pattern, authors have suggested that Dalatiidae would use their luminescence for counterillumination while Etmopteridae could in addition use it as a schooling aid. The aim of this work is to use morpho-physiological techniques to investigate the control and the function of bioluminescence in the velvet belly lantern shark Etmopterus spinax, a common etmopterid species. 15

16

Claes JM & Mallefet J

MATERIALS AND METHODS In February and December 2007, specimens of E. spinax (22.5-52.5 cm TL, total length) were collected in the Raunefjord, Norway. Light microscopy, fluorescence microscopy, and digital imaging analysis software were used to investigate bioluminescence of embryos and free-swimming specimens. We followed the elaboration of the luminous pattern and the development of photophores to determine when they become able to produce light. The density, the size of photophores, as well as the ventral surface occupied by photophores and luminous tissues were calculated for all the sharks. Peroxide-induced luminescence was also recorded from luminous tissues of 30 different sharks, grouped by 10 cm categories, via a luminometer Berthold FB12. Light response was standardized using the maximal intensity of light in megaquanta per second per square centimetre for each luminous zone (Lmax in Mq.s-l.cm-\ A theoretical visual model was equally performed using these data as well as photophore density to estimate maximum visual range of luminous zones and the depth at which these zones match the downwelling light in adult sharks (> 30 cm). A first screening of classical neurotransmitters and hormonal drugs was performed on adult sharks to investigate the control of luminescence in E. spinax. RESULTS AND DISCUSSION We have established the sequential visualization of 9 different luminous zones during E. spinax embryogenesis (Fig.l). We followed the organogenesis of photophores which is a well controlled process whose the last observable event is the apparition of fluorescent vesicles inside the photocytes. These vesicles are also observed in photophores of adult E. spinax and E. lucifer (Fig. 2A). At this moment photophores can emit light after peroxide application. Spontaneous luminescence in embryos confirms that they are able to luminesce before birth (Fig. 2B). During embryogenesis the ventral surface covered by photophore and luminous zone increase, and attains 38% and 82%, respectively. During this period, the diameter of photophores increases while their density decreases. Although the number of tested embryos is limited, it seems that light capabilities induced by peroxide application attained its maximum just before birth (Fig. 3). All these results strongly suggest camouflage by countershading in juveniles, more subject to predation than adults. The maximum theoretical visual ranges were obtained at 700 m when the shark is on its back, a behaviour frequently observed in aquarium. Even though these ranges were relatively weak «1.5 m) they could be an aid for species recognition, for mating, and for schooling in E. spinax. All the zones would match the downwelling light around 600 m, a depth at which adults of this species are found in the Mediterranean Sea which would therefore be also able to counterilluminate. 1o

Bioluminescence of Sharks

17

Fig. 1. Luminous pattern of E. spinax. Numbers correspond to appearance order of zones: I, rostral; 2, ventral; 3, caudal; 4, infra-caudal; 5, mandibular; 6, pectoral; 7, pelvic; 8, lateral; 9, infra-pelvic.

2. (A) Photocytes' fluorescent vesicles (arrow) present in the centre of a photophore of E. lucifer microcospy). Scale bar = 50 J.l.m. (B) Self glowing embryo (11 em TL) of E. spinax. Arrow indicates the insertion of the yolk sac. Scale bar = I em. s:>..

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Fig. 4. Drugs triggering light in E. spinax. SNP = Sodium nitroprusside (NO-donor). Mt 7 (except for Melatonin. Pt = prolactin. N prolactin for whieh N 3). Concentrations: KCI = 0.2 M, others 10-3 M. Control= H20, 0.35 M.

18

Claes JM & Mallefet J

Results of the pharmacological screening studies are shown in Fig. 4. Response to KCI as well as to GABA and 5HT strongly suggests a nervous control of luminescence in E. spinax. Moreover, high responses to melatonin and prolactin are in favour of an additional hormonal control of luminescence, which has never been highlighted in a fish before. NO-donor (SNP), could have a modulator role in control of luminescence of E. spinax as in Argyropelecus hemigymnus, a luminous teleost." ACKNOWLEDGMENTS Research is supported by a F.N.R.S. grant to JM Claes. J Mallefet is Research associate for the F.N.R.S. (Belgium). We would also like to thank EJ Warrant and DE Nilsson for their help in evaluating the luminescence visual range of E. spinax. Contribution to Biodiversity Research Centre. REFERENCES 1. Wilson T, Hastings JW. Bioluminescence. Annu Rev Cell Bioi 1998;14:197230. 2. Reif WE. Functions of scales and photophores in mesopelagic luminescent sharks. Act ZooI1985;66:111-8. 3. Berland B. Copepod Ommatokoita elongata (Grant) in the eyes of the Greenland shark - a possible cause of mutual dependence. Nature 1961;191:829-30. 4. Herring PJ. Tenuous evidence for the luminous mouthed shark. Nature 1985; 318:238. 5. Alcock A. A naturalist in Indian seas. London: Murray 1902:236. 6. Hubbs CL, Iwai T, Matsubara K. External and internal characters, horizontal and vertical distribution, luminescence, and food of the dwarf pelagic shark Euprotomicrus bispinatus. Bull Scripps Inst Oceanogr 1967;10:1-64. 7. Oshima H. Some observations on the luminous organs of fishes. J Coli Sci, Imp Univ, Tok 1911;27:1-25. 8. Seigel JA. Revision of the dalatiid shark genus Squaliolus: Anatomy, systematics, ecology. Copeia 1978;4:602-14. 9. Munk 0, Jorgensen JM. Putatively luminous tissue in the abdominal pouch of a male dalatiine shark, Euprotomicroides zantedeschia Hulley & Penrith, 1966. Act Zool 1988;69:247-51. 10. Coelho R, Figueiredo I, Bordalo P, Erzini K. Depth distribution of the velvet belly lantern shark, Etmopterus spinax, in southern Portugal. Abstract of the 2005 Annual ICES Conference, Aberdeen, UK. 11. Kronstrom J, Holmgren S, Baguet F, Salpietro L, Mallefet J. Nitric oxide in control of luminescence in hatchetfish Argyropelecus hemigymnus. J Exp BioI 2005;208:2951-61.

CHEMIEXCITATION MECHANISM FOR CYPRIDINA (VARGULA) AND AEQUOREA BIOLUMINESCENCE

T HIRANO, 1 H OHBA, 1 Y TAKAHASHI, 1 S MAKI, 1 S KOJIMA, 1 H lKEDA,2 H NIWA 1 JDept of Applied Physics and Chemistry, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan; 2Dept of Applied Chemistry, Grad School of Engineering, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan Email: [email protected]

INTRODUCTION Bioluminescence of the ostracod Cypridina (Vargula) and the jellyfish Aequorea produce light with the substrates, Cypridina luciferin and coelenterazine, which have the imidazo[1,2-a]pyrazin-3(7H)-one (imidazopyrazinone) ring. A remarkable characteristic of the bioluminescence is a high quantum yield of light production (:l..

I

l t-

8000

i

6000

Albumin amount (pg) 2.5 1.7 1.1 NOMORP_ MORP2 ; 8 ,

~

~

4000

•

•

2000 0 2

3

4

6

Albumin Amount (pg)

Fig. 4. Western blots for chicken egg albumin. Left: calibration curves. Right: acquired image from a membrane. In conclusion, the incorporation of an acy lation catalyst in enhancer/lum inol/oxidant HRP substrates is highly advantageous. Although the exact mechanism of action of acylation catalysts has not yet been fully investigated, it is clear from the results of this study that the very significant increase in light output observed in their presence can be translated into a corresponding improvement in sensitivity of chemiluminescent assays.

REFERENCES 1. 2.

3. 4.

5. 6. 7.

Kricka LJ, Voyta JC, Bronstein I. Chemiluminescent methods for detecting and quantitating enzyme activity. Methods EnzymoI2000;305:370-90. Easton PM, Simmonds AC, Rakishev A, Egorov AM, Candeias LP. Quantitative model of the enhancement of peroxidase-induced luminol luminescence. J Am Chern Soc 1996; 118:6619-24. Thorpe GHG, Kricka LJ, Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase. Methods EnzymoI1986;133:331-53. Kricka LJ, Cooper M, J i X, Synthesis and characterization of 4-iodopheny 1boronic acid: a new enhancer for the horseradish peroxidase-catalyzed chemiluminescent oxidation of luminol. Anal Biochem 1996;240: 119-25. Sugiyama M., Method of the chemiluminescence assays of the activity of peroxidase. US Patent No. 5,171,668 (1992). McCapra F. Chemical generation of excited states: the basis of chemiluminescence and bioluminescence. Methods EnzymoI2000;305:3-47. Hatle G, Steglich W, Vorbrilggen H. 4-Dialkylaminopyridines a highly active acylation catalyst. Angew Chern lnt Ed EngI1978;17:569-83.

EFFECT OF SURFACTANTS ON PEROXYOXALATE CHEMILUMINESCENCE REACTION K NAKASHIMA,I K ABE,I S NAKAMURA,I M WADA,I S HARADA,2 N KURODA 1 I Department

of Clinical Pharmacy, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan 2 Lumica Co., 651togaura, Koga-city, Fukuoka 811-3136, Japan E-mail: [email protected]

INTRODUCTION The peroxyoxalate chemiluminescence (PO-CL) method has been widely utilized in pharmaceutical and biomedical analysis due to its high sensitivity and a use of simple instrumentations without a light source. I.2 In a PO-CL system, oxalates or oxamides react with H 20 2 in the presence of a fluorophore to produce a light emission. The reaction has been thought to follow a chemically initiated electron exchange luminescence (CIEEL) mechanism via an high energy intermediate (1,2dioxetane derivative, 1) which forms a charge complex with the co-existing fluorophore. The electron is transferred to the fluorophore which is raised to an excited state and the energy emitted as a light. It has also been reported that surfactant can enhance PO-CL intensity in aqueous conditions via improvement of the fluorescence yield. 3 In order to establish a detection method for a synthetic surfactant by PO-CL, the effects of surfactants on PO-CL were examined using the bis(2,4,6trichlorophenyl)oxalate (TCPO)/rhodamine B/H 2 0 2/imidazoie system.

o

0 II II Ar-O~C~C~O-Ar

Aryloxalates

1 + F _ .........--=0 ..

F*

OH 0 I II

0 II

0 II

0-0

0-0

+ Hz02 -='+' Ar-O~T~T or T~T 1,2-Dioxetane derivatives,

1lo~o H- F~J

-~~..

F* + 2CO 2

--F+ hv

F=fluorophore Fig. 1. Reaction scheme ofPO-CL 135

136

Nakashima K et at.

METHODS Surfactant samples. Fifteen surfactants (a-o) used in this study were commercially available and involved anion-, cation-, zwitterionic- and non-ionic types. Each of surfactant samples at 0.5 or 2 % (w/v) prepared in water or methanol was used. The surfactants examined in this study are listed in Table I. Procedure for CL measurement. Twenty five micro litter of surfactant solution in a test tube were evaporated under N2 gas. To the residue, 25 f!L of 0.5 mM rhodamine B in CH3CN, 5 mM imidazole-HN0 3 (pH 6.5) and 120 mM H 20 2 in CH 3CN were added and mixed for lOs. After adding 25 f!L of 0.25 m M TCPO in CH3CN, CL measurement was performed at room temperature for 120 s in a Luminescencer PSN AB-nOO (Atto Co., Tokyo, Japan).

Sample

a b c

d e f g

h

k I

m n

o

Table 1. Surfactant samples examined in this study Compound Type Alkylsulfate/triethanolamine Sodium poly( oxyethylenetridecylether) laurylsulfate Disodium poly(oxyethylene) laurylsulfosuccinate Anionic Sodium diocty Isulfosuccinate Sodium poly( oxyethylene laurylether)acetate Poly(oxyethylene laurylether)phosphoric acid Cetyl trimethylammonium chloride Cationic Benzalkonium chloride Lauryl dimethylamino acetic betaine Zwitterionic Lauryl dimethylamine oxide Mixture ofpoly(oxyalkylene) and poly(alkylether) Poly( oxyethylene )tridecylether Poly( oxyethylene)tridecylether aqueous solution Non-ionic Fatty acid diethanolamide from Palm Poly( oxyethylene)sorbitane monooleic acid ether

RESULTS The effects of surfactants on the PO-CL reaction were examined. The surfactant solutions were spiked into (I) TCPOlrhodamine BIH 20 2/imidazole-HN0 3 buffer system, (2) TCPO/rhodamine B/H 20 2 system and (3) TCPOIH 2 0z/imidazole-HN0 3 system, respectively. In system (I), the CL intensity of the blank sample was taken as 100. The effect of surfactant was shown as relative CL intensity (RCI) to the CL intensity without surfactant (Fig. 2). Surfactants quenched CL at a 2% concentration and their RCIs ranged from 0.6 to 93.5. One of the reasons for this may be notable change of pH that occurred on adding the surfactant. In contrast, in the system (2), RCIs spiked with several surfactants (B-E, H-L) at 0.5% and CL was enhanced compared to that of the blank (Fig. 3). The RCIs were in range 124-472. This result suggested that several surfactants might playa role as a

Effect oj SurJactants on Peroxyoxalate Chemiluminescence Reaction

137

catalyst. However, their CL intensities were lower than that of the positive control (RCI=700) spiked with 5 mM imidazole-HN0 3 (pH 6.5), and no CL intensity enhancement properties could be observed among the surfactants tested. In system (3), CL intensities were also enhanced by adding surfactants, which might be caused by the fluorescent impurities present in the surfactants. In conclusion, the TCPO/rhodamine BIH 20 2 system was suitable for detection of synthetic surfactants, and the proposed method will be applicable to detect natural detergents.

...... ....... ~

120 100

::i

~

80

...... .......

60

I-;

ro

40

...... U

20

I-;

..0

~

~

~

0 ~~TD, was only 8 x 10.4 and the rate was very slow (rate constant kTD = 1.2 X 10.5 S·I) (Fig. 3). The small value for kTD indicates that 1 underwent typical uncatalyzed TD in p-xylene, as reported for various analogs of dioxetane 1, in contrast to SPD. However, the chemiluminescence for the TD of 1 was observed at a longer wavelength than those for BID and SPD (Fig. 2). The emission of yellow light for the TD of 1 in p-xylene is presumably attributed to ESIPT (excited state intramolecular proton transfer) in which quinone methide 5 was produced as an emitter (Fig. 4). This idea was supported by the fact that methoxyphenyl-analog 6 of 1 underwent TD in p-xylene at 100°C to give weak violet light with Amax TD = 390 nm, c[>TD = 9.2 X 10.4 , and kTD = 4.3 X 10.4 S·1 (Fig. 4).

Solvent-Promoted Chemiluminescent Decomposition of Bicyclic Dioxetanes

141

Fig. 4. Thermal decomposition (TD) of 1 emitting light due to ESIPT and TD of 6

The effects of solvent on the thermal chemiluminescent decomposition of 1 described above are summarized as follows. First, SPD proceeded in an aprotic polar solvent to afford bright light with AmaxSPD = 493-498 nm similar to BID by aCT-induced decomposition mechanism. Second, 1 underwent uncatalyzed TD to give excited 5 which emitted yellow light with Amax TO = 536 nm due to ESIPT in nonpolar p-xylene. It was important to elucidate whether or not SPD is mechanistically different from BID. Thus, the kinetics of the SPD of 1 was investigated in NMP as a representative solvent. SPD should be a consecutive reaction, which consists of the reversible formation of dioxetane bearing an oxidophenyl anion 2 or its solvated species that undergo CT-induced decomposition to give light. However, the reaction proceeded following practically first-order kinetics at 60-100 dc. On the other hand, both AmaxSPD and qJ>PD changed scarcely with the SPD reaction temperature. These results suggested that the thermodynamic profile of the reaction could be simply but meaningfully analyzed in terms of pseudo-first order kinetics. Based on the thus-measured rate constants, 0 PD s, at 60-100 DC, the thermodynamic parameters for the SPD of 1 in NMP were estimated from the Arrhenius plots to be free energy of activation ~G~ = 99.3 kJ mor l, enthalpy of activation ~H~ = 82.4 kJ mor l, and entropy of activation M~ = -56.8 J Klmor'. BID using TBAF in an aprotic polar solvent has been known to proceed following pseudo-first order kinetics independent of the concentration of TBAF when a large excess of TBAF is used. Thus, rate constants J(l1D for 1 were measured using a large excess of TBAF in NMP at 35-55 dc. The Arrhenius plots revealed that the activation parameters for BID were ~G~ = 97.3 kJ mor', ~H+ = 99.8 kJ mor~, and M~ = 8.2 J Klmor'. If we compare these activation parameters with those for SPD, we see that the entropy term of SPD has a large negative value in contrast to that of the BID. These results reveal that SPD proceeded through a transition state with considerably less disorder than that for BID. This is presumably attributed to the participation of hydrogen-bonding between NMP molecule(s) and a phenolic OH of 1. Thus, an intermediary anionic dioxetane 7 was produced through hydrogen bonding as an ion pair with protonated NMP molecule(s) to cause SPD. A model scheme is

142

Tanimura M et al.

illustrated in 5. On the other hand, the intermediary anion 2 should be slightly solvated or naked as an extreme for BID in an aprotic polar solvent such as NMP and DMSO especially with the use ofTBAF.

Solvated 1

7 Ar=

5.

Solvent-promoted decomposition (SPD)

In conclusion, a variety of aprotic polar solvents were shown to promote the (SPD) of bicyclic dioxetane bearing 4-(benzothiazol-2-yl)-3-hydroxyphenyl moiety 1 to emit light as effectively as the in an aprotic polar solvent. SPD caused intramolecular CT-induced BID of chemiluminescence, similar to BID, though the SPD reaction proceeded through a with a large negative entropy of activation in contrast to BID. Furthermore, decomposition of dioxetane 1 was found to occur to light due to ESIPT in ACKNOWLEDGEMENTS The authors (NW and MM) gratefully acknowledge financial assistance provided Grants-in aid (No. 1550043, and No. 17550050) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. REFERENCES 1. Beck S, Koster H. Applications of dioxetane chemiluminescent probes to molecular biology. Anal Chem 2000; 45: 2258-70. 2. Matsumoto M. Advanced chemistry of dioxetane-based chemiluminescent substrates originating from bioluminescence. 1. Photochem. Photobiol. C: Photochem Rev 2004; 5: 27-53. 3. Matsumoto M, Akimoto T, Matsumoto Y, Watanabe N. Bicyclic dioxetanes a 4-(benzoazol-2-yl)-3-hydroxyphenyl moiety: chemiluminescence for base-induced decomposition in aprotic medium and in aqueous medium. Tetrahedron Lett 2005; 46: 6075-8. 4. Baumstark AL. The 1,2-dioxetane ring system: preparation, thermolysis, and insertion reactions. In: Frimer AA ed. Singlet O 2 Vol II. Boca Raton:CRC, 1985: 1-35.

SYNTHESIS AND CHARACTERIZATION OF NEAR-INFRARED CHEMILUMINESCENT PROBES K. TERANISHI Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan Email: [email protected]';p

INTRODUCTION Imidazopyrazinone compounds such as 2-methyl-6-phenylimidazo[1,2-a] and 2-methyl-6-( 4-methoxyphenyl)imidazo[1 ,2-a] pyrazin-3(7 H)-one (CLA) I pyrazin-3(7 H)-one (MCLA)2 have been widly used as chemiluminescent probes for detecting superoxide anions in biological systems (Fig. 1). These compounds presumably react with superoxide anions to form singlet-excited amides to generate amide compounds and light. I have recently developed Red-CLA (Fig. 1) that emits red light (emission A.max 610 nm) by reaction with superoxide anions. 3 Red-CLA emits light with the longest wavelength known for chemiluminescent probes. Detecting luminescence in the near-infrared region (700-900 nm) is advantageous as there is minimal absorption by other biomolecules. Thus, a near-infrared chemiluminescent probe should be important as a reporter molecule for many analytical applications. Herein, I present the synthesis and luminescence properties of chemiluminescent probes that emit the near-infrared light by reaction with superoxide anions.

°nr

d I'"

HlCO

&

1 1 N H

d

CHJ

•

NrN 9"

~

1

so,

~

H

02S"N~N~O H

MetA

1

0'T-i(CHJ N

r

N

N H

,4

0 Red-CLA

Figure 1. Chemical structures of MCLA and Red-CLA

METHODS General measurement of chemiluminescence intensities and spectra. To a mixture of20 mmollL Mops / 0.2 mollL KCl (pH 7.2, 0.1 mL), 0.3 mmollL hypoxanthine (0.1 mL), and probe solution was added xanthine oxidase (XOD) at 20°C. Luminescence intensity and spectra were measured for 1 min using a LumiFlSpectroCapture AB 1850 (Atto Corporation, Tokyo). RESULTS Synthesis. In my system the singlet-excitation energy is generated by the reaction between the MCLA moiety and superoxide anions, followed by transfer of the energy to the indocyanine moiety that in turn emits the near-infrared light via an resonance 143

144

Teranishi K

I MCLA moiety I o

h r N N

R'

---

R,i)" H

R~R 14®....::#~~,/.;'/ N

(6H3)4S0~

o

*1'

o£

N I

1....-::::

R'

trNH R'

N

' ) Energ y

R~R IgffilA,'lC"-:::'''--;:'

N

N

Id

I

(CH3)4S0~

~ndocyanjne moietyJ

+

Near-infrared light

Figure 2. Strategy for the near-infrared chemiluminescence energy transfer (CRET) mechanism (Fig. 2). As shown in Scheme 1, two chemiluminescent compounds 8 and 9 were successfully synthesized. Compounds 3 and 4 were prepared from compounds 1 and 2,4 respectively, followed by amide-coupling with amino compound 7 prepared from 5. 5 Chemical structures of compounds 8 and 9 were confirmed by IH NMR and LC/MS (ESI, positive mode), and their purity confirmed by reverse phase HPLC. The solubility of compound 9 was much higher than that of 8 in 20 mmollL Mops I 0.2 mollL KCI (pH 7.2), due to the additional sulfonate groups. Superoxide anions-induced chemiluminescence. To prove the utility of compounds 8 and 9 on the detection of superoxide anions, I performed experiments on the detection of superoxide anions using the hypoxanthine-xanthine oxidase system as a source of superoxide anions. MCLA (1 flmollL) exhibited superoxide anion-induced chemiluminescence peaks at around 460 nm in the luminescence spectrum (Fig. 3-A). In contrast, chemiluminescence spectra of compounds 8 and 9 (1 flmollL) revealed their luminescence maximum only at 785 nm (Fig. 3-B). The absence of chemiluminescence due to the MCLA moiety indicates that the superoxide anion-induced chemiluminescence of compounds 8 and 9 is generated from the indocyanine moiety. These results clearly indicate that the excitation-energy generated from the MCLA moiety is efficiently transferred to the indocyanine moiety. When instead of compounds 8 and 9 a mixture of MCLA (1 flmollL) and indocyanine compound 4 (1 flmol/L) was used in the test, the chemiluminescence spectrum showed "'-max 460 nm (Fig. 3-C), indicating the conjugation of the indocyanine molecule to MCLA functions for CRET. The luminescence intensity of 9 was about 4 times higher than that of 8. Probably, it is due to improvement of the quantum yield of the indocyanine moiety by the additional sulfate groups on the aromatic groupS.6 Compound 10, which consists of N-ethylisoluminol and indocyanine molecules, showed no luminescence under the same condition. The effect of concentration of compound 9 on the intensity of the superoxide anion-induced chemiluminescence using 0.015 unitlmL XOD is shown in Fig. 4-A; at

Synthesis and Characterization of Near-Infrared Chemiluminescent Probes

145

concentrations less than 5 ~mol/L the chemiluminescence intensity increased with increases in probe concentration. The relationship between the concentration of XOD and luminescence intensity with compound 9 (1 ~mol/L) are shown in Fig. 4-B. Because the light sensitivity of the spectrometer used was low, the weak light emission at low concentration ofXOD could not be detected. In this study I have developed a near-infrared chemiluminescenct probe 9 for the detection of superoxide anions. First, the present study proved that CRET with MCLA and indocyanine molecules is useful to generate near-infrared light. Second, the near-infrared chemiluminescent probe 9 is easily synthesized. FinaJly, the suitability of 9 for the detection of superoxide anions was demonstrated. o

O~OH

d

NyN

I jJ N

I '"

1: R=H 2: R=S03H Pyridine N,N-Oimethylformamide

20°C 40 min

~

H CO ,

H 5

2-Mercapto-2-thiazoline DMAP WSC Pyridine

20°C

88%

4h 44%

Jl

0

O~"Ll

~j(rN

1)~ H3CO

I 6

Ethyle~diamine Pyndine

3: R= H 4: R=S03H

DOC 20 min 100%

2-Mercapto-2-th"18zoline DMAP

20°C 3h 20%

o O~~NH'

l~i)'N

WSC Pyridine N,N-Dimethylformamide

H3

COU

~

H 7

H03S~CH3

H3C~S03H

~ty~~,M

~

(CH')4

~

S0e ,

O~

o

NH

H~~~t~ HN~ o 10

Scheme 1. Synthesis of near-infrared chemiluminescent probes 8 and 9

146

Teranishi K

>,

1400

5

1200

.5

1000

8

800

I

500 , - - - - - - - - - - ,

A

400

.3

200

400

E 350

300 250 ~ 200 c:: 150 .~ 100

5

8

~ 5°UlII~~~~1U 350

9s(

750

550

>-.

1400

.~

1200

.5 "~

'8

600

'§

B

C 450 .~

350

550

750

950

1000 800

'"'

600

.§"

400

j

200

350

550

750

950

Wavelength (om)

Wavelength (om)

Wavelength (om)

Figure 3. Luminescence spectra of MCLA (A), componds 8 and 9 (B), and a mixture of MCLA and compound 4 (C) 2

1600

~

1400

A

600 500

;;

f

~ 1200

"I

2 r-

1000

'§

B 400 300

~ 200

~

400

!l

~

200

"§

j

;!:!

oL-_ _ _ _ _ _~

o

JO

Concentartion of pm be 9 ()lM)

100

j 0.01

C-Oncentration ofXOD (unlts/mL)

Figure 4. Effect of the concentrations of compound 9 (A) and XOD (B) on the intensity of the superoxide-induced luminescence ACKNOWLEDGEMENT I thank Mr. Tsutomu Irie (Atto Corporation, Tokyo, Japan) for spectral measurements. REFERENCES 1. Goto T, Takagi T. Chemiluminescence of a Cypridina luciferin analogue, 2-methyl-6-phenyl-3,7-dihydroimidazo[1 ,2-a]pyrazin-3-one, in the presence of the xanthine-xanthine oxidase system. Bull Chern Soc Jpn 1980;53:833-4. 2. Minakami H, Arai H, Nakano M, Sugioka K, Suzuki S, Sotomatsu A. A new and suitable reconstructed system for NADPH-dependent microsomal lipid peroxidation. Biochem Biophys Res Commun 1988;153:973-8. 3. Teranishi K. Development of imidazopyrazinone red-chemiluminescent probes for detecting superoxide. Luminescence 2007;22:147-56. 4. Lin Y, Weissleder R, Tung C-H. Novel near-infrared cyanine fluorochromes: synthesis, properties, and bioconjugation. Bioconjugate Chern 2002; 13:605-10. 5. Teranishi K, Komoda A, Hisamatsu M, Yamada T. Synthesis and enhanced chemiluminescence of new cyclomaltooligosaccharide (cyclodextrin)-bound 6-phenylimidazo[I,2-a]pyrazin-3(7H)-one. Carbohydr Res 1998;306: 177-87. 6. Mujumdar R B, Ernst L A, Mujumdar S R, Lewis C J, Waggoner A S. Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjugate Chern 1993;4:105-11.

GENERATION OF HIGH-ENERGY CHEMILUMINOPHORES IN AMBIENT LIGHT

YuB TSAPLEV, RF VASIL'EV, AV TROFIMOV Emanuel Institute 0/ Biochemical Physics, Russian Academy o/Sciences, ul. Kosygina 4, Moscow 119334, Russian Federation E-mail: [email protected]@[email protected]

INTRODUCTION Chemiluminescence is a prominent example of converting chemical energy into light and, thus, may be considered as a reversal of photochemistry. Such a symmetry is illustrated by Scheme I, in which Rg denotes the

Scheme 1

Photochemistry:

Rg + hV ~ Rg*

~

Chemiluminescence:

Pr + hV

Eo-

Pr*

Eo-

Int*

~

Int ~ ...

~

Pr

y,""'...... "round-Slale High-Energy Species

Int*

Eo-

Int Eo-

••• Eo-

Rg

reagent molecules, Pr represents the reaction products and Int refers to intermediate species. Apart from "straight-line" photo- (Rg + hv -+ ... -+ Pr) and chemiexcitation (Rg -+ ... -+ Pr + hv) processes, both photochemical and chemiluminescent reactions may involve energy-accumulation channels, in which relatively persistent molecular "depositories of light" (X) are formed. The said X species exist in the ground state, but possess enhanced internal energy, and the excess may be released in the form of a photon emission upon the relevant external influence on the reaction system (e.g., addition of any triggering reagent or heating). These peculiar "light accumulators" may be generated in different ways, in particular, under irradiation of the reaction mixture. Even simple exposure of the chemical system to the ambient light may convert the reagents into high-energy chemiluminophores. These transform upon subsequent addition of pertinent triggers and are exothermal enough to generate electronically excited products, the emitters of chemiluminescence. We term the latter phenomenon as [ight-f.reated f.hemiluminescence (LCCL/ and below discuss our recent findings in this context along with related issues. MATERIALS AND METHODS The chemuluminescence measurements were performed with a photon-counting 147

148

Tsapiev Yu B et ai.

instrument (Hamamatsu photosensor modules H7467, H7360-02 and H7360-03 supplied with the RS-232C interface). Preparation of the samples has been described before. 2 The quantum-chemical calculations were made using the semiempirical PM3 method. RESULTS AND DISCUSSION The material presented herein encompasses the energy accumulation in organic processes through the formation of high-energy chemiluminophores (X in Scheme I), the latter species are furnished by intermediate dioxetanes and pertinent photo isomers and photodimers. Intermediate dioxetane species. The chemiluminescence upon oxidation of unsaturated organic substrates (most prominently, lipids) provides a relevant chemical model for the generation of bioluminescence in living tissues and, thus, merits particular attention. An important feature revealed by our studies is the occurrence of at least two excited-state generation processes. The first chemiexcitation process constitutes the classical free-radical chemiluminescence channel, while the second one is of molecular origin. Addition of antioxidants to the reaction mixture immediately suppresses the radical part of the overall light emission. Then, the remaining molecular component decreases according to an exponential law (the rate constants are of the order of 10.4 S·I). Estimation of the emitter characteristics (lifetimes, quantum yields and radiation constants) leads to values typical for the phosphorescence of carbonyl compounds. Such behavior shows that the observed molecular chemiluminescence is excited in chemical transformations of oxidation products, which are persistent enough to accumulate in the course of reaction. Possible sources of this molecular contribution to the chemiluminescence of unsaturated substrates are dioxetane intermediates. In nature, these high-energy species may be formed through diverse (e.g., enzymatic or photochemical) mechanisms. 3 However, presently we favor the mechanism, which involves cyclization of alkylperoxy radicals. This may be illustrated by considering the oxidation of 1,1,2-trimethylethylene, H 3CCH=C(CH3)2, as a model alkene substrate. In this case, under a constant reaction-initiation rate, several types of radicals exist in their stationary concentrations. These are carbon-centered radicals, the products of addition of initiating radical r' to the double bond, or of the H-atom abstraction by this radical from methyl groups, for instance: H3CCHrC"(CH3 )2, H 3CC"HCr(CH3)2, H2C'CH=C(CH3)2, H3CCH=C(CH3)C"H 2. The fast addition of O2 transforms C radicals into a number of peroxy-radical species: H3CCHrC(CH3)200', H3CC(00")HCr(CH3)2, H2 C(00")CH=C(CH3)2, H3CCH=C(CH3)CH 200·. As a result, various reactions of these radicals occur. Combination of the ROO' radicals is exothermal, but it gives products that are not luminophoric, i.e., it is not a chemiluminescent process. Disproportionation of ROO' gives the emitters of "classic" free-radical chemiluminescence. However, the chain =C-C-O-O' in radicals H 2C(00,)CH=C(CH3)2 and H3CCH=C(CH3)CHzOO' is flexible enough (the bonds are single) for overlapping the unpaired electron with the Jt MO of the double bond, and

Generation of High-Energy Chemiluminophores in Ambient Light

149

so, for forming a four-membered dioxetane cycle. The latter cycle stores large amounts of chemical energy, that may be easily transformed into the electronic excitation upon decomposition of the dioxetane. Intermediate photoisomeric species. Structural photoisomerisation provides another way for the formation of high-energy chemiluminophoric species, which may be noticeable even under low-intensity irradiation of reactants. Upon subsequent addition of certain triggering agents to such photogenerated chemiluminophores, the electronically excited chemiluminescence emitters are formed. This recently observed light-created chemiluminescence (LCCL) phenomenon may be exemplified by the base-triggered chemiluminescent reaction of salicylaldehyde hydrazone pre-irradiated by visible light (400-500 nm) with the intensity of the order of 1 mW/m 2 . The illumination results in isomerisation of the non-chemiluminescent benzenoid reagent form (Rg) into the quinonoid structure (Rg'), the latter is prone to excited-state generation upon reaction with the base (Scheme 2). Scheme 2

The interaction of both Rg and Rg' isomers with bases is exothermic; however, the energy released in the reaction of Rg with HO' is not sufficient for the electronic excitation of the hydrazone anion, the chemiluminescence emitter. The PM3 computations reveal that the enthalpy of the Rg' formation exceeds that of the Rg species by 130 kllmol, which covers the energy deficiency for the excitation of the light emitter. Thus, the Rg' species constitutes the putative high-energy chemiluminophore, which is readily generated in the ambient light. Intermediate photodimeric species. The following example par excellence for the LCCL manifestation is provided by the base-initiated chemiluminescence of preirradiated 9-anthrone? Addition of bases to the 9-anthrone solutions stored in the dark does not trigger a chemiluminescence process. However, exposure of the reactant to ambient light and subsequent addition of base results in excited-state generation as manifested by the chemiluminescence emission. The products of the 9-anthrone photolysis in dioxane were separated by high-performance thin-layer chromatography.

150

Tsaplev Yu B et al.

The analysis of chromatograms has disclosed that the maximum chemiluminescent activity corresponds to the spot of dianthrol (DA, with retention index Rf = 0.25) rather than to that of peroxide species (Rf '" 0.37), whose intervention into the anthrone photochemiluminescence process was suggested in the literature. Thus, DA, which exists in two isomeric forms (Scheme 3), furnishes the relevant high-energy

Scheme 3

*==~hV

~~ H

hj

H

H

KH

EH DA+HO- __ DA-+HzO DA - + HO- - - (E-)* + E- + H 20 (E-)*-- E-+hv

"Head-ta-Head"

"Head-to-TalP'

r

DA

HO-

hv

chemiluminophore species; its base-triggered decomposition results in the electronically excited anthrol anions, K (the chemiluminescence spectrum matches the fluorescence emission of K). It is noteworthy that the DA species may be formed not only under in vitro conditions, but also in nature (the pertinent example is furnished by DA found in st. John's wort [Hypericum perjoratum), as it has been established in the present chemiluminescence studies. At a first glance, the ability of ambient laboratory light to create chemiluminescence seems astonishing. However, under ordinary illumination (only a few mW/m\ more 13 than 10 photons pass through an area of 1 cm z per second, which is 10 9 times greater than the detection limit of laboratory chemiluminometers. Clearly, this gap can be filled by numerous chemical systems, for which the absorption efficiencies mUltiplied by the photochemical transformation and the chemiluminescence yields are no less 9 than 10- . This implies that the list of known chemiluminescent reactions needs to be "revisited" to disclose the systems, which may be particularly influenced by the ambient illumination while preparing the reaction mixtures.

ACKNOWLEDGEMENTS The work was supported by the Russian Foundation of Basic Research, the Russian Academy of Sciences and the Russian Science Support Foundation.

REFERENCES 1. 2. 3.

Vasil'ev RF, Tsaplev YuB. Light-created chemiluminescence. Russ Chern Rev 2006; 75:989-1002. Tsaplev YuB, Vasil'ev RF. The nature of chemiluminescence of the photolyzed anthrone solutions. Russ J Phys Chern 2006;80:795-8. Adam W, Trofimov AV. Contemporary trends in dioxetane chemistry. In: Rappoport Z, ed. The Chemistry of Peroxides (Patai Series). Chichester: Wiley, 2006: 1171-1209.

ALKALINE METAL ION ENHANCED CHEMILUMINESCENCE OF BICYCLIC DIOXETANES BEARING A 3-HYDROXYNAPHTHALEN-2-YL GROUP N WATANABE, F KAKUNO, N HOSHIYA, HK IJUIN, M MATSUMOTO Dept a/Chemistry, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan

INTRODUCTION A dioxetane substituted with an oxyaryl group forms, after deprotonation or deprotection, an unstable dioxetane bearing an oxidoaryl anion which acts as electron donor for the intramolecular charge-transfer (CT) induced chemiluminescence.'·2 The position of the oxido anion relative to the attachment point of the dioxetane on the aryl group influences significantly the chemiluminescence properties. For instance, dioxetanes 1 substituted with a naphthalen-2-yl group bearing an oxido anion at the 4-, 5-, or 7-position ("odd' pattern) give off light more effectively than dioxetane analogs with an oxido anion at the 3-, 6-, or 8-position ("even" pattern) (Fig. 1).3 The phenomenon recognized as the "odd/even" relationship'" has been observed also for dioxetanes substituted with various oxyaryl groups such as oxyphenyl, oxybenzo[b]furan-2-yl, or oxybenzo[b]thiophen-2-yl group? These facts suggest a simple but important question as to whether chemiluminescence with high efficiency can be realized even from a dioxetane bearing an oxyaryl with "even" pattern by modifying the reaction conditions. In the course of our investigation of CTICL-active dioxetanes, we found a rather simple clue to answer the question.

1

: even odd

Fig. 1. Charge-transfer-induced chemiluminescent decomposition of dioxetanes bearing an oxidonaphthalen-2-yl group

RESULTS AND DISCUSSION Bicyclic dioxetane bearing a 3-hydroxynaphthalen-2-yl group 2 has very recently been found to possess a structure in which the 3-hydroxy group forms intramolecular hydrogen bonding with oxygen atom of the dihydrofuran ring, as illustrated in Fig. 2.5 This finding stimulated us to investigate decomposition of 2 in the presence of a metal 151

152

Watanabe Net at.

ion which should coordinate with both 3-oxidonaphthyl anion and oxygen of the dihydrofuran ring to cause expectedly unique chemiluminescence.

3-M

4-M

Fig. 2. Base-induced chemiluminescent decomposition of a dioxetane 2 bearing a 3-hydroxynaphthalen-2-yl group

When a solution of dioxetane 2 in THF (1.0 x 10-4 M, 1 mL) was added to a solution of tetrabutylammonium fluoride (TBAF) in THF (1.0 x 10-2 M, 2 mL) at 25 DC, 2 decomposed through unstable anionic dioxetane 3 to give keto ester 4 with an accompanying emission of orange light with maximum wavelength Amax CTICL = 552 nm and decomposition rate kCTICL = 1.7 X 10-2 S-I. However, chemiluminescence efficiency cpCTICL was quite poor (2.4 x 10-5) and a typical one for "even" pattern, as expected. On the other hand, when 2 was similarly treated with a large excess of sodium t-butoxide in place of TBAF, 2 decomposed to give intense light with TICL Ama/ = 535 nm, chemiluminescence efficiency cpCTICL = 1.5 X 10-3, and decomposition rate kCTICL = 4.0 X 10-2 S-I. This cpCTICL value was more than 60 times of that in TBAF / THF and was comparable to that for naphthyl-substituted dioxetane with "odd' pattern. 3 Comparing AmaxCTICL and kCTICL for sodium t-butoxide in THF with those in TBAF / THF, we see that AmaxCTICL shifted to blue considerably and kCTICL increased. On treatment with potassium t-butoxide instead of sodium t-butoxide in THF, dioxetane 2 decomposed more rapidly (kCTICL = 0.11 S-I) to emit a flash of light with Amax CTICL = 539 nm. The chemiluminescence spectrum is illustrated in Fig. 3 together with chemiluminescence spectra for TBAF system and for the other alkaline metal t-butoxide (vide infra). cpCTICL decreased to 4.5 x 10-4 though it was yet effective 10 times more than that in the TBAF system. Finally, we carried out the decomposition of 2 with lithium t-butoxide in THF. Lithium ion in general coordinates strongly to oxidoaryl anion in an organic solvent because of the smallest ion radius, thus, we did

Alkaline Metal Ion Enhanced Chemiluminescence of Bicyclic Dioxetanes

153

not expected so much that lithium t-butoxide functioned as a base for CTICL. In fact, dioxetane analog bearing a 3-hydroxyphenyl 4 decomposed sluggishly with lithium t-butoxide even in DMSO. However, the decomposition of 2 was effectively induced by lithium t-butoxide in THF at 45°C to give intense light with Amax CTlCL = 535 nm and kCTICL = 4.9 X 10-3 S-I. Astonishingly, the q,CTICL was enhanced more than 200 times of that in TBAF / THF.

450

500

550 600 wavelength / nm

650

700

Fig. 3. Chemiluminescence of dioxetane 2 in THF Effects of alkaline metal ion on the CTICL of dioxetane 2 in THF observed here are summarized as follows. First, all chemiluminescence spectra in the presence of alkaline metal ion shifted 12-17 nm to blue compared to the case in TBAF. Second, the decomposition rate constant kCTICL increased in the order t-BuOK < t-BuONa < t-BuOLi. This order coincides with the order of ion radius decreasing from K+ to Li+. This suggests strongly that alkaline ion coordinates to oxidonaphthyl ion of 3 and Lt does the most tightly among these three alkaline metal ions. Thus, CTICL of 2 proceeded most likely through anionic dioxetane forming chelate 3-M to give chelated keto ester 4-M with the accompanying emission of light. Third, q,CTICL increased in the order TBAF « t-BuOK « t-BuONa «t-BuOLi. This result suggests that conformation of oxidonaphthyl group as an electron donor relative to dioxetane ring affects likely efficiency of singlet-chemiexciation process. Next, we investigated CTICL of a dioxetane bearing a 7-hydroxynaphthalen-2-yl group 5 induced by alkaline metal t-butoxide in THF. Dioxetane 5 belongs to "odd' pattern and is known to emit light effectively, though it cannot form a chelate with a metal ion. When 5 was treated with TBAF in THF, 5 decomposed to give off light effectively with Amax CTICL = 580 nm, chemiluminescence efficiency q,CTICL = 2.3 X 10-2 , and decomposition rate 3 kCTICL = 1.7 X 10- S-I. On the other hand, although 5 decomposed to give very weak light with Ama/TlCL = 564 nm, neither q,CTICL nor kCTICL could not be estimated.

154

Watanabe Net at.

~

o-& 0-0

HO

t-Bu

0

4

HO 5

Fig. 4. BicycIic dioxetanes bearing a 3-hydroxyphenyl4 or 7-hydroxynaphthalen-2-yl group 5

In conclusion, we have discovered that chelation with alkaline metal ion, especially Li+, enhances markedly chemiluminescence efficiency of CTICL for an "even" pattern" hydroxyaryl-substituted dioxetane, i.e., bicycIic dioxetane bearing a 3-hydroxynaphthalene-2-yl group 1 in THF. ACKNOWLEDGEMENTS The authors (MM and NW) gratefully acknowledge financial assistance provided by Grants-in aid (No. 1550043, and No. 17550050) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

REFERENCES 1. Beck S, Koster H. Applications of dioxetane chemiluminescent probes to molecular biology. Anal Chern 2000;45 :2258-70. 2. Matsumoto M. Advanced chemistry of dioxetane-based chemiluminescent substrates originating from bioluminescence. J Photochem Photobiol C Photochem Rev 2004;5:27-53. 3. Hoshiya N, Fukuda N, Maeda H, Watanabe N, Matsumoto M. Synthesis and fluoride-induced chemiluminescent decomposition of bicyclic dioxetanes substituted with a 2-hydroxynaphthyl group. Tetrahedron 2006;62:5808-20. 4. Edwards B, Sparks A, Voyta JC, Bronstein I. Unusual luminescent properties of odd- and even-substituted naphthyl-derivatized dioxetanes. J Biolumin Chemilumin 1990;5:1-4. 5. Hoshiya N, Watanabe N, Ijuin HK, Matsumoto M. Effect of intramolecular hydrogen bonding on thermolysis of dioxetane: unusual instability of bicyclic dioxetanes bearing a hydroxynaphthyl group with vicinal substitution pattern. Chern Lett 2007;36:516-7.

PART 4 APPLIED CHEMILUMINESCENCE


ANALYTICAL CHALLENGES FOR LUMINESCENCE-BASED POINT-OFCARE TESTING DEVICES IN BIOMEDICAL DIAGNOSTICS ARODA,' M GUARDIGLI,' M MIRASOLI,' E MICHELINI,' LS DOLCI,' M MUSIANI 2 JDept of Pharmaceutical Sciences, University of Bologna, Bologna 40126, Italy 2 Dept of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Bologna 40138, Italy, Email: [email protected]

INTRODUCTION Point-of-care tests (POCT) are clinical laboratory tests performed in a self-contained analytical platform that can be operated by non-laboratory healthcare professionals. POCT allow quicker results than centralized laboratory services due to reduced assay time and because the analysis is performed at the site where the sample is drawn or clinical care must be delivered. The recent technology improvements with the possibility to achieve high detectability (down to the micro- and nanomolar levels) for a wide number of analytes allows one to envisage a new generation of diagnostic POCT devices for the detection in biological fluids (blood, serum, saliva) of a panel of biomarkers of a given disease. A POCT device should combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. Microfluidics-based integrated devices relying on biospecific recognition reactions combined with ultrasensitive luminescence detection techniques such as bio-chemiluminescence (BLlCL), electrochemiluminescence (ECL) and photoluminescence, represent one of the most promising options. Indeed, this experimental approach could gain enough sensitivity and selectivity to perform the simultaneous detection of different target analytes present in very low concentrations in complex samples. BIOSPECIFIC RECOGNITION SYSTEMS Different biospecific recognition elements, such as enzymes, antibodies, nucleic acids and receptors, could be used for mUltiplexed assays in a POCT device. Potentially, the device could perform immunoassays (e.g., for hormones, proteins, viruses, bacteria) or nucleic acid hybridization reactions (e.g., for evaluating gene expression and detecting gene mutations, single nucleotide polymorphisms, DNA and RNA sequences). In most cases, the highest detectability is achieved with non-competitive analytical formats, in which the target analyte is captured by a specific probe and revealed by a second labelled probe. Capture probes can be immobilized directly on the surface of the sensor (e.g., on glass, plastic, transparent conductive materials) or, to increase immobilization efficiency, on nanostructured supports (e.g., nanospheres, nanowires, nanofibers). In addition, single or coupled enzyme reactions could be used for the quantification of enzyme activities or enzyme substrates. 157

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RodaA etal.

LUMINESCENCE DETECTION Luminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in ultra-small sample volumes (down to nanoliters) and accurate signal localization and quantification. Detectability can be further increased by passive luminescence signal amplification, i.e., by labeling the probes with nanostructured supports (e.g., nanospheres, molecular dendrimers, biotin-streptavidin scaffolds) bearing a large number of luminescent species. Moreover, various luminescent detection techniques and/or luminescent labels can be combined in a single device to obtain separate quantification of several analytes by spectral- or time-resolved signal acquisition or by independent signal trigger. We have developed luminescent labels with improved characteristics suitable for miniaturized analytical devices and multiplexed assays. For example, we reported a strategy for obtaining BODIPY -based fluorophores for multiplexed assays consisting in the introduction of a common light absorption moiety in the dye molecules. The resulting fluorescent species maintain the emission bands of the parent dyes and can be excited simultaneously at the same wavelength, thus only one excitation source is required.' Time-resolved luminescence is a promising detection technique because it is not affected by sample autofluorescence, but the available labels are often characterized by low quantum yields. We recently described a new chelating ligand that gave water-stable and strongly luminescent lanthanide complexes and demonstrated its suitability for bioanalytical time-resolved luminescence microscopy.' Concerning BL, one of the main limitations of the use of luciferases as labels is their poor stability. To overcome this problem and to facilitate multiplexing, we have obtained new luciferases by cloning the luciferase gene from other species (e.g., Luciola italica), or by producing new mutants characterized by higher thermostability and red-shifted emission spectra.' In addition, it is also desirable to improve the performance of the currently available luminescent labels. We have recently demonstrated that the incorporation of an acylation catalyst in an enhancer/luminol/oxidant CL substrate for horseradish peroxidase resulted in a very significant increase in light output, thus in an improvement of CL assays sensitivity.' In the context of ECL detection, we recently designed a transparent electrochemical cell for ECL imaging (described below) that is also suitable for BLlCL measurements and can thus be employed as a luminescence reading device of general applicability. ANALYTICAL DEVICES The analytical challenge is to achieve multiplexed analysis in the same device, i.e., to perform the biospecific recognition reactions and to reveal them independently by luminescence detection. Multiplexing could be achieved by "position encoding" and "reaction encoding" strategies. In "position encoding" the recognition reactions corresponding to the different analytes take place in different positions within the device and the resulting luminescent signals are detected by a luminescence imaging technique. In "reaction encoding" the various recognition reaction are associated with different luminescence reporting processes and/or labels and the detection rely on the independent trigger of the luminescence processes (e.g., addition of specific BLlCL

,wl,,'u'nl

Challenges Jor Luminescence-Based Point-oj-Care Testing Devices

159

substrates) or on the spectral resolution of light emission. A possible layout of the reading device is shown in Fig. I. The device consists of a on which the various biospecific recognition molecules are immobilized within an array. The analytes are captured by the biospecific rf>C"r.crrIlT,r.n molecules and revealed by a set of bioprobes labelled with luminescent species. The is in contact with an imaging light sensor (CCD, CMOS photosensor array, "avalanche" diodes array) able to localize and quantify the luminescent "Contact" detection, in which the signal is produced on (or very close to) the detection allows it to achieve a much higher optical efficiency than that of conventional camera-based imaging systems. The limitations on the resolution of contact detection can be overcome by a suitable design of the sensor photodiodes can be sized with respect to a given application) and applying suitable cross-talk reduction algorithms. The device will also include a fluidic system for and reagents.

Fluidics system "

Transparent support with biospccific ,wobes immobilized in defined ""cas ........... Imaging light detector (CCD _ or """"y of diodes)

A device for luminescence multiplexed assays based on "position

""",vu ... !;

the device will be suitable for BLlCL measurements. luminescence technique, it will also contain the other elements necessary for generation and measurement, for example excitation sources laser and filters for spectral selection for fluorescence detection or addressable miniaturized electrodes for ECL detection. We have a prototype device to perform ECL imaging detection of biospecific labelled with ECL-active species, such as Ru 2+ complexes. The device is conductive fluorine-doped tin oxide (FTO)-coated which was chemically etched to obtain a three-electrodes electrochemical cell with a silver quasi-reference electrode. Experiments conducted on model systems (micron-sized beads) showed that the generation of the ECL could be switched on/off during the measurement process and that the emitted could be detected with a micrometer-scale resolution by luminescence The characteristics of such device made it suitable also for BLlCL measurements, in which the luminescent signal is triggered by addition of a BLlCL substrate. MICROFLUIDICS A POCT device should be equipped with pumps, valves, sensors,

reservoirs

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Roda A et al.

and all other components required for its operation, comprising an integrated control system for the management of the whole analytical procedure and the acquisition and evaluation of the results. Microfluidics, which is fundamental for POCT development, requires the fabrication of highly integrated devices for performing several different functions on the same substrate chip. In a manner similar to that for microelectronics, the components of a POCT device could be easily produced by microfabrication techniques, which are relatively inexpensive and amenable both to highly elaborate devices and also to mass production. An integrated module for on-line sample pre-analytical treatment and/or clean-up could be also included in the device. With this respect, field-flow fractionation (FFF) techniques, which can separate analytes based on their morphological characteristics (size, shape and superficial properties) can be exploited to develop pre-analytical modules for cells or macromolecules (e.g., proteins, protein complexes or adducts) fractionation, thus providing a selectively enriched fraction for the analysis.

CONCLUSIONS The recent advancements in molecular biology, luminescent labels and detection systems, and micro fabrication techniques would allow the development of POCT devices for multiplexed assays. Biospecific recognition reactions combined with luminescence detection represent the most promising approach due to their high sensitivity, wide range of detectable analytes and possibility of miniaturization. New POCT services would provide numerous advantages and significant patient benefits, but the quality of results could be affected by inadequate training or inappropriate use. All analytical tests, whether performed in the laboratory or not, can run into problems but laboratory staff have more knowledge and experience to recognize and address these situations. Therefore, it is important to get the balance right and maximize the benefits that this exciting technology offers while ensuring that the quality of results and patient safety is not compromised. REFERENCES 1. Weibel N, Charbonniere LJ, Guardigli M, Roda A, Ziessel R. Pyrromethene dialkynyl borane complexes for "cascatelle" energy transfer and protein labeling. Angew Chern Int Ed EngI2005;44:3694-8. 2. Ulrich G, Goze C, Guardigli M, Roda A, Ziessel R. Engineering of highly luminescent lanthanide tags suitable for protein labeling and time-resolved luminescence imaging. J Am Chern Soc 2004;126:4888-96. 3. Branchini BR, Southworth TL, De Angelis J, Michelini E, Roda A. Isolated luciferase gene of L. italica. US Patent 102816-100,2006. 4. Branchini BR, Southworth TL, Khattak NF, Michelini E, Roda A. Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications. Anal Biochem 2005;345:140-8. 5. Marzocchi E, Grilli S, Della Ciana L, Mirasoli M, Prodi L, Roda A. Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalysts. Anal Biochem, accepted for publication.

MOLECULAR IMPRINTED POLYMER· BASED CHEMILUMINESCENCE SENSORS ZHUJUN ZHANG Department of Chemistry, Shaanxi Normal University, Xi' an 710062, China; Email: zzj18 @hotmail.com

INTRODUCTION

Chemiluminescence (CL) is a light emission from a chemical reaction.

CL

analysis has advantages of high sensitivity, wide linear range and simple instrumentation. It has become an attractive analytical tool in biological and chemical analysis. However, CL analysis also has some disadvantages. The main disadvantage is that it has very poor selectivity, which has limited its application. How to solve this problem? One approach is to combine it with a high performance separation method such as high performance liquid chromatography or capillary electrofluorescence. Another approach is to combine it with a specific molecular recognition method such as an enzyme, antibody or receptor, nucleic acid, aptamer, DNA or RNA and molecular imprinted polymer (MIP).l.g Molecular imprinting belongs to the category of host-guest chemistry in super-molecule

chemistry.

It

is

an

interdisciplinary

subject,

involving

macromolecular chemistry, biochemistry, and material chemistry. Molecular imprinting polymerization is a technique of making molecular recognition sites for an analyte molecule in a synthetic polymeric substrate. MIP is synthesized by a polymerization reaction of a mixed solution containing functional monomers, crosslinker and a target molecule in an organic solvent. A complex is formed between the target molecule and the functional monomer through polar interactions. Subsequent polymerization with the cross-linker fixes the positions of the polar groups of the functional monomer. After removing the target molecules, the molecular recognition sites will be made with shape, size and function 161

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complementary to the analyte, and they can rebind the analyte molecules in preference to other closely related structures. MIP is stable and resistant to a wide range of pH, humidity and temperature, so that a sensor modified with a MIP is easily stored and prepared. It also has a long lifetime. Potential target molecules include carbohydrates, amino acids, nucleic acids, alkaloids, drugs, vitamins, proteins, antibodies and bacteria. The target molecule may be covalently or noncovalentIy linked to a functional monomer. Usually, with an increase in binding sites between target molecule and functional monomer, the specific molecular recognition will be increased, but the reversibility of sensor will be decreased. Under certain conditions, we are only interested in specific molecular recognition. For example, we prepared a MIP as an alternative to an antibody in an immunoassay. In this application, the reversibility of MIP is not important. However in other situations, we have to achieve a balance between the specific recognition and reversibility for sensor design. Molecular imprinted polymer-based CL imaging sensors.

When using MIP

as the recognition element in a CL sensor, the selectivity of the CL method can be greatly improved and the interference of some species commonly present in samples can be eliminated.

So, an MIP-based CL sensor can be used directly to

determine the analyte in real samples. Along with the development of CL analysis, significant progress has been made in techniques to measure CL. The CL signal may be detected by both conventional photomultiplier (PMT)-based luminometers and high resolution imaging detectors. Light emission down to the single-photon level can be localized and quantified by CL imaging techniques. Imaging techniques are advantageously used when the spatial distribution of the luminescence signal represents crucial analytical information. MIP combined with CL imaging assay has the advantages of simplicity, high selectivity, high sensitivity and high throughput. Due to the beneficial health effects of trans-resveratrol in grape wine, many methods have been developed for its detection and quantification. 1 In our laboratory, a MIP-based CL imaging sensor for determination of trans-resveratrol in grape wine has been developed. The MIP of trans-resveratrol was prepared by a precipitation polymerization using trans-resveratrol as the target molecule,

Molecular Imprinted Polymer-Based Chemiluminescence Sensors

acid

(MAA)

as

the

functional

monomer,

163

ethylene

dimethacrylate (EGDMA) as the cross linker. The trans-resveratrol in the MIP can be washed out using methanol containing 10% acetic acid (v/v) to uniform MIP microspheres (average diameter -l.5 Ilm) (Fig. 1). Microtiter with 96 wells were coated with trans-resveratrol-MIP, which were fixed in polyvinyl alcohol as glue. The amount of polymer-bound trans-resveratrol was

using the imidazole (IMZ)-catalyzed peroxyoxalate CL reaction.

The

produced was measured with a high-resolution CCD camera. The

exposure time was optimized to 1 min. The intensity of the spots was determined the

FC software, which combines the pixel intensities.

1.

The SEM image of trans-resveratrol-MIP microspheres.

Table 1 shows the tolerable ratios for some interfering substances in grape wine. The trans- resveratrol , cis-resveratroL trans-piceid and cis- piceid have similar structure and in the absence of trans-resveratrol with MIP, the tolerable ratio is 1. When the sensor was prepared with MIP, the tolerable ratio increase to 100. These results show that these substances in grape wine in the normal concentration range did not interfere with the determination of trans-resveratrol. Other substances existing in wine such as fructose, citric acid and many amino acids have no CL

and did not interfere with the trans-resveratrol analysis.

The chiral molecular recognition of neutral molecules has become an important in the fields of analytical, biochemical and pharmaceutical technologies.

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Zhang Z et al.

Table 1. The tolerable ratios for some interfering substances in grape wme. Intelierencc substo'lllces

Without MIP

100 100 100 50

Trt1/IJ -piceid

Cis-resvemtroI Cis-pkeid

1

Vitamin B I. B2. B6. B 12

0.1

With f

-
I x I 0- mollL) caused a decrease in the signal-to-blank ratio. Hence, 2 I x I 0- mollL sodium hydrogen was employed in subsequent experiments. The signal-to-blank ratio continued to increase as the luminol concentration was 7 increased from Ix10- mol/L to 8x10- 7 mollL. It reached a maximum at 8x10- 7 mollL 7 luminol and then decreased. Hence, 8x I 0- mollL luminol was employed in subsequent experiments. The signal-to-blank ratio rapidly increased with the increase in K3Fe(CN)6 concentration in the range of 5xI0-6~6xlO-5 mol/L. Above 6x10-5 mollL, the signal-to-blank ratio decreased slightly. Hence, 6x10-5 mol/L K3Fe(CN)6 was employed in subsequent experiments. Performance of the system for thiamine measurements. Under the selected experimental conditions, the CL intensity was linearly related to the concentration of

Flow Injection Chemiluminescence Determination of Thiamine

223

thiamine over the range of 0.03-1.0 mg/L. The linear regression equation was I (au) = 2 2.40e (10- mg/L) + 13.84 and the correlation coefficient was 0.9936 (n= 8). The detection limit (3s b) was 0.007 mg/L thiamine and the relative standard deviation was 1.1% for 0.5 mg/L thiamine solution (n=II). A complete analysis, including sampling and washing, could be finished in 40 s, giving a sample measurement frequency of 901h. Interference. The effect of some common ions, additives and excipients used in pharmaceutical preparations was studied on the determination of a 0.5 mg/L thiamine solution. A foreign specie was considered not to interfere if it caused a relative error less than ±5% in the peak height. No interference has been observed when including a 1000-fold Na+, K+, SO/-, cr, N0 3- and starch, IOO-fold Ca2+, Zn 2+, glucose and lactose, IO-fold Mg2+. Equal amount of Fe3+, Fe 2+, C0 2+, Cr3+ and Cu 2+ interfered with the determination of thiamine. Determination of thiamine in pharmaceutical prepartions. The proposed method was applied to the determination of thiamine in vitamin BI tablets and vitamin BI injections. The average tablet weight was obtained from the weight of20 tablets. They were finely powdered, homogenized and a portion of the powder, equivalent to about 50 mg of thiamine was accurately weighted and dissolved in water. The resulting mixture was filtered and the filtrate was diluted to 100 mL with water for further sample analysis. Vitamin BI injections were directly analyzed after appropriately diluting with water. The results are summarized in Table 1, which agreed well with those obtained by the Chinese pharmacopoeia method. 9 Tabl~

1. Results for the determination of thiamine in pharmaceutical prepartions

Samples

Nomina I content

Propose d meth 0d

RSD, n=5

Pharmacopoeia method

Tablet 1

10 mg/tablet

9.97 mg/ tablet

1.2%

10.16 mg/ tablet

Tablet 2

10 mg/tablet

10.15 mg/tablet

1.3%

9.74 mg/ tablet

Injection 1

50 mglinjection

46.7 mg/injection

1.2%

48.6 mg/injection

Injection 2

100 mg/injection

97.0 mg/injection

1.1%

98.5 mg/injection

_

Possible CL reaction mechanism. The CL spectra of luminol of K3Fe(CN)6 with and without thiamine were obtained using a modified RF-540 spectrophotofluorimeter. Both CL spectra showed a maximum emission wavelength at 425 nm, which suggested that the excited state of 3-aminophthalate ion JO was still the emitter in the present reaction. When the dissolved oxygen in all solutions were removed by the flow of nitrogen, the CL intensity of the reaction reduced significantly, which indicated that the dissolved oxygen played an important role in the reaction. It was well known that thiamine can

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easily convert to thiol-containing compound in alkaline solution. The thiol-containing compound produced can react with dissolved oxygen in alkaline solution to produce a superoxide anion radical intermediate." Superoxide anion radical then oxidize luminol to produce CL. 12 In the presence of K3Fe(CN)6, the CL signal from the reaction between superoxide anion radical and luminol was enhanced. 13

REFERENCES 1.

2.

3.

4. 5.

6. 7.

8.

9. 10. 11. 12.

13.

Ghasemi J, Abbasi B, Niazi A, Nadat E, Mordai A. Simultaneous spectrophotometric mUlticomponent determination of folic acid, thiamine, riboflavin and pyridoxal by using double divisor-ratio spectra derivative-zero crossing method. Anal Lett 2004;37:2609-23. Alonso A, Almendral MJ, Porras MJ, Curto Y. Flow-injection solvent extraction without phase separation fluorimetric determination of thiamine by the thiochrome method. J Pharm Biomed Anal 2006;42: 171-7. Bohrer D, do Nascimento PC, Ramirez AG, Mendonca JKA, de Cavalho LM, Pomblum SCG, Determination of thiamine in blood serum and urine by high-performance liquid chromatography with dried injection and post-column derivatization. Microchem J 2004;28:71-6. Mrestani Y, Neubert RHH. Thiamine analysis in biological media by capillary zone electrophoresis with a high-sensitivity cell. J Chromatogr 2000;871: 351-6 Mervartova K, Polasek M, Martinez Calatayud J. Recent applications of flow-injection and sequential-injection analysis techniques to chemiluminescence determination of pharmaceuticals. J Pharm Biomed Anal 2007;45:367-81. Grekas N, Calokerinos AC. Determination of thiamine by continuous flow chemiluminescence measurement. Talanta 1990;37: 1043-8. Wasielczuk A, Catala Icardo M, Garcia Mateo JV, Martinez Calatayud J. Flow-injection chemiluminescent determination of thiamine in pharmaceutical samp 1es by on-line photodegradation. Anal Lett 2004;37 :3205-18. Du J, Li Y, Lu J. Flow injection chemiluminescence determination of thiamine based on its enhancing effect on the luminol-hydrogen peroxide system. Talanta 2002;57:661-5. The Pharmacopoeia of People's Republic of China (Part II). Beijing: Chemical Industry Press, 2000:784-7. White EH, Bursey MM. Chemiluminescence of luminol and related hydrazides: the light emission step. 1 Am Chern Soc 1964;86:941-2. Shen 1M, Wu ZQ. Eds. Pharmaceutical Structure and Reagent. Beijing: Chinese Medicine Science and Technology Press, 1989:541. Ohno K, Arakawa H, Yoda R, Maeda M. Development of novel high-sensitivity chemiluminescence assay for luminol using thiourea derivatives. Luminescence 1999; 14:355-60. Du J, Li Y, Lu 1. Flow injection chemiluminescence determination of captopril based on its enhancing effect on the luminol-ferricyanide/ferrocyanide reaction. Luminescence 2002; 17: 169-172.

CHEMILUMINESCENT AND ELECTRON SPIN RESONANCE SPECTROSCOPIC MEASUREMENTS OF REACTIVE OXYGEN SPECIES GENERA TED IN WATER TREATED WITH TITANIA-COATED PHOTOCA T ALYTIC FIBERS C LIN,l,2 K. TANAKA,2 L TANAKA,2 T KAWANO l JGraduate School of Environmental Engineering, The University of Kitakyushu, Kitakyushu 808-0135, Japan 2 K2R Inc., Kitakyushu 807-0871, Japan Email: [email protected].}p

INTRODUCTION Recently, a variety of ultraviolet-driven photochemically active catalysts designated as photo catalysts coated with titanium dioxide (Ti0 2) has been developed and applied for hygiene and antimicrobial purposes. The likely mechanism of such catalysts involves the generation of reactive oxygen species (ROS) on the surface of the catalyst as expected (not fully proven) from the previously proposed models. l However, no attempt to confer long-lasting chemical properties to the waters (e.g., preparation of waters rich in ROS) has been reported, despite of the increasing demands for the use of photocatalysts in various environments including the use in aqueous phase. In the present study, novel water conditioning photocatalytic apparatus (exPCA W-l, K2R Inc., Kitakyushu, Japan) equipped with sheets of TiOrcoated photocatalytic fibers were applied for THE preparation of ROS-containing water. We attempted to detect the generation of superoxide anion and hydroxyl radical as the key members of ROS generated in the water circulated in exPCA W-l by using the superoxide anion-specific chemiluminescent probe Cypridina luciferin analog (CLA), and a spin trapping agent, DMPO (5, 5-dimethyl-I-pyrroline-N-oxide) that readily forms an adduct with hydroxyl radical. Some other factors such as the effects of pH on the superoxide generation were also studied. METHODS Apparatus. For preparation of ROS-containing water, photocatalytic apparatus designated exPCA W-I (fabricated by K2R Inc., Kitakyushu, Japan) was used. It comprised an ultraviolet (UV-A) emitting bulb, ultrasonic wave (USW) generating devices, sheets of titania-coated fiber, a pumping system and gas (0 2 or NO) supply systems. As circulated in the exPCA W-I, water is treated with UV, USW and O 2, enabling the photo-catalytic oxidative conditioning of the water. Detection of superoxide anion. To detect superoxide generated and maintained in the water, a specific chemiluminescence probe, Cypridina luciferin analog (CLA; 2-methyl-6- phenyl-3,7-dihydroimidazo[I,2-a]pyrazin-3-one)2 purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan) was used. Water was circulated in the aparatus for at least 30 min and 0.5 mL was sampled from the reservoir (l L) and added onto CLA a containing reaction mixture (111M in 0.5 mL) in a glass cuvet placed insde the luminometer (CHEM-GLOW Photometer, AMINCO, Silver Spring, MD, USA) or 225

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Luminescencer (ATTO, AB-2200, Japan). Superoxide generation monitored as CLA-chemiluminescence was expressed as relative chemiluminescence units (rcu) as previously described. 3 Detection of hydroxyl radical. For detection of hydroxyl radicals, a spin trapping agent, DMPO was obtained from Sigma-Aldrich. 4 The water containing 500 JlM DMPO was circulated for 30 min in exPCA W-l ,and the water was sampled at 0, 1, 5, 10, 15, 20, 30 min of circulation and exposure to UV, USW and elevated O2 • The samples in a flat-shaped quartz ESR cell were analyzed with an electron spin resonance spectrometer (JEOL-TE200, X-band). DMPO-OH signal were collected with a sweep width of 5 mM, a 100 kHz modulation frequency, 60 s sweep time, a time constant of 0.1 s and microwave power of 10 mW, at room temperature of 20-25°C. RESULTS AND DISCUSSION The superoxide generation in the aqueous phase was monitored using CLA chemiluminescence and a photometer equipped with a pen recorder. The CLA luminescence in the glass cuvet increased following addition of processed water (treated with titania- coated photocatalytic fiber exposed to both UV-C and USW) after sampling from the aparatus. Despite its short life time, superoxide-dependent signal was shown to be long-lasting over 30-60 min even after isolation from the catalyst-equipped, photo-irradiating and USW generating apparatus (Fig. lA). In contrast, chemiluminescence could be no longer detected once Tiron, a superoxide scavenger, was added to the reaction mixture (Fig. 1B), supporting the view that the long-lasting nature of chmiluminescence can be attributed to the continuous generation of superoxide. In addition, the processed water-dependent superoxide generation showed pH-dependency, prefering nutral to alkaline conditions (Fig. 2) .

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Fig. 1. Typical trace of the superoxide-depndent chemiluminescence detected in the presence ofCLA. (A) Lang-lasting nature of chemiluminescence. (B) Inhibition of the chemiluminescence in the presence of Tiron.

Chemiluminescent and Electron Spin Resonance Spectroscopic Measurements

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35

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We also attempted to detect another key member of ROS, hydroxyl radical based on the formation of spin-trapped aduct (DMPO-OH) using ESR (Fig. 3). By circulating the spin trapping agent in the system, we observed the UV-dependent development of DMPO-OH signal with time. While we could detect the presence of hydroxy radicals in the circulating water, no signal could be detected in the water samples isolated from the photochemical reaction apparatus.

REFERENCES 1. Kagenishi,T. Yokawa K, Lin C, Tanaka K, Tanaka L, Kawano T. Chemiluminescent and bioluminescent analysis of plant cell responses to reactive oxygen species produced by newly developped water conditioning apparatus equipped with titania-coated photocatalystic fibers. In: Shen X, Yang X-L, Zhang X-R, Cui ZJ, Kricka LJ, Stanley PE eds. Light emission: Biology and scientiic appliations. Singapore:WorId Scientific. 2009:27-30. 2. Nakano M, Sugioka K, Ushijima Y, Goto T. Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a] pyrazin-3-one, for estimating the ability of human granulocytes to generate O2-. Anal Biochem 1986; 159: 363-9. 3. Kawano T, Kadono T, Furuichi T, Muto S, Lapeyrie F. Aluminum-induced distortion in calcium signaling involving oxidative bursts and channel regulations in tobacco BY-2 cells. Biochem Biophys Res Commun 2003; 308: 35-42. 4. Kawano T, Muto S. Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco suspension culture. J Experi Bot 2000; 51: 685-693.

A SENSITIVE MICELLAR-ENHANCED CHEMILUMINESCENCE METHOD FOR THE DETERMINA TION OF OFLOXACIN BY FLOW INJECTION ANALYSIS HONGY AN MA, Y ANTU ZHANG, LIXIAO MIAO, XUEHUA SUN

College a/Chemistry and Chemical Engineering, Yan 'an University, Yan 'an, 7J 6000, China Email: [email protected]

INTRODUCTION Ofloxacin is a member of the fluoroquinolone class of synthetic antibiotics.' It is widely used in the treatment of a wide range of infection, including respiratory tract, urinary tract and tissue-based infections. Several methods for the determination of ofloxacin have been reported in the literature including spectrophotometric,' fluorimetric,3 chromatographic' and electroanalytic. 5 Francis has reviewed the methodology for the determination of ofloxacin based on chemiluminescence (CL) detection. 6 But to the best of our knowledge, no flow injection CL analysis method for the determination of ofloxacin in the presence of sodium dodecyl sulfate (SDS) surfactant micelles has been described. We investigated the effects of surfactants on emission intensity in the luminol CL system due to the various advantageous properties of surfactants, which have been found to improve CL measurement efficiency,' and reported significant and useful improvements in relative intensity. The method possesses a good accuracy and precision and has been successfully used to determine ofloxacin in pharmaceuticals. EXPERIMENTAL Reagents. All reagents were of analytical grade and all solutions were prepared with doubly distilled water. Ofloxacin was obtained from Beijing Bio Life Science and Technology Co, Ltd, China, Ofloxacin injection was obtained from Ludi Pharmaceutical Ltd, Co" Jiangxi, China, Ofloxacin tablets were purchased from Nanchang Pharmaceutical Factory, Jiangxi, China, The stock standard solution of ofloxacin (I mg/mL) was prepared by dissolving 100 mg of ofloxacin in 0,001 mollL sulphuric acid solution and diluting to 100 mL with the same acid, Working standard solutions were prepared by appropriate dilution of the stock standard solution with water. Luminol was used as supplied to prepare a 0,03 mollL stock solution by dissolving 1.3258 g luminol (synthesized by Department of Chemistry, Shaanxi Normal University, China) with 0.1 mollL NaOH to 250 mL in an amber-colored measuring flask. SDS, H 2 0 2 , NaOH, NaAc-HAc solutions were prepared in water. Apparatus. The flow system used in this work (Fig, 1) consisted of two peristaltic 4 pumps, one of which delivered a sample stream (ofloxacin and 8,Ox 10- mol/L SDS in pH 4.65 NaAc-HAc medium) and H 20 2 stream at a flow rate of 3.4 mLimin, the 229

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other delivered NaOH carrier and luminol solution at the same flow rate. PTFE tubing (O.S mm id) was used to connect all components in the flow system. 100 ilL of a mixture of sample and H20 2 was injected into the NaOH carrier stream by a six-way injection valve and then mixed with luminol solution. The flow cell was a coil of glass tubing (1.3 mm id) spiraled to a diameter of 35 mm with five turns, located in front of the detection window of the photomultiplier tube. The emitted CL was collected with a photomultiplier tube (operated at -SOO V) of the BPCL Ultra Weak Chemiluminescence Analyzer (lnsistitute of Biophysics. Chinese Academy of Sciences, Beijing). The signal was recorded using a computer equipped with a data acquisition interface. Data acquisition and treatment were performed with BPCL WIN software. a

b

c

v d

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Fig. 1. Schematic diagram for the determination of ofloxacin. Pj, P 2 . pump; V. six-way valve; F. flow cell; D. detector; Pc. personal computer; W. waste; a. sample solution (ofloxacin and S.OxI0-4 mollL SDS in NaAc-HAc buffer); b. H 2 0 2 solution; c. NaOH solution; d. luminol solution.

RESUL TS AND DISCUSSION Firstly, in order to obtain an understanding of the mechanism of the sensitized luminol-based CL reaction, the CL emission spectrum was obtained with a F-4500 fluorescence spectrophotometer. The results showed the maximum emission wavelength in the presence of ofloxacin was the same as that in the absence of ofloxacin and the maximum light emission was at about 425 nm. The emitter was 3-aminophthalate, the oxidation product of luminol. Furthermore, the preliminary investigations showed that the sample medium is a very important factor for the determination of ofloxacin with the proposed method. The form of ofloxacin existing in different pH media is different. The experiment indicited that: only when acidic ofloxacin solution was injected into the carrier stream was the enhanced CL signal observed. Thus, we concluded that the possible mechanism is: the ofloxacin may act as a energy source in the CL reaction, the presence of ofloxacin enhanced the luminol-H 2 0 2 CL quantum yield. The maximum CL intensity was obtained from pH 4.0-5.0. Thus, pH 4.65 NaAc-HAc buffer solution was selected as the sample medium. The principles of micellar enhancement including solubilization and solute organization, altering the local microenvironment and changing the light-emitting pathways that affect the quantum yield and reaction rate have already been discussed

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by Townshend et aI.' In order to investigate whether surfactant media enhanced effectively in the present CL reaction system, the characteristics of several different micelles including Tween 80, OP, Triton X-I00, CTMAB, TPB, SDBS and SDS were studied. The results showed SDS enhanced the signal dramatically. SDBS gave a somewhat enhanced signal whereas other surfactants were less effective. Thus, SDS was selected for the present work. The effect of SDS at various concentrations was studied in order to maximize the CL signal. The maximum CL intensity was observed using a concentration of 8.0 x 10-4 mollL SDS. Therefore, this was selected for the present work. The influence of luminol concentration on CL was examined from l.Ox 10- 7 to 6 2.0x 10- mollL. The result indicated that 8.0x 10- 7 mollL luminol gave the highest relative CL intensity and the sensitivity decreased on either side of this value. Therefore, 8.0x 10-7 mollL luminol was chosen for the subsequent experiments. The influence of sodium hydroxide concentration on the CL intensity was investigated at different concentrations from 0.005 to 0.2 mollL and the maximum CL intensity was obtained at 0.01 mollL. Therefore, 0.01 mollL sodium hydroxide was selected for the present work. The effect of H 2 0 2 concentration over the range of 2.0x 10- 4 to 2.0x 10-3 mollL on the CL emission was examined. The peak height increased steeply with increasing H 20 2 concentration up to 8.0xlO-4 mollL, above which CL intensity decreased. Therefore, 8.0x 10-4 mollL H 2 0 2 was used for subsequent work. Flow rate is an important parameter in CL detection. Too low or too high flow rates result in a decrease or even the absence of a CL signal in the flow cell. A rate of 3.4 mLimin was chosen as a suitable condition with superior sensitivity, precision and reduced reagent consumption. Under the selected experimental conditions, a linear calibration graph of oxfloxacin between 4.2x 10- 12 and 3.6x 10-9 g/mL was obtained. The calibration equation was 1=1.3 x 10 12 C + 190.4, r 2=0.9993, with a detection limit of 2.6x 10- 12 g/mL (S/N=3). The relative standard deviation (RSD%) for 11 determinations of l.Ox 10-9 g/mL was 2.0%. The sample measurement frequency was calculated about 60 sampleslh. Table 1. Determination of ofloxacin in a pharmaceutical formulation (n=5) Sample Tablet Injection

Label (mg) 100.0 200.0

Official method (mg) 99.4 197.6

Proposed method(mg) 99.7 198.4

RSD (%) 2.2 2.3

Added (mg) 50.0 50.0

Recovery (%) 98.5 101

The interference of foreign species were tested by analyzing a standard solution of 9 l.Ox 10- g/mL ofloxacin. The tolerable concentration ratios for interference at 5% level were over 5000 for glucose, sucrose, 1000 for magnesium stearate,

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hydroxypropyJcellulose, starch, lactic acid, cr, Ca2+, Mg2+, respectively. Finally, tablets and injections were analyzed by the proposed method. Injectable preparations of ofloxacin were directly diluted with water so that final concentration was in the working range. Ten tablets were weighed to obtain the mean weight, then ground to homogenized powder and an accurately weighed portion of powder corresponding to 100 mg was diluted with 0.001 moUL sulphuric acid fot., the quantitative analysis (see Table 1); the results agree well with those obtained by an official method.' The recoveries varied from 98.5 %-101 %. .

REFERENCES 1.

Schaeffer AI.

The

expanding

role

of fluoroquinolones.

Am

I

Med

2002;113:45-54. 2.

3.

4.

5.

6. 7.

8.

Issa YM, Abdel-Gawad FM, Abou Table MA, Hussein HM. Spectrophotometric determination of ofloxacin and lomefloxacin hydrochloride with some sulphonphthalein dyes. Anal Lett 1997;30:2071-84. Gong QJ, Qiao IL, Du LM, Dong C. Recognition and simultaneous determination of ofloxacin enantiomers by synchronization first derivative fluorescence spectroscopy. Talanta 2000;53:359-65. Halkar UP, Ankalkope PB. Reverse phase high-performance liquid chromatographic determination of ofloxacin and tinizadole in tablets. Indian Drugs 2000;37:585-8. Wu J, Zhao H, Wei L, Ai TZ, Dong XZ. Preparation and application of a poly(vinyl-chloride) membrane coated glass electrode-based ofloxacin ISE. Chin J Anal Chem 2001;29 : 11 06-9. Francis PS, Adcock JL. Chemiluminescence methods for the determination of ofloxacin. Anal Chim Acta 2005; 541:3-12. Townshend A, Youngvises N, Wheatley RA, Liawruangrath S. Flow-injection determination of cinnarizine using surfactant-enhanced permanganate chemiluminesence. Anal Chim Acta 2003;499:223-33. Editorial Committee of the Pharmacopoeia of People' Republic of China. The Pharmacopoeia of People' Republic of China (Part II). Beijing: Chemical Industry Press, 2005 :606-8.

EXCESSIVE EXTRACELLULAR CHEMILUMINESCENCE AND NECROSIS OF NEUTROPHILS IN BOVINE NEONATES AND POTENTIALLY SUPPORTIVE ROLE OF VITAMIN C J MEHRZAD,1 M MOHRI,2 C BURVENICH3 1Department of Pathobiology, F erdowsi University of Mashhad, Mashhad, Iran 2Department of Clinical Science, Ferdowsi University of Mashhad, Mashhad, Iran 3Department of Physiology and Biometry, Ghent University, Merelbeke, Belgium E-mail: [email protected]

INTRODUCTION Neutrophils are the most critical part of the innate immune defense in dairy cOWS. t,2 Their quality in the blood circulation and tissue is crucial during early life of neonatal calves. Vitamin C is one of the most important water-soluble protective agents in mammalian cells. 3 Bovine neonates are unable to synthesize vitamin C. Substantial evidence suggests a link between vitamin C and immunity.t,3 Bovine neutrophils have a potential to produce a substantial amount of reactive oxygen species (ROS) to kill engulfed bacteria. t.6 These ROS production can be both extracellular and intracellular. 2,4,7.9 The neutrophils ROS production and its kinetics can be measured following stimulation with soluble agents, e.g., phorbol 12myristate 13-acetate (PMA) or with particles e.g. zymosan, bacteria, latex beads, using chemiluminescence (CL) assay,2,3,7.9 which was first described by Allen et aJ.1 The different responsiveness of blood neutrophils to PMA stimulation during physiological conditions could result from differences in protein kinase C, NADPHoxidase and myeloperoxidase (MPO) activities. 2,6.9 As these enzyme activities reflect intracellular and extracellular reactions, changes might offer some evidence about the neonates' susceptibility to infections. For example, in dairy cows the maximal animal susceptibility for environmental pathogens coincides with the minimal neutrophil ROS production capacity.l,2,6 Furthermore, dietary vitamin C in dairy cows somehow improves the quality of milk neutrophils 3. Retrospectively, there have been no investigations on the issue of "neutrophils ROS production and antioxidants versus neonates" in cows. Therefore, to obtain a clearer insight into the oxidationreduction reactions of the neutrophils in bovine neonates, the kinetics of PMA stimulated luminol-enhanced CL of blood neutrophils, their viability and the role of vitamin C were investigated in calves immediately after birth. MATERIALS AND METHODS Calves and experimental plan. In total twenty Holstein early neonates were selected; they all were clinically normal. Two separate studies were conducted. In the first study, simultaneous analyses of CL kinetics and viability of blood neutrophils within 12 hours after birth in the healthy calves (n = 10; from Ghent 233

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University dairy farm) were measured. In the second study, at 10 days before anticipated calving, ten healthy Holstein dairy cows from Mashhad University dairy farm were divided in two groups. The cows were fed diets that provided 0 (n = 5) or 50 (n = 5) gm/d of supplemental vitamin C. Within 12 hours after birth, blood samples were collected for isolation of neutrophils, their viability and superoxide anion production capacity. In both studies, isolation of pure blood neutrophils was carried out as described previously.2,4-6 Chemiluminescence, O£ production and viability of blood neutrophils. In the first study luminol-enhanced PMA-stimulated CL was used to assess blood neutrophils CL kinetics. Briefly, CL was performed using a luminometer (LB96P; EG&G Berthold, Germany) at 37°C. A total volume of 200 J.1L was prepared for CL kinetics determination. Immediately after addition of PMA (final concentration of 200 ng/mL) and luminol (final concentration of 0.3 mmoVL) to lOS neutrophils into microtiter plates, CL was measured. The area under the curve (AUC) was determined for registered impulse rate (counts/min) over the entire measurement period of 30 min as previously described. 2,4.6 Viability of neutrophils was evaluated by means of flow cytometry (FACSScan, Becton Dickinson Systems, CA, USA), using propidium iodide exclusion. 2.4.6 In the second study, the superoxide anion (Oi) was measured using superoxide dismutase inhibitable cytochrome C reduction assay. Briefly, after incubation for 30 min the optical density at 550 nm was determined in a Microplate reader, the results were converted to nanomoles of cytochrome C reduced using the extinction coefficient Esso nm = 2.1x104/moVcm. The viability was quantified with microscopic observation of neutrophils using trypan blue dye exclusion. In both studies whole blood and isolated neutrophils were microscopically examined on slides, as described previously.2.4 For statistical analyses of the parameters, the SAS Version 9.1 with analysis of variance was used. Hypothesis testing was done at the 5 % significance level. RESULTS AND DISCUSSION In general, the CL of blood neutrophils in early neonates revealed something interestingly different from their adult counterparts. The CL kinetics of PMAstimulated neutrophils was monophasic pattern. This pattern was more noticeable during day 1 of birth (Figure 1). However, when the neonates drink more colostrum the CL tends to switch towards a biphasic form (Figure 1). Furthermore, microscopic examination of the neutrophils showed the presence of both immature and apoptotic neutrophils in the prepheral blood (Figure 1). From our data in the first study we can conclude that the intensity of neutrophil CL was always lower in early neonates than in the adults. 2.4.6 This discrepancy seems to be somehow related to the existence of immature neutrophils and excessive extracellular ROS in the blood stream. All of this shows the lower effectiveness of the oxygen-dependent intracellular bactericidal mechanism of neonatal neutrophils. 2,4-9 Moreover, as the luminol-dependent system requires hydrogen peroxide (H20 2), 8,9 it is likely that the intracellular H 20 2

Excessive Extracellular Chemiluminescence and Necrosis of Neutrophils

235

pnXitlctlon is low in calves immediately after birth. The absence of a second in blood neutrophils of bovine neonates warrants further study.

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vitamin C potentially improves neonatal neutrophils functions; left is the viability and right panel is the superoxide anion production of Bars represent means and the standard error of the means of 10 calves. In the second stduy, we observed that dietary vitamin C somewhat burst activity or OZ" production (Figure 2; right panel) and 2; left panel) in bovine neonates. Compared with non-vitamin sUJ'plem.enlted group, feeding vitamin C to the pregnant cows around ",,,.tHr,ft neutrophil functions in their neonates. We assume that the

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supplemental vitamin C appears in the colostrum or milk. When the neonates drink this milk the vitamin C is absorbed via gastrointestinal tract into bood circulation. Although statistically the improvement was slight (due to limited number of calves) but immunobiologically this slight imptovement could be vital for the neonates. Overall, these findings suggest that the subsequent intracellular MPO-H 20 2 system is impaired, and may represent an immunosuppressed condition in the very early bovine neonates. Conversely, the slight increase of neutrophil functions with vitamin C application could lead to a better protection of neonatal calves from infectious pathogens. Further studies are in progress to explain these findings.

ACKNOWLEDG EMENTS This study was financially supported by Ferdowsi University of Mashhad (Grant No. 36900) and the Flemish Institute for the Encouragement of Research in the Industry (lWT-grant No. 030784). The authors wish to thank K. Demeyere and A. Shavalian for their excellent technical assistance. REFERENCES 1. Burvenich C, Van Merris V, Mehrzad J, Diez-Fraile A, Duchateau L. Severity of E. coli mastitis is mainly determined by cow factors. Vet Res 2003; 34:521-62. 2. Mehrzad J, Dosogne H, Meyer, E, Heyneman R., Burvenich C. Respiratory burst activity of blood and milk neutrophils in dairy cows during different stages of lactation. J Dairy Res 2001; 68:399-415. 3. Weiss WP, Hogan JS. Effects of dietary vitamin C on neutrophil function and responses to intramammary infusion of lipopolysaccharide in periparturient dairy cows. J Dairy Sci 2007; 90:731-9. 4. Mehrzad J, Dosogne H, Vangroenweghe F, Burvenich C. A comparative study of bovine blood and milk neutrophils functions with luminol dependent chemiluminescence. Luminescence 2001; 16: 343-56. 5. Mehrzad J, Duchateau L, Py6rala S, Burvenich C. Blood and Milk Neutrophil Chemiluminescence and viability in primiparous and pluriparous dairy cows during late pregnancy, around parturition and early lactation. J Dairy Sci 2002; 85:3268-76. 6. Mehrzad J, Duchateau L, Burvenich C. Viability of milk neutrophils and severity of bovine coliform mastitis. J Dairy Sci 2004; 87:4150-62. 7. Allen RC, Stjernholm RL, Steele RH. Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem Biophys Res Commun 1972; 47: 679-84. 8. Briheim G, Stendahl 0, Dahlgren C. lntra- and extracellular events in luminoldependent chemiluminescence of polymorphonuclear leukocytes. Infec lmmun 1984;45: 1-5. 9. Lind J, Merenyi G, Eriksen TE. Chemiluminescence mechanism of cyclic hydrazides such as luminol in aqueous solutions. J Am Chern Soc 1983; 105:7655-61.

CHEMILUMINESCENCE OF 9-BENZYLIDENE-IO-METHYLACRIDANS WITH ELECTRON-DONATING GROUPS BY CHEMICALLY GENERATED SINGLET OXYGEN - APPLICATION TO METAL ION SENSING USING AZACROWNED COMPOUND J MOTOYOSHIYA, T TANAKA, M KUROE, Y NISHII Diivision o/Chemistry and Materials,Faculty o/Textile Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan, [email protected]

INTRODUCTION The thermal decomposition of a 1,2-dioxetane into two carbonyl compounds, one of which is formed in the excited state, often produces chemiluminescence. I The singlet oxygenation of 9-benzylidene-IO-methylacridans to produce chemiluminescent 2 acridan dioxetanes has been occasionally investigated. ,3 The thermal decomposition of the acridan dioxetanes gives the corresponding aldehydes and a fluorescent Nmethylacridone (NMA), the latter of which is the emitter (Scheme I). Although electron donating-substituents attached to the phenyl group of the benzylidene moieties increases chemiluminescence efficiency, the dimethylamino group drastically inhibits the chemiluminescence. 4,5 Such a peculiar effect of the amino group prompted us to investigate this system in more detail and apply it to a metal cation recognition system, Here we report the chemiluminescence behavior of 9-benzylidene-IOmethylacridans bearing electron-donating groups (e,g" alkoxy and amino groups) and describe the potential application to metal cation sensing using an azacrowned compound. {7'/

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238

Motoyoshiya Jet al.

4.0 X 10-5 M), a blue-light flash emission was observed, and the chemiluminescence spectrum was completely in agreement with the fluorescence spectrum of NMA, indicating that the excited NMA was generated from this oxidation reaction. The fluorescence spectrum of the spent solution also agreed with that of NMA and no other fluorescent product was found. A different way to generate singlet oxygen by a combination of alkaline hydrogen peroxide-acetonitrile, a metal ion free system,6 was also carried out [aqueous solution containing hydrogen peroxide (lmL, 1 x 10-5 M) 2 and tetrabutylammonium hydroxide (1 mL, 1 x 10- M) added to acetonitrile solution 7 of la (1 mL, 1 x 10- M)] and this produced a blue-light emission. Table 1. Data of chemiluminescence reaction of benzylideneacridanes (1).

R

:Ea+

1a

p-OMe

-0.27

Ib

m-OMe

0.12

Ie

2.4-(OMe)2

Id

3,4.5-(OMe)3

Ie

p-NMe2

a C

r.Htrm as well as knockdown p22phox expression reduced ROS it is reasonable to suggest that thioredoxin reductase 1 ROS by upregulation of, at least, of the

IIIIII~II~

II-actin

Times (8)

Chemiluminescence detection of the ROS knockdown CONCLUSIONS Thioredoxin reductase upregulates activity of NADPH oxidase expression of its subunit p22phox. Enhanced chemiluminescence as a sensitive method to detect intracellular ROS generation. REFERENCES 1. Shah, AM. NADPH oxidase and endothelial cell function. 09:217-26. 2. A. Thioredoxin. Annu Rev Biochem 1 3. Kricka, Moseley SR, Whitehead TP. Phenols as enhancers of the chemiluminescent horseradish peroxidase-Iuminol-hydrogen reaction: application in luminescence-monitored enzyme Chern 1 31:1335-41.

QUANTITATIVE DETECTION OF SINGLET OXYGEN WITH A CHEMILUMINESCENCE PROBE DURING PHOTODYNAMIC REACTIONS Y ANCHUN WEI, DA XING', SHIMING LUO, WEI XU, QUN CHEN MOE Key Laboratory of Laser Life Science, South China Normal University, Guangzhou, China, E-mail: [email protected]

INTRODUCTION Chemiluminescence is often used to detect qualitatively or quantitatively, microelements, free radical and other biologic or pharmacologic molecules.'·' Active oxygen species, especially singlet oxygen e02), are important cytotoxins.' 10 2 can depress cell activity and even induce cell death by oxidizing lipid, proteins and DNA; for example, in most photodynamic therapies (PDT) 102 is produced as the main cytotoxin; 4 cells often respond to wounding or stress by presenting abnormal 10 2 levels. s Thus 10 2 often needs to be detected as an important measure of the harm to a cell. There are many methods to evaluate whether 10 2 is present and how much was produced. A 102- CL method which uses a chemiluminescence probe molecule to chemically interact with 10 2 and results in the production of excitated energy state products has been reported as a method of 10 2 detection.' However, the CL method also has shortcomings, such as the inefficiency of CL reactions. We have considered ways to improve CL detection. Thus during 10 2 detection with CL, correcting CL was considered. To implement this concept, 102 was measured by FCLA in a photodynamic reaction to achieve correction. We have previously reported that the CL probe, FCLA, which can selectively detect singlet oxygen and superoxide. 7 Here CL is measured and analyzed at different probe concentrations and a method of making the detection precise is discussed. MATERIALS AND METHODS ROS specific chemiluminescence probe FCLA (Tokyo Kasei Kogyo Co., Tokyo, Japan) (100 IlmollL pH7.0) Photosensitizer Protoporphyrin IX disodium salt (PPIX, Aldrich Chemical Co., Milwaukee, Wisconsin) was prepared according to the manufacturer's directions to a concentration of200 IlmollL. The irradiation source for the photosensitization reaction is a custom-built, gated diode laser system (lOOmW, 635 nm, LDC 2000, ThorLab; TEC2000, Wavelength Electronics, USA); and filters (FF500/646Beamsplitter, Semrock Co. USA and 510 nm and 530 nm band-pass filter, Oriel Co., USA.) were used to protect the PMT from scattered irradiation light. The fluorescence was measured using a photon multiplier tube (MP952, PerkinElmer Optoelectronics, Germany) with a counter (PCL-836, Advantech Co., Ltd. Taiwan). The irradiation and fluorescence system is synchronized and controlled by Labview (Labview version 6.1 National Instruments, USA). The laser system is controlled by the TTL level of the counter. 253

254

Wei Y et al.

FCLA was prepared in various concentrations for quenching experiments. PPIX 2 concentration was 10 !AomollL. The laser (10m Wfcm ) irradiated the two reagents at once after mixing them. Signal was record simultaneously. Quenching of FCLA excitation state by FCLA oxide. The photodynamic reaction was performed with PPIX 5 !AomollL, FCLA 2 !AomolfL and a laser setting of 20 mwfcm 2 . Fresh FCLA was used for the first experiment, and then the same reaction was done with added oxidized FCLA 2 !AomollL in solution. The experiment was repeated three times. 2000

1200

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Fig. 1. Relationship of chemiluminescence and probe concentrations in the photodynamic reaction. Fig.2A: CL with 0.075 and 20 !AomollL FCLA; Fig.2B: initial CL with different FCLA concentrations (0.05- 20 !AomollL). RESULTS AND DISCUSSION Fig.lA shows the different probe concentration have distinctive luminescence courses. Both the primary intensity and the decline course are different. In Fig. 1B the CL intensity influenced by FCLA concentrations is shown. The results indicates that the CL declined when the reagent concentration was > 1 !AomolfL. Thus the CL is influenced by the concentration of the FCLA reagent. In other experiments the light absorption at 532 nm and 635 nm was measured. The values were very low although the concentration increased. So the absorbtion influences CL relatively little. As the probe concentration increased the odds of molecular collision increased, thus more energy was transferred intramolecularly. This is the reason for the decline in CL intensity. Fig. 1 indicates fresh FCLA can quench luminescence; but FCLA oxide can also quench CL (Fig. 2). The figure indicates that oxidized FCLA retained its quenching characteristics. Considering the similar molecule structure, the quenching ability of both must be same and the quenching rate should still be stable during the FCLA depletion. Thus the self-quenching to CL can be ignored if using an appropriate proportion to reflect singlet oxygen.

Quantitative Detection of Singlet Oxygen with a Chemiluminescence Probe

CL

corrected.

255

by FCLA oxide in solution. (means±S.E)

the reaction rate which is related with the probe concentration must be Lineweaver-Burk equation, the formulas of correcting CL was ) can be corrected as

and [FCLA]o is t and 0 time FCLA concentration; It is t time CL is solution volume and t> is system proportion coefficient; k is FCLA constant; NA is Avogadro' number. Using the equation, the detected CL was corrected. 3 shows CL and its corrected CL. Comparing the direct and the latter falls slowly with the probe concentration depletion been The result indicates that direct detected CL signal was incorrect due to depletion and could not be precisely related to singlet oxygen. Here the CL accumulation is corrected from 2.96 x 106 to 4.57 X lOti. In conclusion, CL can reflect singlet oxygen in photodynamic reactions; but some factors will affect the CL, including light absorption, self-quenching and concentration

1(JO

150

time(s}

3. FCLA 6Jl mol/L, PPIX 10 Ilmol/L, laser 10 mWlcm 2 . In the correction, the

256

Wei Yet al.

constant k is 10 6 morl-L and the coefficient -L is 1.4 NAV

X

10- 11 mollL

depletion. So some measures have to be taken to correct for these factors. The result indicates that with the correction, more precise singlet oxygen detection can be made to improve CL monitoring.

ACKNOWLEDGEMENTS This research is supported by the National Natural Science Foundation of China (30470494; 30627003) and the Natural Science Foundation of Guangdong Province (7117865).

REFERENCES 1. 2.

3.

4. 5.

6.

7.

Dodeigne C, Thunus L, Lejeune R. Chemiluminescence as a diagnostic tool. A review. Talanta 2000:51 :415-39. Wang J, Xing D. Detection of vitamin C-induced singlet oxygen formation in oxidized LDL using MCLA as a chemiluminescence probe. Acta Biochim Biophys Sin 2002:34: 11-5 Oleinick NL, Morris RL, Belichenko I. The role of apoptosis in response to photodynamic therapy: what, where, why, and how. Photochem Photobiol Sci 2002:1:1-21. Sharman WM, Allen CM, van Lier JE. Photodynamic therapeutics: basic principles and clinical application, Drug discovery today 1999:4:507-17. Chen WL, Xing D, Tan S C, Tang YH, He YH. Imaging of ultra-weak bio-chemiluminescence and singlet oxygen generation in germinating soybean in response to wounding. Luminescence 2003;18:37-41. Qin YF, Xing D, Zhou J, Luo SM, Chen Q. Feasibility of using fluoresceinyl cypridina luciferin analog in a novel chemiluminescence method for real-time photodynamic therapy dosimetry, Photochem Photobiol 2005:81: 1534-8. Wei YC, Zhou J, Xing D, Chen Q. In vivo monitoring of singlet oxygen using delayed chemiluminescence during photodynamic therapy. J Biomed Opt 2007:12:1-7.

FLOW-INJECTION CHEMILUMINESCENCE DETERMINATION OF HUMAN SERUM ALBUMIN BASED ON FLUORESCEINYL CYPRIDINA LUCIFERIN ANALOG- 10 2 REACTION WEI XU, YANCHUN WEI, DA XING,SHINGMING LUO, QUN CHEN MOE Key Laboratory of Laser Life Science, South China Normal University, Guangzhou 510631, China, E-mail:[email protected]

INTRODUCTION The concentration of human serum albumin (HSA) is an important biomarker and quantitative analysis of HSA in urine can provide critical information for early diagnosis and treatment of nephrosis. The most commonly used methods for analysis of micro-concentrations of HSA, are the Lowry, CBBG-250,'" el ectrochemiluminescence, spectrophotometry / fluorospectrophotometry,' Ray leigh light scattering methods,,6 and chemiluminescence.' Among them, chemiluminescence (CL) especially coupled with flow injection analysis (FIA) is considered as the most sensitive and versatile analytical technique. It is characterized by high sensitivity, a large dynamic range, minimum background interference, and good reproducibility. The conventional methods used a quenching effect of proteins for quantitative measurements.' Our proposed technique based on CL enhancement effect effectively determined low concentrations of HSA. In comparison to the CL quenching technique, the method significantly improves the detection sensitivity (ca. 1DO-fold higher). The purpose of this work is to use the CL enhancement technique coupled with FIA to determine HSA in a fluoresceinyl Cypridina lucifer in analog (FCLA)_I02 system, and is based on previous work that CL from the FCLA- 102 system can be strongly enhanced in the presence ofHSA. A simple and fast flow injection analysis has been developed for the determination of HSA, which has been satisfactorily applied to analyze clinical urine samples. METHODS All reagents were of analytical grade or the best grade available. Stock solution of 1 x 10-4 moUL FCLA purchased from Tokyo Kasei Kogyo Company (Tokyo, Japan) was prepared by dissolving 1 mg FCLA in 15 mL water deoxygenated by N2 bubbling and stored at refrigerator (-20°C). The schematic of the flow system used in this work is shown in Fig. 1. There are two peristaltic pumps (A and B). Pump (A) was used to deliver the flow streams of FCLA and hydrogen peroxide, and pump (B) was used to deliver merged stream of either sample or standard of HSA and sodium hypochlorite. FCLA solution (75 JlL) was injected into the carrier stream through an eight-way injection valve equipped with a 75 JlL sample loop, and then it was merged with the mixture solution of sample and sodium hypochlorite, finally reached the flow cell to produce CL emission. The CL signal produced in the flow cell was detected and recorded with a computerized luminescence analyzer MPI-B purchased from Remax ElectronicScience and Technology Company (Xi'an, China). 257

258

Xu Wetal.

Fig. 1. Schematic diagram ofCL flow system. (a) FCLA solution; (b) hydrogen peroxide solution; (c) sample solution or standard solution;(d) sodium hypochlorite; PMT photomultiplier tube; PC personal computer; HV high voltage power

RESULTS Interference studies. We examined the potential interference by basic amino acids, glucose, certain ions commonly found in urine samples (Table 1). The acceptable deviation of the measurement result should be less than 5% of that with HSA only. The results indicated that, up to 20-fold NaCI, MgS04 , NaH 2P0 4 , oxalate, 50-fold glucose, IO-fold KN03 and some amino acids up to the clinically observable maximum concentrations, had no practically significant influence on the CL measurement results. Calibration curve and detection limit. A representative calibration curve characterizing the relationship between CL and HSA concentration is shown in Fig. 2. The calibration curve obeyed a second order equation: M CL = 262.39 + 38.69 IgCHsA + 1.84 (lgC HSA)2 The regression coefficient was R2>0.99. However, the linear dynamic range was 1 x 10- 10 to 1 x 10- 8 mol/L and was expressed by the first-order equation: MCL=116.51 + 5.84 IgC HsA (R 2=0.99, n=7), with a detection limit of 4.5 x 10- 11 mollL (SIN = 3). R.S.D for the consecutive CL detection of without HSA was 3.28%(n=11).

=!

~

T

70

~

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1 "" 1

Log concentration of HSA {mol {.")

8

2

Fig. 2. Calibration curve for HSA. Conditions: FCLA, I x 10- moUL; NaCIO, 3.5 x 10mollL; H 2 0 2 , 1 x 10-2 mollL; PBS 7.4; negative high-voltage, -800 V; flow rate, 2.4 mLimin; injection volume 75 flL.

Flow-Injection Chemiluminescence Determination of Human Serum Albumin

259

Table 1. Effect of common substances in urine samples on CL measurement Interfering substance

Concentration IO·lrnoi/L

Change ofCL (%)

Na'H,PO;

511.8

-4.4%

Na+Cr NH:S,O,'Oxalate

226.7 533.2 1.2 300 200 b 18.5 50 b 480 b JOO b 25 b 8b

-8.0% 4.3% 2.4% 3.1% -4.0% -3.5% -3.3% 2.7% -4.1% 1.2% 4.9%

K~O;

Uric acid Glucose L-Arg L-G1y L-Val L-Tyr L-Trp a

Ion concentration

in urine(IO" mol IL)

PO/ 8.5 Na+ 86.7 cr 113.3 NH; 13.3 0.06 K+34 -------

0.4 -------------

--.-----

---------------

R.S.O' 4.6% 2.5% 3.7% 3.5% 1.0% 3.4% 4.6% 2.8% 4.4% 3.2% 2.5% 4.4%

Each expenment was repeated 4 tImes,. b ,ug/mL

Mechanism of CL enhancement. According to Forster's theory, energy transfer is a distance dependent interaction between the different electronic excited states of molecules in which excitation energy is transferred from one molecule (donor) to another molecule (acceptor). Fig. 3 shows that fluorescence of HSA at 348nm decreased with increasing FCLA concentration. The energy from excited-state HSA maybe be transferred to FCLA or be lost in a non-radiative manner.

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.

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300

350

400

450

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260

Xu Wet al.

Fig. 3 shows fluorescence intensity of FCLA in the presence of HSA (..l.ex=288 nm). Intensity increased greatly in the presence as compared to the absence of HSA in the same conditions as for the studies shown in Fig. 3. This provides strong evidence for the occurrence of Forster type energy transfer from the tryptophan moiety (donor) in HSA to the FCLA molecule (acceptor). We know that the distance from bonded-FCLA to Trp214 is less than 5 nm. Therefore, efficient energy transfer can exist between HSA (donor) and FCLA (acceptor).

ACKNOWLEDGEMENTS This research is supported by the National Natural Science Foundation of China (30470494; 30627003) and the Natural Science Foundation of Guangdong Province (7117865) and the US NIH grant POI-43892 REFERENCES 1. Marion MB. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72; 248-54. 2. Flores R. A rapid and reproducible assay for quantitative estimation of proteins using bromophenol blue. Anal Biochem 1978;88;605-11. 3. Chun Q, Ke AL, Tong SY. Spectrophotometric micromethod for protein determination with tetrachloro tetraiodofluorescein. Anal Lett 1998;31; 1021-36. 4. Li N, Li K A, Tong S Y. A novel protein assay method using tetraphenylporphin tetrasulfonate (TPPS4). Anal Lett 1995;28; 1763-74. 5. Li Y, Dong L, Wang W P, Xingguo Chen. Flow injection analysis-Rayleigh light scattering detection for online determination of protein in human serum sample. Anal Biochem 2006;354;64-9. 6. Feng P, Huang CZ, Li YF. Direct quantification of human serum albumin in human blood serum without separation of y-globulin by the total internal reflected resonance light scattering of thorium-sodium dodecylbenzene sulfonate at water/tetrachloromethane interface. Anal Biochem 2002;308 ;83-9. 7. Huang CB, Zhang K, Ci YX. Sensitization of surfactants on the chemiluminesecence reaction of fluorescein isothiocyanate labeled proteins. J Biochem Biophys Methods 2007;70;341-7. 8. Zhou J, Xing D, Chen Q. Enhancement of fluoresceinyl Cypridina lucifer in analog chemiluminescence by human serum albumin for singlet oxygen detection. Photochem Photobiol 2006;82; I 058-64.

CHARGE-TRANSFER-INDUCED LUMINESCENCE (CTIL) MECHANISMS OF CHEMI- AND BIOLUMINESCENCE REACTIONS K YAMAGUCHI, H ISOBE, S YAMANAKA, M OKUMURA

Dept of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan Email:[email protected] INTRODUCTION Chemi- and bio-Iuminescence phenomena have been attractive not only from the scientific interest conceming with their molecular mechanisms but also from analytical, clinical, and for other various useful applications. Many theoretical studies have been performed to elucidate detailed mechanisms of chemi- and bio-Iuminescence reactions. In past decades, our group has carried out both broken-symmetry (BS) and symmetry-adapted (SA) molecular orbital (MO) studies to clarifY the electronic mechanisms of these reactions. Here, our basic concepts and methodologies, together with computational results, are briefly summarized.

THEORETICAL BACKGROUNDS Symmetry and stability analysis. The semi-empirical unrestricted Hartree-Fock (UHF) method was used for symmetry and stability analysis of chemical reactions at early stage of our theoretical studies. I ,2 The BS MOs for CT diradicals are also expanded in terms of composite donor and acceptor MOs to obtain the Mulliken CT theoretical explanations of their electronic structures. Instability in chemical bonds followed by the BS ab initio calculations is one of the useful approaches for elucidating electronic structures of active reaction intermediates and transition structures. 2 The concept is also useful to characterize chemical reaction mechanisms in combination with the Woodward-Hoffman (WH) orbital symmetry criterion,3 as illustrated in Figure 1. According to the Woodward-Hoffmann rule,3 there are two types of organic reactions: orbital-symmetry allowed and forbidden. On the other hand, the orbital instability condition is the other criterion for distinguishing between nonradical and diradical cases. 2 The combination of the two criteria provides four different cases: (i) allowed nonradical (AN), (ii) allowed radical (AR), (iii) forbidden nonradical (FN), and (iv) forbidden radical (FR). The charge and spin density populations obtained by the ab initio BS MO calculations are responsible for the above classifications as shown in Fig. I, No charge and spin separations appear in the case of AN because of closed-shell character, but spin separation (SS) (1 D is significant for FR case, although the charge separation (CS) (ffi 8) is rather weak, as in the case of homolytic diradical. On the other hand, the zwitterionic (ffi 8) CS is remarkable for FN case, and both SS and CS (1 +, -!) become important for AR case such as electron-transfer diradical reactions. In recent papers, we have performed the symmetry-stability analyses of chemi- and bio-Iuminescence reactions from these theoretical view points. 4,5 261

262

Yamaguchi K et at.

Allowed Radical (AR) CS and SS

:§

:eo

Forbidden Radical (FR) SS but no CS

----_ .. _--------- ----, -----------.-----------_ ..

Allowed Nonradical : Forbidden Nonradical (AN) : (FN) CS but no SS no CS and no SS j

Orbital Symmetry

Fig. 1. Classification of chemical reaction mechanisms by symmetry and stability criteria of molecular orbitals Molecular oxygen reactions. In accord with the classification in Fig. 1, we may expect four characteristic reaction mechanisms of singlet molecular oxygen (10 2) with C-C double bonds to generate dioxetane and dioxetanone, as illustrated in Fig. 2.1 The perepoxide (or 2s + 2a) type cycloaddition of 102 to olefin is regarded as one of the AN reaction paths. On the other hand, 1,4-zwitterion path (B) is regarded as FN, whereas 1,4-diradical path (D) is characterized as FR. The electron-transfer diradical path (C) corresponds to the AR case in our terminology. It has been demonstrated that these mechanisms are very useful for systematic understanding of chemical reactions of singlet molecular oxygen e02) with various olefins. The computational results have been already summarized in one chapter of the book. 1

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40 for Ca2 +, C0 2 +, 30 for Cs +, AI 3 +, tartaric acid, respectively. Applications. The proposed method was applied to determine ascorbic acid in a sample of Yinqiao vitamin C tablets (0.0500 g ground up tablets (20) dissolved in water and filtered and diluted to 500 mL). The recovery rates of the method is 96.6% and 96.3 %; RSD was •

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Superweak Luminescence in Plants and Application to Salt Tolerance in Alfalfa

285

was a significant lag phenomenon in the former condition (Fig. 3b). In 1 % NaCl, the difference appeared 72 h after the seed germination among the 5 varieties. The peak value appeared at 120 h. At this time, the SL value of the tolerant variety was significantly higher than the other 3 varieties.

DISCUSSION SL is a unique method that allows continuous measuring of plant metabolism without destroying the regular growth of plant in biological research work. The mechanism of emission of has not been elucidated. Generally, when an electron in biological system is excited, and the excess energy is released as photons and the system returns to the ground state. Both biological oxidation and cell mitosis may be the source of plant SL and we hypothesize that biological oxidation and cell mitosis co-existed, each with its own effect on SL of the seed. The two peaks in the SL curve of germinating alfalfa seeds supports our hypothesis. There was a significant lag in luminescence of germinating seeds in 1% NaCI compared to distilled water and 0.5%NaCl, and this agrees with the observation that germination of the alfalfa seed under salt stress increases with increase in salt concentration. Under the same saIt stress, the germinating seed of different varieties of alfalfa emitted different SL, and this difference allowed ranking of the degree of salt tolerance of the alfalfa. Our data was the same as the culture experiment data, except that there was some difference in the rank of the sensitive variety and the medium tolerance variety. This may be related to the "flashing phenomenon" of the sensitive variety. Under adverse conditions, especially extreme conditions, some sensitive plants may have disordered metabolism, that is destructive and induces a rise in luminescence that appears as the "flashing phenomenon". In order to find out the salt tolerance limitation of the different varieties, we can increase the salt concentration. This method may be regarded as a useful attempt in researching the "plant flashing phenomenon" under the extreme condition.

ACKNOWLEDGEMENT Supported by Beijing Science Foundation (2007 [N] 16)and Foundation of National Science committee (NQCR-l 0-28) REFERENCES l. Popp FA, Li KH, Qu Q. Eds. Recent advances in biophoton research and it's application. Singapore:Worid Scientific. 1992. 2. Gurwitsch AA. A historical review of the problem of mitogenetic radiation. Experientia 1988,44:545-50.

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Zhou H et al.

3. Inaba H. Photonic Sensing technology is opening new frontiers in biophotonics. Opt Rev 1997;4:1-10. 4. Beloussov L, Popp FA, Voeikov VI, Wijk RV. Biophotonics and coherent systems. Moscow:Moscow University Press, 2000:439. 5. Yang Q, Yu T. Superweak luminescence of pea seedling under Na-salt stress. In: International symposium. Plants under environmental stress, Moscow, Russia. 2001:319-20. 6. Yang Q, Zhou H. The superweak luminescence and its oxidative reaction of plant under nonbiotic stress. 1st Asia conference on photobiology, Japan. 2002:91. 7. Zhou H, Yang Q. A study on the superweak luminescence of different plant seeds at the stage of germination. Acta Biophys Sin 1996,12: 157-60.

DEVELOPMENT AND OPTIMIZATION OF A QUANTITATIVE WESTERN BLOT AND DOT BLOT PROCEDURE FOR THE DETERMINATION OF RESIDUAL HOST CELL PROTEINS PRESENT IN INACTIVATED POLIO VACCINE USING A GZll BASED SIGNAL REAGENT

G ZOMER, M HAMZINK, A DE HAAN, G KERSTEN, K REUBSAET Unit Research and Development, Netherlands Vaccine institute, PO Box 457, 3720AL Bilthoven, The Netherlands Email: [email protected] INTRODUCTION Inactivated polio vaccine (lPV) contains as the active ingredient protective D-antigens. Because the polio virus is grown on a host cell system, residual host cell proteins (HCPs) may be present in the bulk product. During poliovirus cultivation the Vero host cells lyse. Proteins are released into the medium and cell fragments detach from the micro carriers. Cell fragments and host cell proteins are removed during down stream processing (DSP). DSP consists of filtration and chromatographic purification steps. Traces of host cell proteins (HCPs) however will remain in the monovalent pools. We investigated the amount of residual host cell proteins and the effect of the filtration and purification steps on the removal of the host cell proteins. In order to quantify these proteins two approaches were used. Firstly, the HCPs were separated on gel and after incubation with rabbit polyclonal antiserum directed against the HCPmix (capture antibody) followed by incubation with detecting antibody (HRP-labeled mouse anti-rabbit) subsequently visualized and quantified using a GZ 11 based signal reagent. Secondly, a dot-blot approach was followed using the same two incubation steps and detection with GZII signal reagent. MATERIALS AND METHODS Rabbit HCP-mix antiserum. The polyclonal rabbit anti-HCP serum was obtained by a cascade immunization of rabbits with a HCP mix from Vero cells. Three days before immunization blood was obtained from the rabbits. This pooled serum serves as preimmune rabbit serum. The Hcr mix, a blank culture of Vero cells is three times frozen and thawed to induce cell lysis. The culture supernatant was pooled and filtered. The pore size of the filters was comparable with those used during the IPV Vero production process. The HCP mix was divided in small aliquots, rapid frozen using dry ice and stored at -70 °c until use. Rabbit anti-HCP serum: Three rabbits were immunized with the HCP-mix and received a booster with the same HCP mix after 28 days. On day 42 blood samples were collected. Using the antibodies as an affinity column the immuno-dominant proteins were removed from the HCP-mix. The treated HCP-mix was used to immunize the 3 rabbits again on day 56. The same procedure was performed with serum of day 77 to immunize at day 85. The rabbits were given a booster on day 121 followed by collection blood on day 135. The pooled serum of day 135 was used in this study as anti-HCP serum.

287

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Dot blot procesure. Suitably diluted (in PBS) samples or standards (50 ilL) were applied to a nitrocellulose membrane using an ELI FA (Pierce) Unit. After washing with 200 ilL of PBS buffer, the membrane was incubated with polyclonal anti-HCP (1 :300 in assay buffer) for 1 h with gentle shaking. After washing with wash buffer (PBST, PBS containing 0.1 % Tween20), the membrane was incubated with 2nd Ab.HRP (l: 100,000 in assay buffer) for 1 h with gentle shaking. The membrane was washed (4x) with wash buffer. GZll signal reagent (a gift from ZomerBloemen b.v.) was added to the membrane, and after incubating for 5 min, decanted. The glowing membrane was measured in a chemiluminescent imager (Fluorchem 8900) and images were stored as TIF-files and analyzed using Image-l software. Western blot analysis. A suitable amount of sample was first precipitated using a 72% w/w trichloroacetate (TCA)-solution. The samples were thoroughly vortexed followed by centrifugation. The supernatant was carefully removed and 15 ilL of reducing buffer was added. The samples were thoroughly vortexed and placed in a thermostat at 100 DC during 5 minutes. After this the samples were centrifuged for a few seconds, only to let the liquid in the top of the sample container to be spun down. The slots of the precast gel were loaded with 15 ilL of sample and MW -markers (10 ilL). After electrophoresis the gel was washed with water and blot buffer. Via a cold tank transfer in blotting buffer a Western Blotting was performed. After washing with PBS buffer, the membrane was incubated with polyclonal anti-HCP during one hour with gentle shaking. After washing with wash buffer, the membrane was incubated with 2 nd Ab.HRP (1:100,000 in assay buffer) during one hour with gentle shaking. The membrane was washed (4 x) with wash buffer. GZll signal reagent was added to the membrane, and after incubating for 5 minutes, decanted. The glowing membrane was measured in a chemiluminescent imager (Fluorchem 8900). Resulting images were stored as TIF-files and analyzed using Image-l software. Samples. Down stream processing samples that were analyzed consisted of the fraction after concentration (2.1), after size exclusion chromatography (3.1), and after ion exchange chromatography and sterile filtration (5.1). RESULTS AND DISCUSSION An example of a western blot together with a residual protein staining blot is shown in Fig 1. Clearance of the host cell proteins during DSP using a combination of size exclusion and ion exchange chromatography is successful. During the first chromatographic step HCPs are cleared about 50-fold, while after the second chromatographic step no more HCPs can be detected using western blot. Also with respect to the concentration of D-antigen, the vaccine active ingredient, HCPs are cleared efficiently resulting in an increase of specific activity of D-antigen with respect to total protein. From the protein stained blot clearance of HCPs can also be inferred; in the 5.1 fraction only virus related proteins are observed with no indication of HCPs. Although useful in detecting residual HCPs western blot analysis gives only semi-quantitative results (when using serial dilutions of samples, results not shown). I

Ue 1vell)plrlem and Optimization of a Quantitative Western Blot and Dot Blot Procedure

289

Left Western blot stained with HCP polyclonal antibodies. Lane 1 contains mw markers 40,50,60,80, 120,220 kD), lane 2 is a sample of production fraction 1 diluted 250 times, lane 3 contains a sample of production fraction 3.J diluted 10 lane 4 contains the HCP-mix diluted 10 times, lane 5 contains an of production fraction 5.1. Lane 6 contains normal rabbit serum as a control. residual protein staining blot (right, lane 1 mw lane lanes 3-9 correspond with lanes 1-6 of the western blot) on a Because dot blot analysis allows for more samples to be membrane and because dot blots are more easily quantified this technique was also used to assess HCP clearance during DSP. An example is shown in 2. As can be seen from this dot blotting can be performed with reasonable reproducibility and The lowest concentration of the calibrator that can be quantified is about 100 ng/mL. This allows for dilution of 2.1 (6,000-50,000) and 3.1 ~'mll'II'~ (200-2,000) while 5.1 samples were run undiluted. To obtain reliable results were diluted using sample weights instead of sample volumes. Within reproducibility was better than 10% while day to day reproducibility was better than 30%.

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Zomer G et al.

• •• ••

••• •••

2. Dot blot analysis (top left) Dot blot stained with HCP polyclonal antibodies. The top two rows are calibration standards ofHCP mix in duplicate (7 dilutions from 3.8-0.19 /lg/mL plus blank, from left to right). The third and fourth rows are 8 dilutions in duplicate (4&,000-6,000, left to right) of a 2.1 IPV production fraction while the fifth and sixth rows are &dilutions in duplicate {2,000-250, left to of a 3.1 IPV production fraction. Corresponding profile data of the dot blot are shown on the right with the calibration curve (bottom

REFERENCES 1.

Zomer Smitsman C, Arts R, Hamzink M, Kooijman M. blotting using a GZ-ll based chemiluminogenic signal reagent. In: Hill Pl Kricka LJ Stanley PE, eds. Proceedings of the International Symposium on Bioluminescence and Chemiluminescence. World 2006:171-4.

DEVELOPMENT AND OPTIMIZATION OF A FAST AND SENSITIVE ELISA FOR POLIO D-ANTIGEN USING A GZll BASED SIGNAL REAGENT G ZOMER, M HAMZINK Unit Research and Development, Netherlands Vaccine institute, PO Box 457, 3720AL Bilthoven, The Netherlands Email: [email protected]

INTRODUCTION The potency of inactivated polio vaccine (IPV) traditionally is determined by the measurement of the protective D-antigens present in the vaccine. Mostly, a sandwich ELISA test is used for this purpose. l During production of the polio virus and during down stream processing of the vaccine there is a need for a reliable indication of the concentration of D-antigens that is present in the different sample types. Customarily, the ELISA test involves several incubations: Firstly, the binding of the antigens using a type specific antibody coated micro titerplate. Secondly, the antigen is bound by a type specific monoclonal antibody, and thirdly the monoclonal antibody is bound by an HRP-labeled 2nd antibody (conjugate). Each incubation step is followed by washing steps making this assay rather time consuming. In order to speed up the assay the second and third incubation step were combined. Moreover, the incubation steps were performed at 37°C on a shaking incubator resulting in a faster assay. Furthermore, because of the high sensitivity of detection of the HRP-conjugate using the GZ-ll signal reagent much less antibodies (coat, monoclonal, and 2 nd antibody) could be used resulting in a rapid (within 2 hours) and sensitive ELISA. MATERIALS AND METHODS ELISA plates (Greiner, white, high-binding) were coated with type-specific caprylated bovine antiserum diluted 1: 1600 in PBS, overnight at 4°C. Coated plates can be stored dry at -80°C. The plates were washed with wash buffer (PBST, PBS containing 0.1 % Tween20). Samples and standards (100 [lL, diluted in assay buffer (wash buffer containing 0.5% Protifar (Nutricia)) were added to the wells. The sealed plate was incubated at 37°C on a shaking incubator during 30 minutes and washed two times using wash buffer. Incubation with 100 [lL of a mixture of type specific suitably diluted monoclonal (capture antibody) and HRP-Iabeled goat anti mouse (detection antibody) was performed at 37°C on a shaking incubator during 30 minutes and washed four times using wash buffer. GZll based signal reagent (a gift from ZomerBloemen b.v.) was added (100 [lL Iwell) and the glowing plate was placed into a plate luminometer (Berthold Centro LB960). From the raw data calibration curves were constructed using a four parameter fitting routine (MS-Excel) from which unknowns were calculated. Two different protocols were compared during the study (please refer to Table 1 for details). 291

292

Zomer G & Hamzink M

Ta bilL e ayouto f two-steJ Three-step protocol Incubation with antigen (step 1) Wash Incubation with capture antibody (step 2) Wash Incubation with detection antibody (stefl 3) Wash Addition ofOZll signal reagent Measurement

an d tree-step protoco h Two-step protocol Incubation with antigen (step 1) Wash Incubation with capture and detection antibody (step 2) Wash Addition ofOZll signal reagent Measurement

RESULTS Effect of coating dilution. The effect of using different concentrations of coating antibody in the range 1 :200 to 1: 1600 is very small (please refer to Fig. 1 for details). In the final assay a dilution of 1: 1600 was used. 25.0 20.0 -+-200 : ....... 400

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Fig. 1. The effect of coating dilution on calibration curve for type 1 D-antigen using sequential incubations with monoclonal antibody and conjugate. Effect of shaking. Shaking the micro titerplate during the first incubation (with antigen) results in a much faster establisment of equilibrium (see Fig 2 for details). Equilibrium was reached after shaking the plate during 30 minutes, and this time period was adopted for all incubation steps.

Development and Optimization of a Fast and Sensitive ELISA for Polio D-Antigen

293

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Effect of two-step vs three-step incubation In order to combine the incubations with capture and detection antibody the effect of diluting the capture antibody concentration was studied. Figure 3 shows the results which clearly indicate that diluting the capture antibody greatly improves the specific binding. When more concentrated capture antibody concentrations are used the detection antibody binds less to the sandwich resulting in a lower signal. 45000 40000 35000 30000 =0 ..l

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Fig. 3. Effect of diluting the monoclonal antibody in the two-step protocol results in improved assay performance (detection antibody dilution - 1 :500,000) When the capture antibody is further diluted, down to 1:50,000 the calibration curves for the two-step protocol and three-step protocol became virtually superimposable (please refer to Fig 4 for details).

294

Zomer G & Hamzink M

60000 50000 40000 :> ..J

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Fig. 4. Comparing two-step vs three-step protocol using 1:50,000 dilution of capture antibody (detection antibody dilution 1:500,000) DISCUSSION In this study we have shown that by combining two incubation steps and by performing all incubations at 37 DC on a shaking incubator together with the use of the GZl1 signal reagent a sensitive and rapid ELISA method has been developed. The use of GZll signal reagent with its inherent great sensitivity for HRP detection allowed for the dilution of coating, capture and detection antibodies, resulting in much less aspecific binding. The signal reagent is a two-component stable formulation which when mixed 1:1 can be used at least during one working day. REFERENCES 1. Rezapkin G, Dragunsky E, Chumakov K. Improved ELISA test for the determination of potency of Inactivated Poliovirus Vaccine (lPV). Biologicals 2005;33:17-27. 2. Zomer G. Development of a chemiluminescence immunoassay for clara cell protein at sub-pM levels. In: Case JF, Herring PJ, Robison BH, Haddock SHD, Kricka LJ, Stanley PE, ed. Proceedings of the 11 th International Symposium on Bioluminescence and Chemiluminescence. Singapore:World Scientific, 2001: 377-80.

PARTS APPLIED ELECTROLUMINESCENCE


DETECTION OF XANTHOMONAS ORYZAE Pv. ORYZICOLA BY ELECTROCHEMILUMINESCENCE POLYMERASE CHAIN REACTION METHOD JIE WEI, LINGRUI ZHANG MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631,China Email: [email protected]

INTRODUCTION

Xanthomonas oryzae pv. Oryzicola (Xoo) is a gram-negative, rod-shaped bacterium. It is emerging as an important pathogen of rice and is a recognized biosecurity threat

to most of the rice growing countries. Thus, a highly sensitive, yet simple and safe approach for Xoo detection is required. The electrochemiluminescence polymerase chain reaction (ECL-PCR) method has high sensitivity in nucleic acid analysis, which we have described in our previous articles. '4 We have now developed a new ECL-PCR method and used it to detect Xoo for the first time. The principle of this ECL-PCR method is shown in Fig. I. 16-23s rDNA of Xoo was amplified by PCR. At the end of 3' terminal of the primers, a pair of universal sequences is added so that all PCR products contain these sequences. These PCR products are used to hybridize with a TBR-probe and biotin-probe, which is complementary to the universal

sequence.

streptavidin s.7 ,

Through the

specific

interaction

between

biotin

and

the hybridized products are captured by magnetic beads that are

coated by streptavidin. After magnetic separation, the samples are mixed with TPA and detected by ECL. This method is simple and highly sensitive; it can significantly reduce costs by employing the universal probes. EXPERIMENTAL The forward primer was 5'-TAACTGAATAGACTAAGACGCATGACGTCAT CGTCCTGT -3'.

The

reverse

primer

was

5'-CTAATCAACGACCTTGTATCCTC GGAGCTATATGCCGTGC-3'. The TBR probe was 5'-TBR-TAACTGAATAGA CTAAGAC-3'. The biotin probe was

5'-biotin-GATACAAGGTCGTTGATTAG-3'.

297

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cetyltrimethyl

298

Wei J & Zhang L

ammonium bromide (CT AS) method for sample extraction and purification reported by Lipp. et aZ. was used in this study.8 The amplification protocol consisted of 2 min at 94°C for initial denaturation, 30s at 63°C for primer annealing and I min at 72 °c for extension. After amplification, biotin-probe and TBR-probe were added to. The mixtures were incubated for 5 min at 94°C and I h at 63°C. Then, streptavidin coated magnetic beads were added. The mixture was then shaken at room temperature for 30 min. After washing and removing the supernatant, the samples were added to the detection cell of ECL analyzer (built in our lab).9

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Fig. 1. The principle of this EeL-peR method for detection of Xanthomonas oryzae pv. Oryzicola RESULTS AND DISCUSSION

Fig. 2a shows the EeL detection results of both healthy and infected samples. From this figure we can see that, the EeL intensity of infected samples and un infected samples has such a striking contrast that we can clearly distinguish them. In order to verify the feasibility of this method, 1% agarose gel electrophoresis analysis for peR products was performed in the experiment (Fig. 2b). condition is 1% agarose gel at 80Y for an hour.

The electrophoresis

Lane I and lane 2 are all the peR

products of infected samples. Lane M represent markers (100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp). Lane 1-2 has a band between 400 and 500bp which was consistent with the expected peR product of size

oryzae pv. Or,vzicola by Electrochemiluminescence peR Method

Detection

299

electrophoresis are consistent with the results of ECL

The results of detection.

In

we have developed a sensitive ECL-PCR method for Xoo detection. to the traditional detection methods, this ECL-PCR is a

low

noise and specific technique. Due to its sensitivity and simplicity, this will have an enormous potential for detecting plant pathogenic bacteria.

time(s)

(b)

ECL detection results of both healthy and infected samples and the agarose electrophoresis analysis results of the PCR products.

ACKNOWLEDGEMENTS This research is supported by the National Natural Science Foundation of China 307001

the National High Technology Research and

of China (863 Program) (2007 AA lOZ204», and the Natural Science Foundation of

,imm2:00IH!

Province (7005825).

REFERENCES . Liu

Shen XV, Zhu DB.

Detection of genetically modified

electrochemiluminescence PCR method. Biosens Bioelectron Shen XY, Liu JF. A method to quantitatively detect H-ras point mutation based on electrochemiluminescence. Biochem Biophys Res Commun

300

Wei J & Zhang L

3. Liu JF, Xing D, Shen XY. Electrochemiluminescence polymerase chain reaction detection of genetically modified organisms. Anal Chim Acta 2005;537:119-123. 4. Zhu DB, Xing D, Shen XY, Liu JF, Chen Q. High sensitive approach for point mutation detection based on electrochemiluminescence. Biosens Bioelectron 2004;20:448-53. 5. Blackburn GF, Shah HP, Kenten JH. Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. Clin Chem 1991;37:1534-39. 6. Jong MD, Weel JFL, Schuurman T, Quantitation of Varicella-Zoster Virus DNA in whole blood, plasma, and serum by PCR and electrochemiluminescence. J Clin MicrobioI2000;38:2568-73. 7. Collins RA, Ko LS, Fung KY. A method to detect major serotypes of foot-and-mouth disease virus. Biochem Biophys Res Commun 2002;297:267-74. 8. Schutzbank T, Smith 1. Detection of human immunodeficiency virus type 1 proviral DNA by PCR using an electrochemiluminescence-tagged probe, J Clin MicrobioI1995;33:2036-41. 9. Zhu DB, Xing D, Shen XY, Yan GH. High sensitive detection of presenilin-l point

mutation

2003 ;48: 1741-44.

based

on

electrochemiluminescence.

Chin

Sci

Bull

A NOVEL ELECTROCHEMILUMINESCENT SENSOR BASED ON CATiONIC POLYMER/CHITOSAN FOR ULTRASENSITiVE DETECTiON OF HYDROGEN PEROXIDE XIAOPING WU,* YOUMEI WANG, HONG DAI, GUONAN CHEN Ministry of Education Key Laboratory of Analysis and Detection Technology for Food Safety and Department of Chemistry, Fuzhou University, Fuzhou 350002, Fujian, China; *E-mail: [email protected]

INTRODUCTiON Electrochemiluminescence (EeL) is the most sensitive detection technique for EeL-based sensors have emerged as powerful tools for ultrasensitive analysis. I ,2 The electron transfer rate between the electrode and as well as the reactivity and interaction of the target analyte with modified are key considerations for biosensor fabrication. the the modified and selectivity of biosensor largely depend on the formation and immobilization of a functional modified film on the base surface. have gained interest in industrial applications and because of their macromolecular size exclusion effect and We found that poly(diallyldimethylammonium chloride) used cationic polyelectrolyte,4 could enhance the EeL intensity of luminol. Therefore an EeL-based sensor - PDDA-chitosan modified glassy carbon electrode was developed and applied for ultrasensitive analysis (Fig. 1), of peroxide via reaction with luminol (detection limit of 0.85 nmoI/L). The combination of highly specific biological reaction and the sensitive EeL detection provides a powerful analytical tool for clinical application. MATERIALS AND METHODS Luminol, poly(diallyldimethyl ammonium chloride) and chitosan were obtained

1. EeL generation on the PDDA-chitosan modified GeE. 301

302

WuX etal.

from Sigma. ECL detection was performed by using a BPCL Ultra-Weak Chemiluminescence Analyzer (Institute of Biophysics, Chinese Academy of Sciences) with a CHI 620B electrochemical analyzer (Shanghai Chenghua Instrument Co., China) as potential controller. A three-electrode system was used, including a PDDA/chitosan modified GCE as the working electrode, a platinum wire as the counter electrode and Ag/AgCI (sat. KCI) electrode as the reference electrode. Electrode preparation. A 0.5 % w/w chitosan solution was prepared according to Burchardt et al. 4 The PDDA-chitosan film modified electrode was prepared by immobilizing 5 /-lL of PDDA-chitosan solution [60 /-lL PDDA (10 % w/w) and 40 uL chitosan (0.5 % w/w)] on the surface of glassy carbon electrode. After drying at room temperature, it was used directly for ECL detection. RESULTS AND DISCUSSION Electrochemical chracterization of PDDA-chitosan modified GCE. Fig. 2 shows cyclic voltammograms (CYs) of 1.0 mmoliL ferricyanide at differently modified electrodes. Compared with the response at bare GC (curve a), the peak current obtained at the chitosan modified electrode was increased (curve b), owing to the absorbtion of the negatively charged ferricyanide to the highly positively charged chitosan on the electrode surface. When the electrode was modified with PDDA-chitosan solution, the continuing increase of peak current became obvious (curve c). That can be attributed to the replacementofa part of chitos an by PDDA, which has highly positive charge density and permselectivity. The PDDA-chitosan modified film is a better mass transfer layer, which is useful as an ion-to-electron transducer. 120 80

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Potential I V

Fig. 2. Cyclic voltammograms offerricyanide at different electrodes: (a) bare GCE; (b) Chitosan modified GCE; (c) PDDA-chitosan modified GCE. Supporting electrolyte, 1.0 mmollL Fe(CN)63- + 0.2 moliL KCI; scan rate, 50 mY·s- 1 .

A Novel Electrochemiluminescent Sensor Based on Cationic PolymerlChitosan

303

Cyclic voltammetry and ECL of luminol-H 202 system at different electrodes. Cyclic voltammograms of luminol at different electrodes in 0.1 moUL phosphate buffer (PBS, pH=7.5) were obtained (Fig. 3A). Similarly, an enhancement of redox current from the analyte was observed at the PDDA-chitosan modified GCE, compared with the response at the bare GCE. This is due to the good permselectivity and highly positive charge density of the PDDA-chitosan composite layer. The negatively charged luminol could be easily absorbed on the surface of modified GCE through electrostatic interaction, which was supported by the linear increase of oxidation current vs. scan rates. The ECL response of luminol at bare GCE and modified GCE were obtained (Fig. 3B). It was observed that the ECL intensity of luminol system increased remarkably (Fig.3B-c) when the PDDA-chitosan modified GCE was employed, that indicated the rapidly enrichment ofluminol on the surface of modified GCE. 2000

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Fig. 3. (A) Cyclic voltammograms from mixing 2x 10- moUL luminol and 1 xl 0- 7 moUL H 20 2 in 0.1 mol/L PBS buffer solution (pH=7.5) at (a) bare GCE; (b) 5 PDDA-chitosan modified GCE. (B) ECL response from mixing 2 xl 0- moUL 7 luminol and I x 10- moUL H 2 0 2 in 0.1 mollL PBS buffer solution (pH=7.5) at (a) Bare GCE; (b) Chitosan modified GCE; (c) PDDA-chitosan modified GCE. The effect of some experimental parameters on the ECL intensity of luminol-H 2 0 2 system were studied. The optimum conditions for the detection of H 20 2 were scan 5 mode, CV; scan rate ISO mV/s; buffer, 0.1 mollL PBS, pH 7.5; luminol, 2 x 10moUL. Reproducibility and stability of PDDA-chitosan modified GCE. The proposed method was reproducible, and the relative standard deviation (RSD) of ECL from mixing I xIO- 7 moUL H 2 0 2 and 2.0xIO-5 mol/L luminol solution was 2.6% for 10 independently prepared electrodes, and 0.97 % for one electrode. The PDDA-chitosan modified GCE showed good repeatability and long-term stability. The electrochemical and ECL response of the modified electrode remained unchanged even when it had been stored at 4 °c for two weeks.

304

Wu X et al.

Linear range and detection limit for H 20 2 detection. Under the optimization condition, a calibration curve for the determination of H 2 0 2 was obtained in aqueous solution. The ECL intensity (IECL) was linear with the concentration of H 20 2 (CH202 ) in the range of 1.0xlO-9-5xlO-5 moVL [IEcd (a.u.) =3374.5 + 2748.2C H202 1 (moI/L) R2=0.9984]. The detection limit (S/N=3) was 8.5xl0- l omollL. Owing to the size exclusion effect and permselectivity of polyelectrolytes, the PDDA-chitosan modified GCE was free of interference that may co-exist in biological samples, such as K+, Na+, SO/-, N0 3 -, lactose, amylum, sucrose, fructose, maltose, citric acid, uric acid and ascorbic acid. The results indicated a good selectivity to the detection of hydrogen peroxide.

CONCLUSION The PDDA-chitosan modified glassy carbon electrode greatly enhanced the ECL response of luminol-H2 0 2 system and showed good selectivity and repeatabilty for detection of H 20 2 (detection limit ca. 8.5 x 10- 10 mollL) and a five orders of magnitude dynamic working range. The performance of this sensor makes it very attractive for future application, since hydrogen peroxide is not only an essential mediator but also a by-product of several highly selective oxidases in biological processes. ACKNOWLEDGEMENTS This project was supported by the National Nature Sciences Foundation of China (20735002, 20575011), Program for New Century Excellent Talents in University (NECT-06-0572) and Fujian Provincial Natural Science Foundation of China (00510006). REFERENCES 1. Richter M. Electrochemiluminescence (ECL). Chern Rev 2004; 104:3003-36. 2. Marquette C, Leca B, Blum LJ. Electrogenerated chemiluminescence of luminol for oxidase- based fibre-optic biosensors. Luminescence 2001;16:159-65. 3. Yang M, Yang Y, Liu B, Shen G, Yu R. Amperometric glucose biosensor based on chitosan with improved selectivity and stability. Sens Actuat B 2004; I 0 1:269-76. 4. Burchardt M, Wittstock G. Kinetic studies of glucose oxidase in polyelectrolyte multilayer films by means of scanning electrochemical microscopy (SECM). Bioelectrochem 2008;72:66-76.

CAPILLARY ELECTROPHORESIS-ELECTROCHEMILUMINESCENCE DETECTION OF CIPROFLOXACIN IN BIOLOGICAL FLUIDS XIAOMING ZHOU: Ll JIA

MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China, Email: [email protected]

INTRODUCTION Ciprofloxacin (CIP) is a potent second generation fluoroquinolone drug (Fig. 1) widely used both in human and veterinary medicine to treat infectious diseases. High-performance liquid chromatography (HPLC) based methods have been em-ployed for its deter~inationY Capill~ry electrophoresis (CE) may have potential for CIP analysis. Advantages of CE for CIP analysis include its speed and cost of analysis, and the possibility of rapid method development. Electrochemiluminescence (ECL) is a type of chemiluminescence produced as a result of electrochemical reactions. ECL detection has many advantages including its simplicity, inexpensive instrumentation, low background noise, high sensitivity, 3 good ~electivity, ~nd wide dynamic linear range. - We have dev~loped a ne~ simple and sensitive CE-ECL method for CIP analysis in biological fluids. The method is based on the CE separation and the detection of secondary amino moieties in CIP with end-column tris(2.2-bipyridyl)ruthenium(II) electrochemiluminescence.

o

o

I

F

OH N

L Ciprofloxacin Fig. 1. The molecular structure of CIP 305

306

Zhou X & Jia L

EXPERIMENTAL Reagents were of analytical grade. Solutions for CE were stored at 4°C. The solutions were all passed through 0.22 mm filters before being injected into the CE system. The CE-ECL system was described previously.4 A standard stock solution of CIP was dissolved in methanol (1 mg/mL) and stored at 4°C. Working standard solutions were prepared daily by diluting the stock standard solutions in 0.1 % acetic acid. For preconditioning, the capillary was pretreated by rinsing at high pressure with 1 MNaOH for 10 min, pure water for 10 min, and phosphate electrolyte for 15 min. In order to obtain better reproducibility, between runs, the capillary is rinsed at high pressure with 0.1 M NaOH 1 min, pure water for 2 min, and buffer for 3 min. The injection was done electrokinetically and CE was performed at room temperature. Blood samples from a healthy volunteer were collected and immediately centrifuged at 3000 rpm/min for IS min. Serum was spiked with CIP at different concentration levels aliquotted and stored at -20°C. Before use, the serum was diluted 10-fold with 0.1 % acetic acid to decrease the interference of the ionic strength of the sample matrix. Fresh urinary samples from a healthy volunteer were spiked with CIP at different concentration levels, and were filtered through a membrane (0.22 ~m). After that, the samples were diluted 20-fold with 0.1 % acetic acid. The filtrate was injected into the CE-ECL system and analyzed. RESUL TS AND DISCUSSION Optimization of CE-ECL conditions. The detection potential was firstly optimized because ECL is dependent on the potential applied to the electrode. It was found the highest ECL intensity was obtained for a potential of 1.15 Y. The ECL reaction of Ru(bpy)/+ with alkylamine was a pH-dependent process and the maximum ECL emission was observed in pH 8.5 for crp. The concentration of phosphate buffer in the ECL detection reservoir was also carefully examined. The corresponding ECL intensity increased remarkably when the concentration of phosphate buffer was changed from 20 to 100 mM, but the baseline was unstable > 100 mM. Therefore, 100 mM phosphate buffer was selected. The effect of the concentration of Ru(bpy)/+ on ECL intensity was investigated. The experimental results showed that the ECL signals for crp increase almost linearly with the increase of the Ru(bpy)/+ concentration in the range from 2 to 8 mM. While the background noise also increases with the increased of the concentration of Ru(bpy)/+. Considering the signal to noise ratio,S mM Ru(bpy)/+ was selected. Separation voltage was investigated in the range of 8 to 20 kY. When separation voltage increased, ECL intensity increased and reached a maximum at 14 kY. When the separation voltage exceeded 14KY, the ECL intensity decreased. This is due to the strong flow of effluent from the capillary decreased the concentration of Ru(bpy)3 2 + at the working electrode surface, thereby reducing the efficiency of ECL reaction. Thus, 14 kY was selected.

,au"ua,' v

2.

Electrophoresis-Electrochemiluminescence Detection of Ciprofloxacin

pr()or~lm"

307

of serum samples (A), urinary samples (8), and standard CIP samples (C).

Detection limit of CIP. Calibration was linear in the range 0.05-1.5 (y=810 (±26) x + 45 (±18) and R=0.997). Detection limit of 15 with a signal-to-noise of 3 was achieved for CI P. The repeatability of the method was studied by six consecutive injections of standard solution of CIP at 1 Relative standard derivations of the ECL intensity and the migration time were 3.25 and 0.84% for CIP, respectively. to human urine and human serum. The proposed CE-ECL method to the determination of CIP in urinary samples and blood In both urinary samples and blood samples were spiked with different concentrations levels of CI P. Due to the inherent excellent selectivity and of the CE-ECL method, the samples were prepared without extra some simple procedure such as concentration, filtration, and dilution. was the electropherograms of the blank serum sample (a), and 50 CIP standard solution was spiked into serum sample as shown in (b).

308

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was the electropherograms of the blank urinary sample (a), and 10 ""giL CIP standard solution was spiked into urinary sample as shown in (b). The peak of CIP was verified according to the electropherograms of standard samples as shown in Fig. 2 (C). Reported concentrations ofciprofloxacin varied from 0.1 to 0.65 mg/L in CE-ECL is sensitive enough for the measurement of CIP in biological fluids. We noted that although some unknown peaks were found in the electropherograms of aqueous and from 0.17 to 0.51 mg/L in vitreous after the oral administration of various doses of the antibiotic to humans. s Therefore the current results indicate this both urinary and blood samples, no interferences were found co-migrating with CIP thus showing the proper specificity of the proposed method.

ACKNOWLEDGEMENTS This research is supported by the National Natural Science Foundation of China (30600128; 30700155), the National High Technology Research and Development Program of China (863 Program) (2007AAI0Z204)), and the Natural Science Foundation of Guangdong Province (7005825). REFERENCES 1. Liang H, Kays MB, Sowinski KM, Separation of levofloxacin, ciprofloxacin, gatifloxacin, moxifloxacin, trovafloxacin and cinoxacin by high-performance liquid chromatography: application to levofloxacin determination in human plasma. I Chromatogr B 2002;772:53-63. 2. Zotou A, Miltiadou N, Sensitive LC determination of ciprofloxacin in pharmaceutical preparations and biological fluids. I Pharm Biomed Anal 2002;28:55-68. 3. Zhu DB, Xing D, Shen XY, Liu IF. A method to quantitatively detect H-ras point mutation based on electrochemiluminescence. Biochem Biophys Res Commun 2004;324:964-9. 4. Gao WD, Liu JF, Yang XR, Wang E. New technique for capillary electrophoresis directly coupled with end-column electrochemiluminescence detection. Electrophoresis 2002;23:3683-91. 5. Lesk MR, Ammann H, Marcil G, Vinet B, Lamer L, Sebag M. The penetration of oral ciprofloxacin into the aqueous, vitreous and subretinal fluid of humans. Am I Ophthalmol. 1993; 115:623-628.

PART 6 BIOMEDICAL APPLICATION OF FLUORESCENT PROTEINS


A NOVEL MULTICOLOR FLUORESCENT PROTEIN FROM THE SOFT CORAL SCLERONEPHTHYA GRACILLIMA KUEKENTHAL YUKO KATO,I MITSURU JIMBO,2 CHITOSHI SATO,3 T ASTUY A TAKAHASHI, I YUKIMITSU IMAHARA,4 HISAO KAMIY A2 1Department of Environment, Tohwa University, Fukuolw City, 815-8510, Japan 2Department of Marine Biosciences, Kitasato University, iwate, Japan ~ 3Junshin Junior College, Fukuolw City, 815-8510, Japan Walwyama Prefectural Museum of Natural History, Japan Email: [email protected]

INTRODUCTION In recent years marine organisms have been considered as potential sources for useful substances such as medicines and biotechnological reagents. Green fluorescent protein from Aequorea victoria is well known for its usefulness in biotechnology. I In our survey of the fluorescent proteins in marine animals, Scleronephthya gracillima (Kuekenthal) was found to possess multicolored fluorescent proteins. This paper deals with the separation of a new fluorescent protein named "Akane" and also its fluorescent spectroscopic properties. The fluorescence dependency on pH suggests that the fluorescence derives from protonation of the chromophore.") The cDNA cloning of "Akane" was also conducted. METHODS Extraction and separation of fluorescent protein. The soft coral S. gracillima Kuekenthal was first fractionated by a gel filtration, then the fluorescent protein was separated by anion exchange column chromatography on an anion Q-Sepharose High Performance column (Amersham Biosciences) after ultra-filtration with a USY-J membrane (Advantec). All the fractions were analyzed by spectrofluorophotometer (RF-5300 PC Shimadzu). Fluorescent spectroscopic analysis. The fluorescent spectroscopic analysis of the fraction from the anion exchange column was performed using J0 mM-Tris buffer pH 8.5, changing O.lM to 0.15M NaCI. RESULTS AND DISCUSSION The fluorescence intensities at each wavelength (430, 505, 570, and 660 nm) and absorbance (280 nm) of the fraction are depicted in Fig. I. Multiple emissions were observed at one excitation wavelength 298 nm. Fluorescent spectra of Fr (2-10) and Fr (3-6) were examined at different sodium chloride concentrations. Emission peaks were observed at 430 480, 505, 570, 636 and 663 nm. The remarkable result was that the 570 nm emission peak only appeared in 0.15M-NaCI (Fig. 2). pH dependency of the fluorescent spectra were analyzed by changing the pH from 7.0 to 8.5. Results are shown in Fig. 3. 311

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A Novel Multicolor Fluorescent Proteinfrom the Soft Coral S. gracillima Kuekenthal Fluorescence -pH8.S Int.

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Fig. 3. Emission spectra of Fr (3-6) (left) and Fr (2-10) (right) in pH range 7.0 to 8.5. left: Fr (3-6) 0.15M-NaCI was excited by 298 nm, changing the pH from 7.0 to 8.5. It was remarkable that the 566 nm emission only appears at pH 8.5, then 477 nm emission appeared at pH 8.0 to 7.0, while the shape of 663 nm and 503 nm emissions did not show significant change. right: Fr (2-10) 0.10 M-NaCI was excited at 298 nm. The 475 nm emission appeared at pH 8.0 to 7.0 not at pH 8.5, while the 436 nm emission only appeared at pH 7.0. LC-ESI-MSIMS analysis and eDNA Cloning. In-gel trypsin digestion of the component followed by peptide fragment sequence analysis by LC-ESI-MS/MS LCQdecaXP gave the amino acid sequence of YPADLPDYFK. The homology search based on MASCOT on the NCBInr Database revealed that the component showed homology to a cyan fluorescent protein Acropora aculeus. Then cDNA was cloned by the RACE method using a gene specific primer designed from amino acid sequences obtained by LCIESI/MS/MS. Four closely related cDNAs were obtained. The ORFs of the cDNAs are composed of 225 residues. Deduced amino acid sequence of "Akane" is contained the putative chromophore sequence ofGFP family at His62-Tyr63-Gly64 for (Table 1). Table 1. Complete gene sequence of "Akane" 1 41 81 121 161 201

MNPIKEDMKV KVYLEGNVNG HAFAIEGEGK GNPLDGTQTM NLTVKEGAPL PFSFDILTTS LHYGNRVFTK YPADIPDYFK QSFPEGFSWE RTMTYEDKGI CTIRSDISLQ GDCFIQKVRF HGINFPSNGP VMQKKTLKWE PSTERMYVRD GVLVGDINNA LLLEGGGHYV CDFKTTYKAK KVVQLPDYHF VDIRIEILSH DRDYNKVKLY EHAVARHSLV PSQAR *

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CONCLUSION The fluorescent protein was separated from the extract of the soft coral by gel filtration and anion exchange column chromatography. In the fluorescent spectroscopic analysis of the purified protein, multiple emissions from the protein were observed (using one excitation wavelength) and these emissions depended on the pH (7.0 - 8.5) and also on the sodium chloride concentration. The complete gene sequence of "Akane" was gained from cDNA cloning. The partial sequence 081SFPEGFSWER91 is similar to the DsRed sequence' and the 71 80 y pADLPDYFK is a homology to a cyan fluorescent protein Acropora aculeus. Thus, cDNA of "Akane" had strong similarities to green fluorescent protein families especially to Dendronephthya sp." in the sequence of y71_T92MTYEDKGICTIRI04.

ACKNOWLEDGEMENTS We are grateful to Sachiko Matsuhashi, Faculty of Medicine and the Graduate School of Saga University, and Susumu Watanabe, Hitachi High-Tech Manufacturing & Service Corporation Proteome Analysis Laboratory for their involvement in this research.

REFERENCES 1.

2.

3.

4.

5. 6.

George TH, Tim BM, Eun SP, et al. Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application. Biochemistry 2002;4l:l54 77-88 Brooks R, Ofelia R, Anna VP et al. pH-Dependent fluorescence of a heterologously expressed Aequorea Green Fluorescent Protein mutant: In situ spectral characteristics and applicability to intracellular pH estimation. Biochemistry 1998;37:9894-901. Marc AE, Rebekka MW, George TH, et al. Structural and spectral response of Green Fluorescent Protein variants to changes in pH. Biochemistry 1999;38:5296-301. Ahmed AH, Samuel TH, Watt WW. Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity. Chern Phys 2001;274:37-55. Mark A W, Michael S, Rama R. The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat Struct Bioi 2000;7:1133-38. Pakhomov AA, Martynova NY, Gurskaya NG, et al. Photoconversion of the chromophore of a fluorescent protein from Dendronephthya sp. Biochem (Mosc) 2004 ;69 :90 1-8.

FLUORESCENCE FROM S2-LEVEL OF COMPLEXES OF TRYPTOPHAN WITH EUROPIUM (III) IN WATER-ETHANOL SOLUTION 10 OSINA, S OSTAHOV, V KAZAKOV Institute a/Organic Chemistry, USc, RAS, Pro Oktybrya, 71, U/a,450022, Russia Email: [email protected]

INTRODUCTION The participation of higher excited singlet states (Sn' n > I) of molecules in photophysical (Sn ~ So fluorescence (FL)) or photochemical (photoinduced electron transfer (PET), isomerization, etc.) processes, which compete with radiation less deactivation, manifests itself in the dependence of the quantum yield (cp) and FL spectra on the wavelength of the exciting light (the violation of the Vavilov law). Such processes were first shown for the FL of azulene solutions due to the transition from the second excited level to the ground state S2 ~ So . I METHODS The FL spectra were recorded on a Hitachi MPF-4 spectrofluorimeter. The absorption spectra were recorded on a Specord M-40 spectrophotometer. The tryptophan (Trp) and EuCI 3 '6Hp were purified by double recrystallJration from twice distilled water and dried in vacuum. The Trp concentration was 10 moUL. RESULTS The FL spectra of Trp in water and dry ethanol are independent of the wavelength of the excitation light (I\.exc). The introduction of europium chloride, which forms complexes with tryptophan,2 (log PI = 4.82 ± 0.07 (HP, 298 K)) quenches FL through PET,1-4 also it has no effect on the FL spectra of Trp in individual solvents. Taking into account the strong Stokes shift of the FL spectra of Trp in H 20 (Amax = 353 nm) relative to the Trp ethanol solutions (Amax = 337 nm), we may assume that an increase in the H 20 content in the water-ethanol solvent will be accompanied by a "red" shift of the FL spectra. However, in 90% C 2H sOH, excitation into the second and shorter-wavelength absorption bands of Trp does not lead to the bathochromic shift, rather, a shortwavelength "shoulder" emerges in the FL spectrum of Trp (Fig. 1, spectrum 1). In the presence of europium chloride, the "shoulder" at 295 nm transforms into a peak (Fig. 1, spectra 2 and 3), which is assigned to the S2 ~ So radiative transition. 315

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Osina IO et al.

I

I

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,

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270 310 350 390 A,nm Fig. 1. FL spectra ofTrp: (1) without EuCb'6H zO, (2) c(Eu(III» = 1O-5 mo I/L, and (3) c (Eu(III» = 6 x 10-5 mol/L ("-exc = 245 nm, 90% CZH 50H, 298 K). The long-wavelength SI ~ So FL of Trp (Aexc = 245 nm) in 90% CZH 50H IS efficiently quenched by europium chloride, according to the Stern-Volmer equation: loll -1 = K[Q] (Fig. 2, curve 1) with the quenching constant K = 12000 Llmol (298 K). Quenching of the SI ~ So FL ofTrp (Arnax = 337 nm) by Eu(III) chloride makes it possible to clearly observe the short-wavelength Sz ~ So component (Arnax = 295 nm). The latter becomes dominant at a Eu(lII) concentration of 6x 10-5 mollL (Fig. 1, 3). At the same time, we observe the rise of Sz ~ So FL of Trp with an increase in the Eu(III) concentration (Fig. 2, curve 2). The Sz ~ So FL of Trp in aqueous alcohol solutions depends not only on the complexation of the amino acid but also on the excitation energy. Fig. 3 shows the FL spectra and the dependence ofl(295 nm)/I(337 nm) (1(295 nm) and 1(337 nm) are the intensities of the Sz ~ So and SI ~ So transitions, respectively) on the Aexc at a -5

Eu(III) concenration of 6 xl 0 mol/L. In the excitation wavelength range L'1 Aexc = 210 - 260 nm, the intensities of S I ~ So FL and short-wavelength Sz ~ So FL of Trp are redistributed. At 210 nm, the S\ ~ So FL is dominant and the short-wavelength component is observed as a poorly pronounced peak (Fig. 3, spectrum 1).

Fluorescence from S2-Level of Complexes of Tryptophan with Europium (III)

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("'exc =

245 nm, 90% C2HPH, 298 K).

As the "'exc increases, the S2 -7 So FL intensity increases and achieves a maximum value at 245 nm (Fig. 3, curve 4 and spectrum 2). In the wavelength range 245 - 260 nm, the short-wavelength S2 -7 So FL decays (Fig. 3, curve 4), and at "'exc ~260 nm, only ordinary S[ -7 So FL ofTrp is observed (Fig. 3, spectrum 3).

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=

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Osina 10 et al.

The /(295 nm)//(337 nm) ratio of the intensities of the Sz -7 So and SI -7 So transitions increases with the concentration of europium chloride upon excitation at a wavelength in the range l1A..xc = 210 - 260 nm. At the same time, at any Eu(III) concentration, the maximal /(295 nm)//(337 nm) value is always observed at 245 nm (Fig. 4, curve 4), which can be naturally attributed to the position of the Sz-Ievel with a minimal vibronic energy. Assuming that E(Sz) = 5.06 eV, we may estimate the -I

energy gap width I1E(Sz - SI)::::: 9400 cm (E(SI) = 3.9 eV \ Despite long-term studies of the photophysics and photochemistry of Trp, our findings are the first observation of its FL due to the transition from the S2 to the Solevel.

ACKNOWLEDGMENTS This work was supported by the Division of Chemistry and Materials Science of the RAS in the framework of program no. 1-0kh and Russian Science Support Foundation. REFERENCES I. Beer M, Longuet-Higgins H. Anomalous light emission of azulene. J Chern Phys 1955;23:1390-1. 2. Batyaev I, Fogileva R. Thermodynamics of tryptophan complexation with rare earth elements. Zh Neorg Khim 1976;21:1199-1201. 3. Ermolaev V, Sveshnikova E, Shakhverdov T. Study of complexation between organic molecules and rare earth elements ions in solutions by method of electronic energy transfer. Usp Khim 1976;45:1753-81. 4. Kazakov V, Ostakhov S, Alyab'ev A, Farrakhova G. Reversible photoinduced electron transfer from tryptophan to Eu(fod)3, Hfod 11 EuCh'6H 20 in the liquid and frozen etanol solutions. High Energy Chemistry 2005;39:97-9. 5. Horrocks W, Bolender J, Smith W, Supkowski R. Photosensitized near infrared luminescence of ytterbium (III) in proteins and complexes occurs via an internal redox process. J Am Chern Soc 1997;119:5972-3.

IDENTIFICA TION OF DEVELOPMENTAL ENHANCERS USING TARGETED REGIONAL ELECTROPORATION (TREP) OF EVOLUTIONARILY CONSERVED REGIONS CU PIRA, SA CALTHARP, K KANA Y A, SK MANU, LF GREER, KC OBERG Department of Pathology and Human Anatomy, Loma Linda University, 24785 Stewart St, Evans Hall Rm B09, Loma Linda, CA 92350, USA Email: [email protected]

INTRODUCTION During development, precise temporal and spatial regulation of critical genes is required to orchestrate body plan morphology. Preservation of a generalized developmental process and body plan across divergent species suggests that regulation has also been conserved. Thus, evolutionarily conserved regions (ECRs) in association with developmentally important genes are likely candidates as regulatory elements. Screening of ECRs has recently been described during early chick development, using in vitro whole embryo electroporation of ECR constructs containing green fluorescent protein (GFP).' The major limitation of this technique is that the chick embryos only survive in vitro for about 48 hrs after electroporation and thus enhancers involved in later development and organogenesis cannot be determined. We previously reported on a method to deliver expression vectors at targeted locations during in ovo development by confined microelectroporation (CMEP).2 We have modified this technique to broaden the targeted region of electroporation and vector delivery, i.e., targeted regional electroporation (TREP). Herein, we demonstrate the ability of this technique to screen for ECR activity at later stages of development. MATERIALS AND METHODS Construction of pTK-EGFP ECR constructs. Evolutionarily conserved regions (ECRs) were identified using the VISTA genome browser. To test ECR activity, we generated expression constructs with pTK-EGFP plasmid (a gift from Dr. Uchikawa, Osaka University), which contains the minimal HSV TK promoter linked to an enhanced GFP reporter gene (Fig.! ).' ECRs were isolated by PCR from genomic mouse (Emx2) or chicken (SHH) DNA. Each was ligated into pTK-EGFP at KpnI and XhoI. Plasmids were isolated and purified using the EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA). pCAGGS-RFP plasmid (gift from Dr. Tickle, University of Dundee) was co-e!ectroporated to verify transfection. Electroporation. Whole embryo electroporation in vitro was performed as previously described.' For targeted regional electroporation (TREP) chick embryos were stained (neutral red) and staged according to Hamburger and Hamilton (HH).3 The vitelline membrane overlying the embryo was removed and a small slit was cut on the yolk membrane near the heart. Platinum electrodes (0.3 mm diameter, at 2.5 mm distance) were mounted on a micromanipulator and positioned parallel to the embryo. 319

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11 origin

B

c

L Targeted Regional Electroporation (TREP). ECRs were ligated into pTK-EGFP plasmid. (B) DNA solution (hash was into the embryonic coelom of the presumptive wing region of HH14 chick The cathode (-) was placed underneath the embryo in the yolk while the anode (+) was placed above the embryo. The cathode was slid through the slit and into the yolk. DNA solution (2 0.25 flg/flL pCAGGS-RFP with phenol red and TE was into the embryonic coelom of the lateral plate mesoderm. The anode was above the embryo and 3-5 drops of PBS were used to promote conductivity. was performed using the CUY-2! Electropol'ator San Antonio, TX) at 8 volts, with 3 pulses of 50 msec ONIlOO msec OFF. After each the electrodes were cleaned in bleach, RNase-free water, and PBS to remove residual yolk. Fluorescence was visualized by a fluorescent MZ-8) and digitally recorded (Sony DKC-5000).

2. Identification of Evolutionarily Conserved Regions of the Emx2 locus across divergent species using VISTA Genome Browser revealed mUltiple evolutionarily conserved regions (ECRs; shaded columns).

ldt'ntificcuic'n of Developmental Enhancers Using Targeted Regional Electroporation

321

RESULTS ECR enhancer activity in embryos. Non-coding evolutionarily Fig. 2) may retain regulatory function. Whole could conceptually be used to screen the potential role development, but mUltiple attempts at in ovo EP of early embryos were unsucessful (early embryonic disk and associated membranes were too to survive even slight disruption during electrode placement). electroporated and grown in vitro. The embryo can survive in vitro for but distortion of embryonic growth is evident. We examined ECR associated with Emx2, a transcription factor linked to brain In vitro whole embryo EP demonstrated enhancer of brain development (Fig. 3). With additional manipulations, extended to 60 1m, however, limb buds were still not present. limb development could not be determined by this method.

of an Emx2-related ECR in the after whole embryo EP and 60 hrs of growth in vitro should Hll 18, but under incandescent light (light) it more closely resembles of Efficient transfection is noted by uniform expression) in the forebrain is indicated to the Emx2 expression domain within the brain. To overcome the difficulty of limited survival with whole embryo EP, we developed EP We evaluated TREP using a known limb-specific enhancer ECR (1.8 sonic hedgehog (SHH) expression in the limb's zone of the native limb-specific SHH expression domain done by whole embryo we performed TREP at to limb outgrowth. Adequate transfection was determined by RFP ZPA-related GFP expression 48 hrs after TREP confirmed enhancer of this However, the Emx2-related ECR that showed enhancer in the brain was not detected in the limb.

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Fig. 4, SJOT-related ECR Enhancer Activity is Detected in the ZPA after TREP. Broad RFP expression is detected in the limb bud 48 hrs after TREP; in contrast, GFP expression is restricted to the region of the ZPA (arrowheads). No enhancer activity is detected in the limb-related Emx2 expression domain (ISH) by the £mx2-related ECR CONCLUSION Targeted regional electroporation (TREP) extends the capacity of whole embryo electroporation to identify regulatory ECRs that participate at any point in the process of development. Although we targeted the limb, other organs, regions or even later stages could readily be examined. Developmental pathways are frequently re-utilized during development and thus, require tissue/organ specific regulation. We anticipate that this technique will be of great value in the identification of conserved tissuespecific regulatory elements that participate in the process of development and morphogenesis. REFERENCES 1. Uchikawa M, Ishida Y, Takemoto T, Kamachi Y, Kondoh H. Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals. Dev Cell 2003;4:509-19. 2. Oberg KC, Pira CU, Revelli J-P, Ratz B, Aguilar-Cordova E, Eichele G. Efficient ectopic gene expression targeting chick mesenchyme. Dev Dyn 2002;224:29i~302. 3. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol 1951;88:49-92.

PART 7 DEVELOPMENT AND BIOMEDICAL APPLICATIONS OF QUANTUM DOTS AND OTHER INORGANIC FLUORESCENT MATERIALS


QUANTUM DOTS AS FLUORESCENT RESONANCE ENERGY TRANSFER DONORS IN ANTIBODY-ANTIGEN SYSTEM HU SHAN,l YANG HAI,l CAl RUXru,u ZHANG QI,l YANG, XIANGLIANG 1* JCollege 0/ Life Science and Technology, Huazhong University o/Science and Technology, Wuhan, Hubei, China; 2Col/ege o/Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei, China. Email: [email protected]

INTRODUCTION Fluorescence resonance energy transfer (FRET) is a powerful technique to study the structures and interactions of individual biomolecules. Quantum dots (QDs) have gained widespread application as energy donors and acceptors in a variety of FRET based biological studies. They have been used as energy donors to study the FRET process, 1 to sense glucose,2 and to detect oligonucleotide hybridization. 3 Streptavidin-biotin4 as a bridge have been applied in most FRET systems because of its high affinity and small volume. Immunocomplexes of antigen and antibody as a bridge 5 in FRET systems have been studied and FRET processes has been observed. Su's studies 5 proved the size of immunocomplex was small enough to fulfil the need of FRET. Because the combination between antigen and antibody is relatively slow and the reaction is reversible, FRET in an antigen-antibody system differs from the streptavidin-biotin system. Competitive reactions based on reversible combination of antigen and antibody are widely used in ELISA. It also has the potential for a novel quantitative analysis method based on FRET and reversible reaction of antigen-antibody. In this paper, a novel competitive immunoassay to detect the concentration of protein (IgG) based on FRET and is described. EXPERIMENTAL Instrumentation and reagents. Fluorescence experiments were recorded by a Hitachi F-4500 fluorescence spectrophotometer and Perkin Elmer 1420 multilabel counter. QDs were purchased from Invitrogen. ImmunoPure F(ab')2 Preparation Kit that is used to prepare F(ab')2, was obtained from Pierce. Preparation of bioconjugate QD-F(ab')z. Rabbit anti-mouse IgG F(ab'h fragments were prepared following the experimental procedure provided by Pierce. QDs and F(ab')z were conjugated following the experimental procedure provided by Invitrogen. FRET assay between RBITC-IgG and QD-F(ab'h Experiment 1: The fluorescence spectrum of the mixture of diluted QD-F(ab')2 and RBITC-IgG was monitored (excitation 300 nm) every 0.5 h and detected at 2 h. The same procedure was done to the mixture of diluted QD-F(ab'h and PBS. Experiment 2: The fluorescence spectrum of the mixture was detected every 0.5 h. 0.33 mg/mL and Img/mL mouse IgG was added respectively to the mixture above and the fluorescence spectrum was monitored. Experiment 3: PBS and RBITC-IgG were 325

326

Set at.

fluorescence was monitored. Experiment 3: PBS and were added to the diluted QD-F(ab')2 respectively. The fluorescence of the mixtures was detected (excitation 300 nm) every 0.5 h. 1.5 /-lL 1mg/mL mouse was added to both the mixtures and the fluorescence spectrum was detected in the was same way. 3.5 Img/mL mouse IgG and then 10 !!L 1 mg/mL mouse added and the above experiments repeated.

RESULT AND DISCUSSION of the conjugates. Fig. I (a) shows the fluorescence bioconjugate excited at 300 nm and emitted at 562 nm. shows the fluorescence spectrum of the RBlTC-IgG conjugate excited at 562 nm. This indicated that the emission of the QD would excite RBlTC which means that RBITC could be used as the FRET acceptor for the of RBlTC is low and an increase in the fluorescence of RBITC the was not observed. FRET effect of and F(ab')z. Experiment 1: The fluorescence decreased and emission at 562 nm of the mixture of QD-F(ab')2 and became stable after .5 h. The same experiment was done to compare the mixture of and and it was found that the fluorescence was stable but decreased. The reason could be that FRET occurred between QD and RBITC when mouse and rabbit-anti-mouse F(ab')z formed an immunocomplex. 40

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To validate whether FRET occurred between and was added as a competitor. QD fluorescence intensity decreased when was added. Intensity increased when IgG was added showing that FRET occurred. The reason could be that free JgG competed with conjugated to so the number of RBITC-IgG-F(ab')Z-QD complexes was reduced and the of FRET reduced accordingly. Therefore, the fluorescence intensity of recovered. Additionally, the increasing fluorescence with the increase of indicated that the method could be used to determine the concentration of "rA'''''''C

Dots as Fluorescent Resonance

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of the fluorescence intensity of the mixture with different concentrations of at different times. influence of fluorometer, the fluorescence ratio was calculated. 100%

2 indicated that the concentration of fluorescence recovery of the mixture of QD-F(ab'h and mixed with the fluorescence intensity of mixed decreased significantly. The fluorescence intensity increased after

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mouse IgG was added to the mixture, while that of QD-F(ab')2 and PBS did not change, which indicated that FRET caused the fluorescence decrease of QDs. To eliminate the influence of the fluorescence change of QDs, QD-F(ab'h plus PBS was taken as blank and compared with QD-F(ab')2 plus RBITC. Fig. 4 shows that fluorescence was reduced over a period of time, which indicated that FRET occurred based on the combination of F(ab')2 and IgG. The experiment also showed that there is a correlation between the extent of fluorescence increase and the concentration of IgG. Therefore, this method is convenient to determine the concentration of proteins.

CONCLUSION This is the first demonstration of the feasibility of determining IgG concentration utilizing FRET in a system comprising dye labeled IgG and QDs labeled F(ab')2. F(ab')2 was prepared to shorten the distance between energy donors and acceptors in favor of FRET occurring. The resu Its showed that the system was suitable for FRET and provided a new way to detect protein simply and quickly. Another aspect of this work was that fluorescence quenching has been used to detect the target, whereas fluorescence enhancement has been widely used in many other FRET systems. A kinetic method may be adopted to study the relationship between the fluorescence and the concentration of protein. Additionally, the fluorescence of QDs was enhanced when IgG was directly added to QD-F(ab')z. The reason may be that FRET occurred between IgG and QDs, the QDs being excitated by the fluorescence oflgG at 340nm. Further research is required to investigate this mechanism. ACKNOWLEDGEMENTS This work was financially supported by the NSF of China (No: 30670552). REFERENCES I.

2. 3.

4.

5.

Clapp AR, Medintz IL, Mauro 1M, Fisher BR, Bawendi MG, Mattoussi H. Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors. 1 Am Chern Soc 2004;126:301-10. Duong HD, Rhee n. Use of CdSe/ZnS core-shell quantum dots as energy transfer donors in sensing glucose. Talanta 2007;73:899-905. Algar WR, Krull UJ. Towards multi-colour strategies for the detection of oligonucleotide hybridization using quantum dots as energy donors in FRET. Anal Chim Acta2007;581:193-201. Hildebrandt N, Charbonniere LJ, Beck M, Ziessel RF, Lohmannsroben HG. Quantum dots as efficient energy acceptors in a time-resolved fluoroimmunoassay. Angew Chern Int Ed 2005;44:7612-5. Li Y, Ma Q, Wang X, Su X. Fluorescence resonance energy transfer between two quantum dots with immunocomplexes of antigen and antibody as a bridge. Luminescence 2007;22:60-6.

SYNTHESIS AND PHOTOLUMINESCENCE OF GREEN-EMITTING 3 XdY,GdhSiOs:Tb + PHOSPHOR UNDER VUV EXCITATION ZHANG ZH 1,2 WANG YH 2 LI XX2

of Materials Scien~e and Enginee~ing, Wuhan Institute of Technology, Wuhan 430073, P.R. China; 2Department of Materials Science, Lanzhou University, Lanzhou 730000, P.R. China, Email: [email protected] 1School

INTRODUCTION Recently, attention has been paid to phosphors under vacuum ultraviolet (VUV) excitation due to the demands of plasma display panels (PDPs) and possible generation of mercury-free fluorescent lamps. For green-emitting phosphors for PDPs application, the most widely used is Zn2Si04:Mn2+, but its decay time is so long that it is difficult to exploit the fast response of PDPS. I,2 Therefore, a new green-emitting phosphors with short decay time under VUV excitation must be found. Previously,3 we studied the luminescent properties ofXz-Y19TbolSiOs under VUV excitation, and surveyed its feasibility for PDPs application. The results revealed that XZ-Y19Tbo.ISiOs presented stronger emission intensity and lower decay time than commercial Zn2Si04:Mn2+ phosphor. However, systematic investigations on the VUV photoluminescence of X2 - Y2SiOs doped with different concentration of Tb 3+ have not been reported. In this paper, in order to optimize the performance of Xz- Y2SiOs:Tb3+ and evaluate the effects of Gd 3+ on the photoluminescence ofX 2-Y 2SiOs:Tb3+, X 2-(Y,GdhSiOs:Tb 3+ samples were prepared and their luminescence properties were investigated in VUV regions in detail. EXPERIMENTAL Xz-(Y,Gd)2SiOs:Tb3+ samples X2-Y2_x_yGdyTbxSiOs (0.05 :s; x :s; 0.4,0 :s; Y :s; 0.5) were prepared by a co-precipitation process. 3 Y20 3 (99.99%), Gd 20 3 (99.99%), Tb 40 7 (99.99%) and tetraethyl orthosilicate (TEOS) (AR) were used as starting materials. Crystallinity of the sample was analyzed using Rigaku D/max--2400 X-ray diffractometer with Ni-filtered Cu Ka radiation. Excitation and emission spectra were measured at room temperature by FLS-920T fluorescence spectrophotometer with a VM-504-type vacuum monochromator (deuterium lamp source). Excitation spectrum was corrected with sodium salicylate. The decay time of the samples was examined under 147 nm excitation. The same equipment conditions were adopted for all as-prepared samples and commercial Zn2Si04:Mn2+ RESUL TS AND DISCUSSION All the powder X-ray diffraction patterns of as-prepared X2-Y2_x_yGdyTbxSiOs (0.05 :s; x :s; 0.4, 0 :s; y :s; 0.5) samples could be recognized as a single phase and readily indexed to monoclinic symmetry. Excitation spectra of X2-Y16Gdo.2Tb02SiOs (a) and XrYLSTbo.2SiOs (b) monitored at 542 nm (Fig. 1) consist of a direct Tb3+ 329

330 Zhou L-Yet al.

excitation region for wavelengths> 190 nm and a host lattice excitation region for wavelengths Silicates are taking more and more attention as useful luminescent hosts because of the stable crystal structure, and high physical and chemical stability.? Sol-gel method is an efficient technique for preparation of nano-sized phosphors due to good mixing of starting materials and relatively low reaction temperature: In this article, Na2Ca4Mg2Si40Is:Tb3+ nanoparticles were synthesized for the first time by sol-gel method.

MATERIAL AND METHODS Reagents were of analytical grade. Stoichiometric tetraethoxy- silicane [(CH3CH20)4Si, TEOS] and ethanol were mixed with stirring to obtain a TEOS solution. Magnesium nitrate, calcium nitrate, sodium nitrate aqueous solution and Tb(N0 3)3 solution were then added dropwise to the required amount of TEOS solution with vigorously stirring. Then, appropriate amount of HN0 3 was applied as the catalyst for the hydrolysis of TEOS with stirring continued for 4 h. The resultant gel was dried at 120°C for 3 h. The precursor particles were put into a furnace and pre-calcined at 500°C for 2 h, then calcined at 1000 °c for 5 h to obtain phosphor samples. Powder X-ray diffraction (XRD, 40 kV and 35 rnA, Cu Ka = 1.5406 A RigakulDmax - 2200, Japan) was used for crystal phase identification. Field emission scan electron microscopy (LEO-1530 FE-SEM, Germany) was used to observe the morphology and size of the calcined particles. Near UV excitation and emission spectra were measured on a HITACHI F-4500 fluorescence spectrophotometer (Japan).

RESULTS AND DISCUSSION The powder XRD patterns for the particles prepared by the above processes are shown in Fig. 1, and panels a, b, and c show the patterns for the samples calcined 333

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for 5 h in air at 900 1000 °c and 1100 DC, respectively. When the precursor was calcined at 900°C, the characteristic peaks of Na2Ca4Mg2Si4015 42appear with the existing peaks of CaO (ICDD 28-0775). At 1000 form without impurity phase. When the is increased to 1100 the intensity of the peaks does not change significantly, and no new are observed. So the optimum firing temperature is about 1000 "C. 400-,-------c----------, 350 300 250

50

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2tJ(degree)

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of the Na2C34Mg2Si4015 : 4 mol % Tb 3 + phosphor calcined at 900°C, (b): 1000 °c and (c): 1100 for 5 h

ofNa2C34Mg2Si40J5 : 4 mol % Tb3+ phosphor calcined at 1000 °c for 5 h

shows the FE-SEM images of Na2Ca4Mg2Si401S: 4 mol % showed a narrow size-distribution of about 80 ~ 100 nm with There was only minor aggregation ofthe particles. 3 shows the room temperature near UV excitation and emission of the phosphor. When the detection wavelength is monitored at 545 nm, the excitation spectrum consists of four bands, the 4f8 transition at the shorter wavelength (245 nm and 285 nm) with a higher intensity and transition at the longer wavelength (352 nm and 370 nm) with weaker excitation with 245 nm UV light, the characteristic luminescence is due to 4, 3) and 5 D4 -> 7FJ (J 6, 5, 4, 3) line emissions of the

Luminescent Properties ojNa2 Ca4Mg2Si4015: Tb 3 + Nano-Sized Phosphor

335

ions. The strongest emiSSIOn is located at 545 nm corresponding to S D4 --> 7 Fs transition, while thef- ftransition lines from the higher level sD 3 are not observed due to the increased concentration of Tb 3 +., In order to verify the best Tb 3+ doping ratio to Na2Ca4Mg2Si401s, a series of doping experiments with Tb 3 + doping ratios to CaO from 4 mol % to 10 mol % were carried out. The luminescence intensity is enhanced as the increasing of the Tb 3 + doping ratio and reaches a maximum at 8 mol % of Tb 3 +. When the Tb3+ doping ratio is higher above 8 mol %, the 3 luminescence intensity reduces contrarily. As the Tb + concentration increases, the Tb-Tb distance decreases. Tb ions strongly cross-relaxation interact resulting in a decrease of the lifetime. lo Based on a single exponential method, the decays time 3 of the Na2Ca4Mg2Si40ls : 8 mol %Tb + phosphor monitored at 545 nm and excited at 245 nm is 2.96 ms, which was short and suitable for PDP application.

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The emission color was analyzed with the help of CIE chromaticity coordinates. The coordinates of Na2C'4Mg2Si401s:Tb3+were found to fall in the yellowish green region of the CIE chromaticity diagram and with an decrease ofTb 3+, the phosphor exhibited deeper green emission, as shown in Fig. 4.

CONCLUSIONS 3 Na2Ca4Mg2Si401S :Tb + phosphors with a spherical shape and a size about 80 100 nm were prepared using sol-gel method. Upon excitation with near UV light excitation, the phosphor showed strong green-emission peaked at around 545 nm, corresponding to the SD4 -> 7Fs transition of Tb3+, and the highest PL intensity at 545 nm was found at a content of about 8 mol % Tb 3+. With a decrease ofTb 3+, the phosphor exhibited deeper green emission. All the characteristics indicated that the 3 Na2Ca4Mg2Si4015 :Tb + would be a promising green phosphor for PDP application. ACKNOWLEDGEMENTS Financially supported by grants from the Science Foundation of Guangxi Province (No. 0731014), the Natural Science Foundation of Guangxi University (X051107), the large-scale instrument of Guangxi cooperates and shares network. REFERENCES 1. Liang HB, Tao Y, Su Q, Wang SB. VUV-UV Photoluminescence spectra of strontium orthophosphate doped with rare earth ions. J Solid State Chern 2002; 167:435-40. 2. Rao RP, Devine DJ. Re-activated lanthanide phosphate phosphors for PDP applications. J Lumin 2000;87-89:1260-263 3. Moine B, Bizarri G. Rare-earth doped phosphors: oldies or goldies? Mater Sci Eng B 2003;105:2-7. 4. Ronda CR. Recent achievements in research on phosphors for lamps and displays, 1. Lumin 1997;72-74:49-4. 5. Itoh K, Kamata N, Shimazu T, et al. An improved emission characteristics of 3 Tb +-doped sol-gel glasses by utilizing high solubility of terbium nitrate. J Lumin 2000;87-89:676-8. 6. Kim CH, Kwon E, Park CH, Hwang YJ, Bae HS, Yu BY, et al. Phosphors for plasma display panels, J. Alloys Comp., 2000;311 :33-39 7. Lin YH, Tang ZL, Zhang ZT, Nan CWo Luminescence of Eu2+ and D/+ activated R3MgSi20s-based (R=Ca, Sr, Ba) phosphors. J Alloys Comp 2003;348:76-9. 8. Patra A, Baker GA, Baker SN. Effects of dopant concentration and annealing temperature on the phosphorescence from Zn2Si04: Mn2+ nanocrystals, J Lumin 2005;111:105-11. 9. Kim GC, Park HL, Kim TW. Emission color tuning from blue to green through cross-relaxation in heavily Tb 3+-doped YAI0 3, Mater Res Bull 2001 ;36: 1603-8. 10. Duhamel-Henry N, Adam JL, Jacquier B, Linare C. Photoluminescence of new fluorophosphate glasses containing a high concentration of terbium (III) ions. Opt Mater 1996;5:197-207.

PART 8

BIOLUMINESCENCE, CHEMILUMINESCENCE AND FLUORESCENCE IMAGING


THE MEASUREMENT OF CYTOSOLIC ATP DURING APOPTOSIS: BIOLUMINESCENCE IMAGING AT THE SINGLE CELL LEVEL RYUTARO AKIYOSHI, HIROBUMI SUZUKI Research & Development Division, Olympus Corporation.2-3Kuboyama-cho Hachioji-shi, Tokyo, 192-8512, Japan Email: ryutaro _ [email protected]

INTRODUCTION Apoptosis is a process of genetically programmed cell death. This distinct form of cell death plays an essential role in embryonic development and metabolic homeostasis. At present, it is known that apoptosis demands energy supplied by ATP for caspase activation, enzymatic hydrolysis of macromolecules, chromatin condensation, and apoptotic body formation. 1-4 The total amount of ATP during apoptosis has been measured by firefly luciferin-Iuciferase assay system using a luminometer. 5 Beetle luciferases (including those of the firefly and the click beetle) have been used for the highly sensitive detection of ATP because beetle luciferases catalyze the oxidation of D-Iuciferin in the presence of ATP, Mg2+ and molecular oxygen. 6 However, this assay system detects only a total amount of luminescence of the cell population. In order to analyze cellular activity using bioluminescence at single cell level, we developed a luminescence microscope, 7 and attempted to measure cytosolic ATP during apoptosis at the single cell level. MATERIALS AND METHODS The measurement of cytosolic ATP during apoptosis. He La cells were plated on a 35 mm culture dish with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and were transfected transiently with Emerald Luc control vector (Toyobo), click beetle luciferase under the SV40 promoter. For 24 h after transfection, the medium was replaced with Opti-Mem (Invitrogen), and D-Iuciferin was added into the medium at the final concentration of 500 11M. After the 30 min incubation, apoptosis is induced by the addition of the following reagents to the medium at the final concentration; 4 11M staurosporine (STS), 30 11M carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 10 Ilg/mL cycloheximide (CHX). The luminescence was continuously monitored using a luminometer (Kronos, ATTO) after addition of apototic inducers by lOs integration of photons with 1 min interval at 37°C. Bioluminescence imaging. Luminescence images were acquired using the Luminoview (LV200, Olympus) luminescence imaging system attached to an electron multiplier charge-coupled device camera (ImagEM, Hamamatsu Photonics). The dish was kept at 37°C in the humidified chamber during observation. Each image was taken by 40 x objective lens [numerical aperture (NA) 1.30] at 10 s exposure, 15 s interval, binning 1 x 1 and EM-gain 255, or 60 x phase contrast objective lens (NA 1.25) at 60 s exposure, 90 s interval, binning lxl and EM-gain 255. Duration time of observation 339

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was 10 h. Luminescence intensity from single cells was measured as an average value in a region of interest (ROI) enclosed for each cell by MetaMorph software (Universal Imaging). RESULTS AND DISCUSSION Cytosolic ATP dynamics of HeLa cell during apoptosis was measured using a luminometer (Fig.I). Total amount of ATP increased within 20 to 120 min and decreased gradually within 10 h via three types of apoptotic induction (STS, FCCP and CHX). As CHX is an inhibitor of protein synthesis, ATP elevation after apoptotic induction is not due to elevation of luciferase synthesis. On the other hand, ATP elevation is suppressed by glucose-free medium and 2-deoxy-D-glucose. It leads to a hypothesis that glycolysis pathway contributes to an elevation of cytosolic ATP. 5 This hypothesis is supported by the result using FCCP, an inhibitor of mitochondrial A TP production.

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Time (hour) Cytosolic ATP measurement of HeLa cells during apoptosis by luminometer (Kronos, ATTO).

Fig. 2(a) shows the luminescence image of HeLa cells before apoptotic induction. Cytosolic ATP dynamics during apoptosis induced by STS is analyzed at single cell level [Fig. 2(b)]. ATP elevation within 30 to 45 min was also observed and it paralleled the result obtained using a luminometer (Fig. 1). FCCP and CHX yield similar results (data not shown). Moreover, we observed a phenomenon whereby some of cells emit a flash of light after 4 to 8 h. Fig. 3 shows detailed morphology of the cells emitting flashes of light in bright field and luminescence images. The flash of light was observed just before the cell shrinks [Fig. 3(b) Arrow]. As shown in Fig. 3, most of cells shrink before cell death, but the flash of light occurs at different times in

Measurement ofCytosolic ATP During Apoptosis

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each cell. This stereotypical morphological change occurs with formation of membrane-bound apoptotic bodies that contain cytosolic and nuclear fragments. The apoptotic body is known not to release its contents, but it is considered that intracellular conditions, such as pH, D-luciferin concentration and ATP level are We that the flash of light is caused by some of these But it is uncertain whether the flash of light is due to increased cytosolic ATP or not at the present. Such result cannot be obtained by conventional luminometric assay The bioluminescence imaging assay can detect not only cytosolic ATP level but also of an individual cell. This assay system will make a new window in cell biology.

3.E+07 2.E+07 2.E+07 1.E+07 5.E+06 O.E+OO

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Time (hour)

2. Bioluminescence image captured by Luminoview and cytosolic ATP measurement at the single cell level. (a)Bioluminescence image of He La cells Emerald Luc obtained by 40 x objective lense. (b) Time course of cytosolic ATP after addition of STS (4 J.1M) for 7 cells selected in 2(a).

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3. Flash luminescence observed before cell Luminoview. Phase contrast image; (b) Bioluminescence image of HeLa Cell lens. were obtained by 60 x phase contrast apoptosis and emits flash luminescence. but its luminescence intensity does not increase. REFERENCES 1. Richter C, Schweizer M, Cossarizza A, Franceschi C. Control of cellular ATP level. FEBS Lett 1996;378:742-52. a switch in Nicotera P, Leist M, Ferrando-May E. Intracellular between apoptosis and necrosis. Toxicol Lett 1998; I 02-103: 139-42. 3. Hu Benedict MA, Ding L, Nunez G. Role of cytochrome c and in Apaf-l-mediated caspase-9 activation and 8:3586-95. Eriksson JE, Weis M, Orrenuis S, Chow condensation during apoptosis requires ATP. Biochem J 1 Sabirov Maeno E, Ando-Akatsuka Zamaraeva Okada. Y. Cells die with increased cytosolic ATP bioluminescence study with intracellular luciferase. Cell Death Differen 390-7. 6. Lam McElroy WD. Introduction to beetle luciferases J Biolumin Chemilumin 1989;4:289-301. 7. Suzuki H, Dosaka S, Ohashi-Hatta Y, Sugiyama T. Luminescence Hill PJ, Kricka for assay of single live cells. In: Szalay PE. eds. Bioluminescence and Chemiluminescence. 2006:53-6.

BIOLUMINESCENCE IMAGING OF BACTERIA-HOST INTERPLAY: INTERACTION OF E. COLI WITH EPITHELIAL CELLS LY BROYKO,I H WANG,I J ELLIOT,I R DADARWAL,I 0 MINIKH I,2 MW GRIFFITHS I JCanadian Research Institute for Food Safety, University of Guelph Guelph ON N1G 2W1, Canada 2Department of Chemical Enzymology, Lomonosov Moscow State University Moscow 117899, Russia Email: lbrovko@uoguelphca

INTRODUCTION According to the CDC, foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year. I The estimated cost for five major food-borne bacterial pathogens (Campylobacter (all serotypes), Salmonella (nontyphoidal), Escherichia coli 0157, E. coli non-0157 STEC, and Listeria monocytogenes) in year 2000 was 6.9 billion dollars. 2,3 The best defense against food-borne illnesses is the early detection and/or identification of contamination events. Currently, the most sensitive and reliable assays for bacterial pathogens are based on specific recognition by antibodies (ELISA) and nucleic acids (PCR, in situ hybridization). However, these methods are relatively time-consuming, may require costly equipment and often involve pre-treatment of samples prior to performing these assays. The time needed to perform the assay and its cost are almost prohibitive for routine, everyday screening of food and environmental samples. Both bacterial and eukaryotic cells are active participants in the infection process. Recently molecular and cellular mechanisms of bacterial pathogenesis were defined for some members of the Enterobacteriaceae family that showed similarity, namely Salmonella, Shigella, enteropathogenic and Escherichia co!i.4-9 The host cell plays an active role in bacterial adhesion, following which bacterial pathogens activate host cell signal transduction pathways. Many pathogens use the same signal molecules (namely Ca2 +, protein kinases, inositol-3-phosphate, etc.) that participate in signal transduction 2 in mammalian cells after the action of different stimuli. Influx of Ca + and/or a subsequent increase in protein phosphorylation were detected in mammalian cells after adhesion of several enteric pathogens. These changes occur within minutes or even seconds after cell-cell interaction. Bioluminescent methods provide researchers with unique opportunities to track growth and movement of cells in real-time and to non-destructively monitor fast metabolic changes with high sensitivity and specificity directly in a live animal or ceIJ. IO- 13 Despite these evident advantages, few results have been reported on the application of bioluminescent methods for monitoring the processes of bacterial infection. I I The goal of our investigation was to apply available bioluminescent techniques for investigation of the early stages of interaction of bacterial pathogens with the host cell 343

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to gain knowledge that could be applied to the detection of infective organisms. As a model system, interaction of Escherichia coli with epithelial HeLa cells was studied. E. coli represents a wide group of related bacteria including harmless, or even beneficial species that produce vitamin K, as well as highly pathogenic strains that cause serious disease. They commonly reside in the gut of warm-blooded animals, including humans, where they colonize intestinal mucosa. Thus, interaction with HeLa cells may provide a good insight into events that occur within the gastrointestinal environment. MATERIAL AND METHODS Cell cultures and their cultivation. All strains of E. coli were obtained from the CRIFS culture collection, University of Guelph. Bacteria were grown at 3rC in LB broth or agar supplemented with 50 Ilg/mL of ampicillin where indicated. Enumeration of bacteria was performed by plating counting and presented in colony forming units (CFU). Construction of the bioluminescent bacterial strains was performed by electroporation of parent strains with plasmid pT7 carrying the full lux operon of Photorhabdus luminesens as described previously.14 CCL-2TM HeLa cells were obtained from ATCC and grown in Eagle's minimal essential media (EMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO 2 • Assessment of the attachment of E. coli to HeLa cells by bioluminescent method. HeLa cells were grown in 24-well microtiter plates until 80-90% con fluency was reached. The monolayer of HeLa cells was then inoculated with the bioluminescent bacteria, incubated for 1 h (37°C 5% CO 2), washed twice and fresh medium was added. The plate was later incubated under the same conditions and bioluminescence was monitored using a Multilabel Plate Reader Victor™ (Perkin Elmer) and/or Night Owl (Berthold EG&G) imaging device. From the time-course of the bioluminescence, lag periods for growth of bacteria attached to epithelial cells were estimated. These were compared with the lag periods of the growth curves obtained under similar conditions for bacterial suspensions in tissue culture media containing a known initial number of cells (range 1 - 106 CFUlmL). On the basis of this comparison, the level of attached bacterial cells was estimated. RESULTS Bioluminescent E. coli. Fifteen strains of E. coli were transformed to give a bioluminescent phenotype and these included 5 non-pathogenic E. coli, 5 enterohemorrhagic E. coli 0157:H7 and 5 enteropathogenic E. coli (EPEC) of different O:H serotypes. Bioluminescence in Relative Light Units (RLU) of bacterial cell dilutions was measured and standard curves (RLU vs CFUlml) were obtained for each strain. Surprisingly, there were significant differences observed between investigated strains. There was no correlation between pathogenicity of the strain and its bioluminescence. The observed difference between the brightest and dimmest was more then one order of magnitude. In further experiments each individual standard curve was used to assess numbers of that particular strain.

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Attachment of E. coli to HeLa cells. Attachment at different levels of inoculation 3 6 (10 _10 CFU/mL) and physiological state of bacteria was investigated as described above. Typical results are presented in Table I. There was no significant difference in attachment of actively growing and starved cells to the monolayer of epithelial cells. The difference in attachment between strains did not correlate with pathogenicity and was not statistically different for all investigated strains. Table 1. Attachment of E. coli bacteria to HeLa cells as assessed by bioluminescence. Attachment, cfu per well Inoculation level 105 cell per Inoculation level 10 6 cell per well well

Strain

IE. coli O157:H7 92-192, 94-37, 666H8, 92-355,

PT67 PT34 PT23 PT23

IEHEC Ol32NM 0103:H2 O153:H25 1N0n-pathogenic E. 'coli ATCC 10798, KI2 ATCC 15597, EC 3000

Growing cells Starved cells Growing cells 10 5 5

10 >5x10 4 >5x104

5xl0 >10 5

4

>5x10 4

ACKNOWLEDGEMENTS This work was partially supported by SENTINEL Bioactive paper network, NSERC, Canada; and NCFPD, USA. REFERENCES 1. Mead PS, Shutsker L, Dietz Y, et al. Food-related illness and death in the United States, 1999. Emerging Infect Dis 1999;5 :607-25. 2. Anon. Economics of food-borne diseases: Overview. http://www.ers.usda.gov/Briefing/FoodborneDisease/overview.htm 3. Stinson T. The national economic impacts of a food terrorism event: Preliminary estimates, Proceedings of The Institute of Food Technologists' I sl Annual food protection & defense research conference, November 2005, Atlanta, Georgia, http://www.ift.org/fooddefense/3-Stinson.pdf

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Finlay BB, Cossart P. Exploitation of mammalian host cell functions by bacterial pathogens. Science 1997;276:718-25. Pace J, Hayman MJ, Galan JE. Signal transduction and invasion of epithelial cells by S.typhimurium. Cell, 1993;72:505-14. Oehio C, Prevost MC, Sansonetti PJ. Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of contract in by a pp60 c-src -mediated signalling pathway. EMBO J 1995;14:2471-82. Nisan I, Wolff C, Hanski E, Rosenshine I. Interaction of enteropathogenic Escherichia coli with host epithelial cells. Folia Microbiol 1998;43:247-52. Wadsworth, S.1., Goldfine, H. Listeria monocytogenes Phospholipase C dependent Calcium signaling modulates bacterial entry into J774 macrophage-like cells. Infect Immun 1999;67:1770-8 Shin S, Kur GH, Kim YB, Park KJ, Park YM, Lee WS. Intracellular calcium antagonist protects cultured peritoneal macrophages against anthrax lethal toxininduced cytotoxicity. Cell Bioi ToxicoI2000;16:137-44. Rider TH, Petrovick MS, Nargi FE, Harper JO, Schoebel EO, Mathews RH, et al. A B Cell-Based sensor for rapid identification of pathogens. Science 2003 ;30 1:213-5. Hammermueller J, Gyles CL. The development of a rapid bioluminescent Vero cell assay. In: MA Karmali MA and AG Goglio AG, eds. Recent advances in Verotoxin producing Escherichia coli infections. Amsterdam:Elsevier 1994:11316. Allue I, Gandelman 0, Oementieva E, Ugarova N, Cobbold P. Evidence for rapid consumption of millimolar concentrations of cytoplasmic ATP during rigorcontracture of metabolically compromised single cardiomyocytes. Biochem J 1996;319:463-9. Brovko L, Young 0, Griffiths MW. Method for assessment of functional activity of antibodies for live bacteria. J Microbiol Methods 2004;58:49-57. Meighen EA, Szittner RB. Multiple Repetitive elements and organization of the lux operon of luminescent terrestrial bacteria. J Bacteriol 1992; 114: 5371-8l.

UL TRASENSITIVE CHEMILUMINESCENT IMMUNOCHEMICAL LOCALISATION OF PROTEIN COMPONENTS IN PAINTING CROSS-SECTIONS LS DOLCI,I G SCIUTTO/ M RIZZOLI/ M GUARDIGLI,I R MAZZEO,2 S PRATI,2 A RODA 1 1Dept of Pharmaceutical Science. University of Bologna. Bologna 40126, Italy 2Microchemistry and Microscopy Art Diagnostic Laboratory, University of Bologna. Ravenna 48100, Italy Email: [email protected]

INTRODUCTION Identification and localization of binding media and different organic components in multilayer paint samples is crucial in the study of manufactured techniques and for authentication and conservation purposes. To this end, use of immunological techniques has been proposed many years ago based on the unambiguous reaction between antibody and protein target (antigen). These techniques would allow to distinguish between different proteins and also to determine the biological source of a protein (e.g., bovine vs. rabbit collagen). Such techniques are extensively employed in bioanalytical and clinical chemistry,1.2 but only few results are reported in literature concerning the characterization of organic materials in paint crosssections using antibodies labeled with fluorescent markers.'·4 These first applications are characterized by strong interferences due to the presence of painting materials, which can show an intense autofluorescence. We have developed a new method for the localization of ovalbumin (chicken eggwhite albumin, a protein often present in binding media and varnishes) in paint cross-sections based on chemiluminescence (CL) imaging detection. Thanks to the absence of an excitation source, no interference was observed from painting materials. The CL immunochemical method for the localization of ovalbumin relied on the binding to the target protein of a specific primary antibody, which was then detected by a horseradish peroxidase (HRP)-Iabeled secondary antibody and a CL enzyme substrate. The imaging of the CL signal produced by the enzyme-catalyzed reaction allowed the detection and the stratigraphic localization of the target protein. After evaluation of the performance of the method on standard samples, several real samples collected from old paintings were analyzed. MATERIALS AND METHODS Reagents. Anti-chicken egg albumin antibody (whole antiserum, produced in rabbit), HRP-conjugate polyclonal anti-rabbit IgG antibody (produced in goat), albumin from chicken egg white (ovalbumin), and bovine nonfat dried milk were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Primary and secondary antibodies were diluted 1:2000 and 1:4000 (v/v), respectively, in PBS/milk 347

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(phosphate buffer saline, pH 7.4, containing 1.25% dried milk). The luminol-based HRP CL detection reagent Westar Supernova was from Cyanagen (Bologna, Italy). The smalt (ground glass colored with cobalt(II) salts), azurite and malachite (CU3(C03h(OHh), hematite (FeZ03), cinnabar (HgS), and minium (Pb 30 4 ) pigments were obtained from Zecchi (Florence, Italy). Polyester resin for sample embedding (SeriFix Resin and SeriFix Hardener) was purchased from Struers A/S (Ballerup, Denmark). Instruments. Chemiluminescence imaging microscopy experiments were performed using a BX60 epifluorescence microscope (Olympus Optical, Tokyo, Japan) connected to an ultrasensitive CCD camera (LN/CCD Princeton Instruments, Roper Scientific, Trenton, NJ). The microscope was enclosed in a dark box to avoid interference from ambient light. Live color images of the samples were obtained by acquiring separate grayscale images corresponding to the RGB colors by means of a RGB filter (CRI Inc., Woburn, MA) and a white LED light source. Image processing and quantitative analysis were performed using the image analysis software Metamorph v. 4.5 (Universal Imaging Corporation, Downington, PA, USA). Sample preparation. Standard samples were obtained by the application of a thin layer of whole-egg tempera (a mixture of egg white, yolk and water in 1: 1: 1 (v/v) ratio, mixed or not with pigments) on a ground layer of gypsum (purchased from Zecchi) and rabbit glue (purchased from Phase, Bologna, Italy). The following pigment/egg ratios were adopted: 4: 1 for azurite, malachite, smalt and hematite; 3: 1 for cinnabar and minium. Small samples were collected and embedded in polyester resin, following the conventional procedure. The transverse section were obtained by abrasion of the resin and polishing the surface using fine silica abrasive papers (4000- to l2000-grade, purchased from Micro-Surface, Wilton, lA, USA). Experimental procedure. Sample cross-sections were treated for 1 h at room temperature with the blocking solution (5% dried milk in water) then, after washing (3 x) with PBS/milk, incubated overnight at 4°C with the anti-ovalbumin antibody (primary antibody). Afterwards, the samples were washed (5 x) with PBS/milk, incubated for 4 h at 4°C with the HRP-Iabeled anti-rabbit IgG antibody (secondary antibody), and washed again (5 x) with PBS/milk. Then the HRP CL detection reagent was added to cover the cross-section and the CL images were acquired using an integration time of 120 s. RESULTS AND DISCUSSION Due to the porosity of the cross-sections, optimization of blocking and incubation steps was critical to avoid non-specific adsorption of the immunoreagents, which would cause high background signals and decrease the detectability of the target protein. The non-specific binding of the immunoreagents could be reduced with a dry polishing method, thereby decreasing the heterogeneity of the surface, and by lowering the incubation temperature. The specificity of the assay was assessed by performing the immunolocalization of ovalbumin in standard samples with or without the primary antibody. As expected, in the absence of the primary antibody

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no CL was detected. In addition, no cross reactivity of the primary antibody was observed in standard samples containing other organic binding media (fish intelferences due to metal ions contained in pigments we oil). To standard of whole-egg tempera with different common inorganic pigments malachite, hematite, cinnabar, and minium). The experiments in the absence of the immunoreagents did not show any catalysis of the and for all the pigments we were able to observe CL emission from the I). We plan to include in further experiments other inorganic and of historical interest, also performing investigations on "~''''-'''''' to assess the effect of degradation processes .

. Chemiluminescent immunolocalization of ovalbumin in a standard with smalt and tempera. Left: live image; right: CL image. Bar: 200 Jlm. The CL immunochemical techniques was applied on old in order to evaluate its performance in samples with a natural and riPfrn";lc'pm collagen, casein, etc). Different proteins could be also detected in by employing a mixture of analyte-specific nrl1m>llCV one by a secondary antibody labeled with a d iffurent enzyme HRP and alkaline phosphatase) detectable with a specific CL substrate. REFERENCES Roda Pasini P, Musiani M. Girotti S, Baraldini Carrea Suozzi Chemiluminescent low-light imaging of biospecific reactions on macro- and a videocamera-based luminograph. Anal Chem 2.

3.

4.

Marangi M, Casanova S, Grigioni Roda E, Roda A. Chemiluminescence quantitative immunohistochemical determination of MRP2 in liver biopsies. J Histochem Cytochem 2005;53:1451-7. Ramirez Barat de la Vina S. Characterization of proteins in media immunofluorescence. A note on methodological Stud Conserv 2001 A, Millay V, Quick M. The use of immunofluorescence and enzyme-linked immunosorbent assay as for proteins identification in artists' materials. J Am Inst Conserv 05.

DEVELOPMENT OF A NEW DEVICE FOR ULTRASENSITIVE ELECTROCHEMILUMINESCENCE MICROSCOPE IMAGING LS DOLCI,I M RIZZOU,I E MARZOCCHI,2 S ZANARINI,3 LOELLA CIANA,2 A RODA I I Dept of Pharmaceutical Sciences, University of Bologna, Bologna 40126, Italy 2Cyanagen s. r.l. , via Stradelli Guelfi 401c, Bologna 40138, Italy 3 Dept of Chemistry G. Ciamician, University of Bologna, Bologna 40126, Italy Email: [email protected]

INTRODUCTION Electrogenerated chemiluminescence (ECL) is a highly sensItIve analytical technique '" widely used in biosensors',3 and clinical chemistry.IS." Until now no applications have been reported on the use of ECL for ultrasensitive low-light imaging microscopy of immunohistochemical (IHC) and hybridization (ISH) methods on cells or biological tissues. The topographic 20 localization of an analyte down to a few molecules is extremely important in bioanalysis since the evaluation of its distribution in cells is a key factor in physiology, physiopathology and therapeutics. To perform the ECL detection of a biospecific reagent labeled with an ECL-active marker we designed and developed a transparent electrochemical cell using FTOcoated glass compatible with optical microscopy. The cell allows the generation of a luminescence signal which can be activated and switched off during the measurement process. As a model system we used heavy micron-sized beads to simulate biological cells. MATERIALS AND METHODS [Ru ( 4 (4'-methyl-2,2'-bipyridin-4-yl) butan-l-aminium (2,2'-bipyridine)2] (CI0 4 )3 (Ru(bpyh 2+ -NH 2), bis(2,2' -bipyridine)-[ 4-[ 4' -methyl-2,2' -bipyridin-4-yl)butanoic acid] ruthenium bis(hexatluorophosphate) (Ru(bpy)/+ -COOH), 3-sulfo-Nhydroxysuccinimide (s-NHS), and biotin-cadaverine-TFAc were purchased from Cyanagen (Bologna, Italy). Tris(2,2' -bipyridine)dichlororuthenium(lI) (Ru(bpyh 2+), streptavidin from Streptomyces avidinii, MES, tripropylamine, N-(3dimethylaminopropyl)-N' -ethyl-carbodiimide hydrochloride (EDC), and N,N'dicyclohexylcarbodiimid (DCC) were purchased from Sigma-Aldrich Co. (St. Louis, MO). The heavy-core carboxylated beads (diameter 8 flm) were purchased from Spherotech (Libertyville, IL, USA). Fluorine-doped tin oxide-coated glasses (20 Q) were purchased from Flexitec Electronica Organica (Curitiba Parana, Brasil). For microscopy imaging a BX60 epitluorescence microscope (Olympus Optical, Tokyo, Japan) connected to a liquid nitrogen-cooled ultrasensitive CCD camera (LN/CCD Princeton Instruments, Roper Scientific, Trenton, NJ) was used. The microscope was enclosed in a dark box to avoid interference from ambient light and equipped with an OptiScan ES 103 motorized microscope stage (Prior Scientific Instruments Ltd., Fulbourn, England) for sample positioning. 351

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Glass electrochemical cell. A microscope FTO-glass was chemically etched 7 to produce a standard three-electrodes cell. Quasi-reference electrode was prepared flash galvanic deposition directly on the FTO electrode. Electrochemical activity was monitored by cyclic voltammetry using a Fc-MeOH 10-4 mollL solution in PBS 0.1 mollL Electrodes shapes and distances have been optimized to obtain the shown in Fig. t, which produced experimental curves compatible with ext)ected for first oxidation (+0.2-0.3 V vs. Ag).

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1. ECL optical imaging system. A: side view; B: top view (R: quasi-reference electrode; W: working electrode; C: counter electrode) profile for a Ru(bpy)/+rrPA solution. Test solution was 10.4 mol/L Ru(bpy)2+3 and 3x1O·2 mol/L TPA in 0.1 M PBS (pH 7.6). voltammetry and ECL measurements were carried out with an AUTOLAB electrochemical station (Ecochemie, Holland) at 0.2 Vis scan speed. The ECL signal a was collected by a photomultiplier tube (Hamamatsu R255, biased at 750 V) few millimeters in front of the working electrode. To register ECL signal, the photomultiplier output signal was sent to an ultralow noise current preamplifier (Acton Research model 181, 10.5 AN). Method I: Direct of Ru(bpy)/+-NH 2 to carboxylate beads. 200 IlL of 8/lm beads suspension were washed three times in 0.1 mollL borate buffer (pH 9.6) and two times in 0.1 mol/L MES (pH 5.5) buffer. The beads were finally resuspended in 250 ofMES buffer. Carboxylic groups were activated adding EDC and s-NHS to a final concentration of 50 mmollL and 2 mmollL, respectively. The reaction mixture was gently mixed for 1 h at RT. After a washing cycle (see above) 500 IlL of a 0.75 mmollL Ru(bPY)32+-NH2 solution in 0.1 mol/L borate buffer (pH 8.6) were added and the suspension incubated overnight at 4°C. The beads were finally washed three times in PBS. Method II: Indirect binding using biotin-streptavidin interaction. of -COOH to streptavidin. Ru(bpy)/"-COOH was dissolved in DMF to obtain 70 IlL of a solution with a concentration of 7.1 xl 0.3 mollL. 1.5 equivalents of DCC and NHS were added and the reaction solution was gently mixed for 4 h at RT. 630 ~L of a 2.0x1O· 5 mollL streptavidin solution in 0.1 mollL borate buffer (pH 9.4) were then added (activated Ru(bpy)/+-COOH:streptavidin 40:1 molar ratio). The solution was incubated overnight and the labeled protein was subsequently purified with dialysis against 5 L of PBS.

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of biotin to carboxylate beads. Beads were washed and activated using the described procedure. After 1 h of RT incubation 500 J-lL of 9 mmollL biotin-cadaverine in 0.1 mol/L borate buffer (pH 8.6) were added. The mixture was incubated overnight at 4°C and the beads were finally washed three times in PBS. Labeled beads complex. 50 J-lL of biotinylated beads were centrifuged and after buffer removal 50 J-lL of labeled streptavidin were added. The mixture was gently mixed for 2 h at RT then three washing steps "Pl·~"'·nH'rI centrifuging and resuspending with PBS. The characterization of the was carried out using a fluorescence imaging of non-treated and treated beads.

of the new glass cell was evaluated by electrochemical measurements of a Ru(bpy)/+/TPA solution in PBS. A typical light/current/voltage in 2. The cyclic voltammetry shows the of vV''' .... 'VA oxidation potential at about +l.l V vs Ag. Along with the redox reaction concurrent strong light emission was detected confirming the cell was then to the ECL process. The reproducibility of the measurements on the same cell, collecting the ECL solution.

Left: light/current/voltage profile (recorded at 0.2 Vis) for a live image and (B) ECL image of Ru(bpy)/+-conjugated core micron-sized beads was obtained from the second to the tenth cycle of measurement a cathodic cell-cleaning step before each measurement a constant was imposed for 30 s). The satisfactory results obtained by the FTOcell allowed the development of ECL low-light microscope with a highly sensitive nitrogen-cooled CCD. The glass cell was thus to investigate the heavy core micron-sized beads as a model to simulate cells. The Ru-coupled beads (see Method I) were poured onto the cell and a first fluorescence microscope image was acquired exciting the beads with a UV source to cont1rm the Ru complex-beads reaction. Then both the ECL image and the transmitted light (live) image were acquired (Fig. 2). Tn these images each bright

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spot represents a single bead carrying several Ru(bpy)/+ mOIetIes. This result confirms that this technique allows the spatial localization of the ECL signal. The subsequent step focused on a biological assay model in which biotinylated beads were recognized by ruthenium labeled streptavidin (see Method II). Images were acquired from sample solutions containing the purified adduct. Although the observed signal intensity was lower with respect to the previous experiment, the collected images showed a very similar trend. The glass cell described in this work proved to be a successful tool for ECL-based imaging. Once coupled with low-light microscope it was possible to acquire both the live image (thanks to the cell transparency) and the ECL-generated image. These features make the whole system very promlsmg for ultrasensitive immunohistochemical and in situ hybridization techniques in which the comparison of the two images is a critical factor to localize the analyte. As for the near future perspectives, the technique presented has to be further improved and fully investigated using real biological samples. The final goal is to use the glass cell system to detect and precisely localize the binding of labeled antibodies or DNA on a biological sample. Moreover the method can be used in multiplex analysis employing different ECL labels and/or different luminescent principles (BL, CL) to simultaneously localize different targets in the same sample.

REFERENCES 1. Bard AJ. Electrogenerated chemiluminescence. New York: Marcel Dekker, 2004. 2. Knight A. A review of recent trends in analytical applications of electrogenerated chemiluminescence. Trends Anal Chern 1999;18:47-62. 3. Gerardi RD, Barnett NW, Lewis SW. Analytical applications of tris(2,2'bipyridyl)ruthenium(III) as a chemiluminescent reagent. Anal Chim Acta 1999;378: 1-41. 4. Gorman BA, Francis PS, Barnett NW. Tris(2,2-bipyridyl)ruthenium(II) chemiluminescence. Analyst 2006;131 :616-39. 5. Corgier BP, Marquette CA, Blum LJ. Direct electrochemical addressing of immunoglobulins: immuno-chip on screen-printed microarray. Biosens Bioelectron 2007;22: 1522-6. 6. Marquette CA, Blum LJ. Electrochemiluminescent biosensing. Anal Bioanal Chern 2008;390: 155-68. 7. www.tlexitec.com.br. 8. Zanarini S, Rampazzo E, Bich D, et al. Synthesis and electrochemiluminescence of a Ru(bpY)3-labeled coupling adduct produced on a self-assembled monolayer. J Phys Chern 2008;112:2949-57.

VISUALIZATION OF SEQUENTIAL RESPONSE IN INTRA CELLULAR SIGNAL TRANSDUCTION CASCADE BY FLUORESCENCE AND LUMINESCENCE IMAGING IN THE SAME LIVING CELL

Y. HATTA-OHASHI, T. TAKAHASHI, H. SUZUKI Research & Development Division. Olympus Corporation. Hachioji. Tokyo 192-8512. Japan, Email: [email protected]

INTRODUCTION Cells recognize changes in their environment through the cell surface receptors, resulting in initiation of the intra-cellular signal transduction followed by gene expression of the downstream transcription factor. We have observed protein-protein interaction in cell signaling by fluorescence imaging, and have detected gene expression by reporter assay with luminescence detection. Until now, it had been impossible to observe both of the processes in the same living cell. To observe the two processes sequentially, we developed a luminescence imaging system that is also applicable to fluorescence imaging and we applied it to (I) observation of translocation ofPKC (Protein Kinase C) e from cytoplasm to the cell membrane and following gene expression of the downstream transcription factor, NF-KB, in HeLa cells and (2) Raf-l activation and following gene expression of API in PCI2 cells. MATERIALS AND METHODS Visualization of PKC activation and monitoring NF-K BI gene expression. The pPKCe-EGFP vector contains a fusion of PKCe and EGFP (enhanced Green Fluorescence Protein) under the control of the SV40 promoter. The pGL4-NF-KBI contains the GL4 luciferase under the control of the NF-KB I promoter. HeLa cells were plated on a 35 mm culture dish with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and were co-transfected transiently with the pPKC6-EGFP and the pGL4-NF-KBI vectors. At 48 h incubation after transfection, the medium was replaced with DMEM containing 1% FCS and 10 mM HEPES (pH 7.2). Cells were treated with PMA (phorbol myristate acetate 5ng/mL), and then 500 !!M O-Iuciferin (Promega) was added before the imaging experiment. Visualization of Ras activation and monitoring API gene expression. The pCI-EGFP-RBO+RSV-HA-Ras vector contains the human RBO (Ras-binding domain: 1-149 amino acid) of the Raf-I controlling expression of EGFP and Ras coding sequence. I The pGL4-APl contains the GL4 luciferase under the control of the API promoter. PCI2 cells were plated on a 35 mm culture dish with Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FCS and were cotransfected transiently with the pCI-EGFP-RBO+RSV-HA-Ras and the pGL4-API vectors. At 48 h incubation after transfection, the medium was replaced with RPMIl640 containing 1% FCS and 10mM HEPES (pH 7.2). Cells were

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treated with EGF (epidermal growth factor 50 ng/mL), and then 500 f-tM D-luciferin was added before the imaging experiment. ' . .Hit"."" _.. ___ ......... '" of fluorescence and luminescence. Images were acquired the luminescence imaging system Luminoview (LV200, Olympus) attached with cooled charge-coupled device (CCD) camera, ImagEM (Hamamatsu Photonics) under dark conditions. The operating temperature of the CCO camera was set to -65°C. In this system, the optical parameters such as numerical aperture (NA) of and tube lens, total magnification were optimized for luminescence of a single cell. 2 The dish was kept at 37°C in the humidified chamber of f'rr,c("·r>np> during observation. For fluorescence imaging, the excitation and emission filters were used (conditions of observation described in figure legends).

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Luminescence intensity from single cells was measured as an average value in a region of interest (ROI) encircled for each cell by using MetaMorph software (Universal imaging). RESULTS AND DISCUSSION We successfully observed the translocation of PKCe by fluorescence imaging from the cytoplasm to the cell membrane after PMA stimulation, and following gene expression of the downstream transcription factor, NF-KB, by luminescence imaging in the same HeLa cells (Figs. I A and I B). We monitored the chronological response of the NF-KBI promoter activity in each individual cell, and found that the response was different among cells (Fig. IC). Accordingly, the two processes of protein localization and transcription in signal transduction could be sequentially measured in the same cell. We next examined the Ras activation after EGF stimulation in PCI2 cells. As shown in Fig. 2A, the translocation of Raf-l could be visualized by fluorescence imaging. In Fig. 2B, the line analysis clearly indicated that Raf-I was translocated from the cytoplasm to the plasma membrane with EGF stimulation. The luminescence images of the API gene expression were shown in Fig. 3A. In the time course analysis, the API promoter activity revealed the heterogenetic response in each cell (Fig. 3B). Although the protein-protein interaction in cell signaling and the following gene expression have been observed separately so far, the present method enables us to measure the different stages of the signaling pathway in real-time in living cells. Recently, the single-cell analysis of the gene expression with bioluminescence imaging was reported in several studies 3,4 and they provide insight into mechanisms of gene regulation that could not be obtained by averaging effects in cell population. In addition, with the visualization combined with the fluorescence detection, bioluminescence imaging can be applied to a wide range of kinetic imaging applications. Thus, our imaging system will be helpful in understanding gene regulation in signal transduction,

REFERENCES I.

2.

3. 4.

Kao S, laiswal RK, Kolch W, Landreth GE. Identification of the mechanisms regulating the differential activation of the MAPK cascade by epidermal growth factor and nerve growth factor in PCI2 cells. 1 BioI Chem 2001;276:18169-77. Suzuki H et al. Luminescence microscope for reporter assay of single living cells, Proceedings of the 14th International Symposium on Bioluminescence and Chemiluminescence. 2006;53-56. Welsh DK et al. Bioluminescence imaging in living organisms. Curr Opin Biotechnol. 2005;16:73-8. Ukai H Kobayashi TJ, Nagano M, et al. Melanopsin-dependent photo-perturbation reveals desynchronization underlying the singularity of mammalian circadian clocks. Nat Cell BioI. 2007;9:1327-34.

BIOLUMINESCENCE IMAGING OF INTRACELLULAR CALCIUM DYNAMICS BY THE PHOTO PROTEIN OBELIN MA Y MAW THET, T SUGIYAMA, H SUZUKI Research & Development Division, Olympus Corporation, Hachioji, Tokyo 192-8512, Japan Email: [email protected]

INTRODUCTION The bioluminescent system (luciferase reporter assay system) is widely used for the study of gene expression, signal transduction and other cellu lar activities. The luciferase assay is conventionally performed by the photon-counting luminometer method. In this system, light emitting from cells is measured as integrated value through all cells. Recently, we developed a luminescence microscope to monitor expression activity of genes of interest in each cell spatially and temporally as images, 1.2 and demonstrated heterogeneous response of c-fos gene promoter activity in each cell by ATP stimulation. ATP stimulation leads to calcium release from intracellular membranes, mitochondria and endoplasmic reticulum. The c-fos promoter contains a calcium response element region and responds to numerous environmental changes from outside the cell. We tried to visualize the process from calcium signalling to gene expression of c-fos at the single cell level using the calcium-regulated photo protein, obelin, and firefly luciferase as a c-fos reporter. MATERIALS AND METHODS Reporter gene construction. Apoobelin gene 3 was inserted into mammalian expression vector, pcDNA3.1 (Invitrogen). The complete region of c-fos gene promoter 4 was inserted into the firefly luciferase vector (pGL3 basic promoter vector, Promega). HeLa cell was co-transfected the apoobelin and c-fos promoter constructs by FuGene HD (Roche). Ca2+ imaging. HeLa cell co-transfected apoobelin and c-fos promoter constructs were incubated in Dulbecco's modified Eagle's medium containing coelenterazine (Renilla luciferase assay system, Promega) for 4 h to reconstruct obelin. The cell was stimulated by 500 ~MATP, and the luminescence image captured on a luminescence microscope (Luminoview, LV 200, Olympus) equipped with iXon EM-CCD camera (Andor). Binning of the CCD was 2x2, and the exposure time was 25 s with 30 s interval. The cell was re-stimulated by I 0 ~M ionomysin at 20 min after A TP stimulation. c-fos imaging promoter assay. After Ca2+ imaging without ionomysin stimulation, 1 mM luciferin was added into the medium, and luminescence image was captured by 5 min exposure time at 12 min interval. The c-fos signal light was separated from Ca2+ signal light by an optical long pass filter (610 nm), and the promoter activity was monitored sequentially after ci+ response of the same cells. 359

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RESULTS AND DISCUSSION Luminescence image of obelin regulated by and its field in HeLa cells are shown in Fig. I, and intracellular dynamics in some chronologically (see Fig. 2). responses are observed selected cells are

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£ 275 1--------------------"--""',,£'0"'--'-"'-.1 250

------1

!lEAl§;-

~~f-~c----------------------~

361

~ 1100 .~ 1000

f-Mc+T-------------

~900 1i BOO

~3oo ~~----~~~~~~~--~--~

~

~ 250 H------------------'-=----~

-

I~= \~

""/\

.J

"-

-~

~

~~--~

"& 700

.. .""

~ - ----"_._---

j

\

I I

\-~ \

Cell 2

600

1 2 3 4

o

5 6 7 8 9 10 11 12 13 14 15 16 17 1819 20 T line after A1P sUnulation (min)

Time after A1P stinulation ( hr )

425~----·---

.~4oo

~

! ::: V~--~-~~------------I

I E ---------------------/,---__1

375 1

~ 1500 ~---4 ~---------i ~ 1350 ~-- -I------~

~350 f-~---------~--__1 ~ 325 I-----ct-T~F

i

j!

i

2xlO'

lx10'

.."Oxl0.8

6..0:&:10"'

Concentration ofbenserazide, M

Fig. 2. Calibration curve for benserazide obtained by micelle-enhanced fluorescence intensities as function of concentrations ofbenserazide. 1I. em 318 nm and pH 5.0.

392

Lee SH et al.

Sample 1

. Ma dopar tabl ets. T a bl e. 1 A nalytlca I . I resu Its III Benserazide Itablet (mg) Found Itablet (mg)

2

25 25

3

25

25.21 25.15 24.91

CONCLUSION A simple fluorimetric method has been developed for the determination of benserazide. The anionic surfactant of SDS showed a strong sensitizing effect for the fluorescence of benserazide in a pH 5.0 buffer. The proposed method can be applied to the assay of benserazide in real sample with good results.

REFERENCES 1.

Wolters EC. Deep brain stimulation and continuous dopaminergic stimulation in advanced Parkinson's disease. Parkinsonism Relat D 2007;13:S18-3. 2. Fanali S, Pucci V, Sabbioni C, Raggi MA. Quality control of benserazide-Ievodopa and carbidopa-Ievodopa tablets by capillary zone electrophoresis. Electrophoresis 2000;21 :2432-7. Treseder SA, Rose S, Summo L, Jenner P. 2003. Commonly used L-amino 3. acid decarboxylase inhibitors block monoamine oxidase activity in the rat. J Neural Transm 2003;110:229-38. 4. Tang Y, Cao L, Qian X, Zhang R. Quantitative analysis of technical samples of benserazide by reversed-phase high-performance liquid chromatography. Sepu 1985;2:56-8. 5. Di P, Yin F, Mao H. Determination of dl-benserazide by the 2.5th order differential anodic stripping voltammetry. Fenxi Huaxue 1992;20:1416-8. 6. Dinc E, Kaya S, Doganay T, Baleanu D. Continuous wavelet and derivative transforms for the simultaneous quantitative analysis and dissolution test of levodopa-benserazide tablets. J Pharmaceut Biomed 2007;44:991-5. 7. Pistonesi M, Centurion ME, Band BSF, Damiani PC, Olivieri AC. Simultaneous determination of levodopa and benserazide by stopped-flow injection analysis and three-way multivariate calibration of kinetic-spectrophotometric data. J Pharmaceut Biomed 2004;36:541-7. 8. He WW, Zhou XW, Lu JQ. Capillary electrophoresis-chemiluminescence detection of levodopa and benserazide in Madopar tablet. Chin Chern Lett 2007;18:91-3. 9. He WW, Zhou XW, Lu JQ. Simultaneous determination of benserazide and levodopa by capillary electrophoresis-chemiluminescence using an improved interface. J Chromatogr A 2006; 1131 :289-2. 10. Wang C, Huang P, Liu Y. Simultaneous determination of levodopa and benserazide in madopar tablets by HPLC-ECD. Zhongguo Yiyuan Yaoxue Zazhi 2003;23:517-9.

IMPROVEMENT IN CARBARYL ASSA Y BY FLUORESCENCE IN A MICELLAR MEDIUM SH LEE,! CW JEON,2 WH KIM,! HY CHUNG,! SM WABAIDUR,! HW PARK,! YS SUH,! MA KHAN! I Department o/Chemistry, Kyungpook National University, Taegu, 702-701, Korea 2Korea Institute o/Geoscience & Mineral Resources, Dafjon, 305-350, Korea e-mail: [email protected]

INTRODUCTION Carbaryl (I-naphthyl methylcarbamate) is a chemical in the carbamate family used chiefly as an insecticide. It is a colorless white crystalline solid. Carbaryl disrupts the nervous system by adding a carbamyl moiety to the active site of the acetylcholinesterase enzyme, which prevents it from interacting with acetylcholine.! It is classified as a likely human carcinogen by the EPA. The pesticide is used indiscriminately, so the toxicity has raised public concern about the ecosystem and human health. Carbaryl is lethal to many non-target insects such as the honeybee. Accumulation of the pesticide occurs in many aquatic organisms such as catfish and algae. 2 Due to public health and ecosystem concerns a number of analytical procedures have been used to determine carbaryl concentrations. Most of analytical methods employed for quantification of pesticides are based on spectrophotometry,3.4 separation by chromatographic techniques such as thin-layer chromatography (TLC),5 gas chromatography (GC)6 and high pressure liquid chromatography (HPLC).7 Chromatographic methods have high sensitivity and accuracy but have disadvantages such as high cost, high volumes oftoxic solvents and complex operation and this limits their application. Fluorescence spectroscopy can be used for determination of carbamate pesticides residues, and the micellar medium fluorescence enhancement method has been exploited in recent years because of its inherent sensitivity. EXPERIMENTAL Apparatus. SPEX Fluorolog-2 spectrofluorometer (Edison, NJ, USA)., 450-W xenon lamp, R 928 photomultiplier tube powered at 950V (Hamamatsu Co.), SPEX DM 3000F spectroscopy computer. A pH meter (Model Orion 520A, USA) was used for pH adjustment. Reagents. Carbaryl (Fluka, USA), Sodium dodecyl sulfate (SDS) (Fluka, USA), Ethyl alcohol (Duksan, Korea), Sodium hydrogen phosphate (Na2HP04) (Merck, Germany), Sodium dihydrogen phosphate (NaH 2P0 4) (Merck, Germany). Basic procedure. Carbaryl standard stock solution was prepared in distilled water through ultrasonication for 6 h at 40 to 50°C. Carbaryl solution (0.1 flmollL - 0.1 mmollL) was diluted with distilled water in a 10 mL calibrated flask containing 2 mL phosphate buffer solution (pH 7), 2 mL ethanol (20%) and 2 mL SDS solution (0.1 mmollL). The fluorescence measurements were performed at "'em = 349 nm and "'ex = 281 nm. 393

394

Lee SH et at.

RESULTS AND DISCUSSION Spectral characteristics and selection of surfactant. Increase in the fluorescence intensity of the carbaryl solution was observed using SDS as surfactant at excitation and emission wavelengths of 281 nm and 349 nm respectively (Fig. 1). As can be seen from Fig. 1, Triton X-100 and dodecyl pyridinium chloride (DPC) decreased the fluorescence. This phenomenon clearly indicates that cationic surfactant (DPC) and neutral surfactant (Triton X-100) have negative effects on the fluorescence intensity for carbaryl while anionic surfactant (SDS) enhances the fluorescence intensity of carbaryl. This surfactant-enhanced phenomenon by SDS was used for the spectrofluorimetric determination of carbaryl. Parameter optimization. For maximum fluorescence various parameters, e.g. alcohol, pH and SDS concentration were optimized for the spectrofluorimetric determination of carbaryl. By addition of ethanol an increase in fluorescence was observed. Further it was found that increasing the ethanol percentage up to 20 % increased fluorescence. Beyond 20 % ethanol a decrease in fluorescence was found. Therefore, 20 % ethanol was used in further experiments. Phosphate buffer pH 7 was found to be the optimum buffer. The optimum concentration for SDS was found to be 1.0 x 10-4 mol/L. Above this concentration of SDS, a decrease in response was observed.

•.AdO·

1

L(,A+VU-huftfl"t$f)S Z. CA ~ pH .. butf..]'

4. ('A "" pU - buffn t-llihm X· ~ pH .... bldft't··~ [,pC

11)()

3, ('A

I..hl0'

1.0dO·

Wavelmglb, am

Fig. 1. Fluorescence excitation (Eem = 349nm) and emission (Eex = 281nm) spectra of carbaryl. Conditions: carbaryl, 1.0 x 10-4 M, pH 7.0.

Improvement in Carbaryl Assay by Fluorescence in a Micellar Medium 2.0xlO·

395

R:O.99965

_ 1.5x:lO'

flo

0.0 " ' - - _ - ' -_ _---'_ _ _-'--_ _-"'-_ _ _-'-----_ 0.0

2.0sl0·'

4.01= ilia I=Cdm,1>+C 2 Im,2>+---+C d (m)lm, d(m», From the eigen function equation

He 11.fJ m >= Em 11.fJ m >

we can obtain

(6)

412

Pang X-F

(7)

where

em = Col .(Cl, C

2,

"',

Cd(m)), Hm is a d(m) X d(m), real, symmetrical

matrix with the diagonal elements. Applying the above formulae, we calculate numerically the quantum energy levels of

a

-helix protein molecules by using

generally accepted experimental data of physical parameters, which are'-" W=(39~ 58.5) N/m; M=(3.51 ~ 5.73) X 10-25 kg is mass of amino acid,

XI =(56 ~

o

62)PN,X2 = (10 -15)PN, J=7.8 em-I, ro=4.5 A, L,=14.63 em-I, L2=12.45 cm-',

G2 =49.73 em-I for Pang's model. The calculated results of quantum vibrational energy levels from the Davydov's model 4 and Pang's model 7-'2 together with the experimental data are presented in Table 1. We see from the Table 1 that the energy-spectrum of the protein is quite complicated, but its distribution has many particularities: (1) The vibrational energy spectra consist of a series of manifolds or energy-bands, i.e., there are several energy-levels corresponding each vibrational quantum-number m.

For example, the first excitation state (m=l) (from

1610-1678cm-') contains 8, there are 44 and 164 in m=2 (from 3179-3358cm-'), respectively. Hence, as m increases, the energy discrepancy, 6. E, between energy-levels decreases gradually. For instance, the discrepancy, 6. E, changes from 6cm-' to 23cm-' at m=l, but 6. E is 0-14cm-' at m=2 , respectively. We can suppose that there is individual energy-bands at large m. (2) The vibrational spectra have strong local mode pattern, or the discrepancy between the energy-levels depends strongly on nonlinear interaction, Y, as we see from Eqs. (12) and (14). This is due to the fact that

Y

is much greater than J,

fl,

f2

and

f3.

(3) The local-mode

degeneracies of energy levels appear at higher-lying vibrational states which begins to occur at m?=2, e.g., degeneracy at 3242cm-' and 3259cm-' at m=2, etc.). Therefore, the degeneracy increases with increasing m. We can see from the energy-spectra shown in Table 1 that the protein molecules can absorb or radiate the

Mechanism and Properties of Bio-Photon Emission and Absorption of Protein Molecules

413

Table 1. Vibrational energy-spectra of protein with three channels in cm- 1

M

exp

cal

M

I

1610.42

I

1612.01

I

1627.64

I

1630.11

I

exp

cal

I

1650

1653.81

I

1666

1667.65

I

1678.73

2

3150

3179.40

2

3203.19

2

3205

3204.71

2

3211.85

3212.95

2

3213.21

3216

3216.84

2

3218.19

3242.48

2

3242.45

2 2 2 2

3250

1662

1661.98

3249.68

2

3258.78

2

3259.87

2

3261.77

2

3260.95

2

3262.97

2

3263.67

2

3269.43

2

3278.89

2

2

3282.84

2

3283.97

2

3285.44

2

3286.49

2

3287.44

2

3290.49

2

3298.96

2

3300.09

2

3301.15

2

3302.13

2 2

3279

3267

3267.39 3277.71

3280

3280.21

2

3309.47

2

3310.21

2

3312.91

2

3313.37

2

3321.54

2

3322.49

2

3323.56 3329.16

2 2

3327.96 3333.91

2

*Where "exp" is experimental values, cal is calculated values in Pang's theory . bio-photons with a wavelength of 5-7

~m

and < 3

~m,

which are infrared emissions.

Very clearly, the bio-photon emissions of proteins are caused by the transitions of energy levels of the quanta or excitons after absorbing the bio-energy released from

414

Pang X-F

ATP hydrolysis.

ACKNOWLEDGEMENTS The authors would like to acknowledge the National "973" project of China for financial support (grant No:2007CB9361 03).

REFERENCES 1. Popp FA, Li KH, Gu Q, eds. Recent advances in biophoton research and its application. Singapore:Worid Scientific, 1992:47-154. 2. Gu Q, Popp FA. Biophoton physics: Potential measure of organizational order. In: Ernst G, Jung M, Holick F, eds. Biological effects of light. Berlin:Walter de Gruyter, 1994:425-44. 3. Pang X-F. The theory of non-linear quantum mechanics, Chongqing: Chinese Chongqing Press, 1994, 567-634. 4. Davydov AS. Space-periodical excitations in nonlinear systems. In: Solitons in molecular systems. DordrechtReideel, 2nd edn, 1991: 132-87. 5. Pang X-F. Phys Rev E Improvement of the Davydov theory of bioenergy transport in protein molecular systems Phys Rev E 2000;62:6989-98. 6. Pang X-F. The lifetime of the soliton in the improved Davydov model at the biological temperature 300 K for protein molecules Eur Phys J B 2001 ;19:297-316. 7. Pang X-F, Feng V-Po Quantum mechanics III nonlinear systems. Singapore:Worid Scientific 2005,:471-576. 8. Pang X-F Soliton physics. Chengdu: Sichuan Technology Press, 2003:673-723. 9. Pang X-F, Yu J-F, Luo V-H. Influences of quantum and disorder Effects on soliton excited in protein in improved model. Commun Theor Phys 2005;43:367-76. 10. Pang X-F. Vibrational energy-spectra of protein molecules and non-thermally biological effect of infrared light. J Int Infr Mill Waves 2001 ;22:291-306. 11. Pang X-F, Zhang HW, Luo YH. The influence of the heat bath and structural disorder in protein molecules on soliton transported bio-energy in an improved model J Phys Cond Mat 2006;18:613-27. 12. Pang X-F. Quantum vibrational energy spectra of molecular chains in crystalline acetanilide. J Phys Chern Solids 2001 ;62:793-6.

THE MECHANISM OF PHOTON EMISSION OF BIO- TISSUES AND ITS PROPERTIES

PANG XIAO-FENGY CAO XIAN-YU 1 1Institute

of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, PR.China and 2Internationai Centre for Materials Physics, Chinese Academy ofSciences, Shenyang I 10016, China Bio-photon emission from tissues in human beings, animals and plants is a general biological phenomenon, every bio-tissue can radiate bio-photons with a certain strength and frequency.I-6 It can provide an insight into the state of activity in living systems. However, the mechanism of bio-photon emission is not clear. It is known that bio-photon emission is a result of energy transition of electrons or atoms, therefore, it is necessary to relate this to various biological activities, such as, the changes of structures and conformations and states of bio-tissues, including cells and bio-macromolecules. We think that the bio-photon emission arises from the growth and development of biological tissues after absorbing the energy, material and information from the environment through bio-organs. In these processes the states of bio-molecules, atoms and bonded electrons in these bio-tissues are changed and these changes and transitions of states result in the emission of bio-photons. In these biological processes, the interactions of light-carried information plays an important role in the conversion and transfer of bio-energy and biomaterial as well as the processing of bio-information. We know from experiments that 90% of the bio-information needed for living organisms comes from this process. Although this process is complicated we can study only the interaction of the absorbed photons with the small biomolecules and their final variations of state resulting from the interactions In general, the small biomolecules vibrate or move around their equilibrium positions, we here refer to them as "localizors". Hence living systems are composed of a large number of localizors, but the latter consist also of many atoms and electrons including bonded electrons that can interact with the light. Then the states of the localizors will be changed and can transit to other quantum states. In the meantime, the bio-entropy of the systems will also be changed. We can calculate the change of bio-entropy of the living systems by 415

416

Pang X-F & Cao X-Y

using quantum statistical-theory of the non-equilibrium state. 9- 13 In such a case, the changes of number and states of particles arising from the energy, material and information absorbed from the environments can be represented by: 3-6

where n i is numbers of localizer with the energy E i ' mv is number of photon with the energy E v ,the n i and mvare the functions related to time, I denotes the type of bound. The gl in Eq. (1) is a function related to time and represents the conversion of material, energy and information, and the features of non-equilibrium state of the living system, thus it embodies the open characteristics of living systems due to the interaction of the system with the incoming photons, energy and material. In the combined system, the numbers of microscopic states of this great system can be represented by:

where N is totality of the localizors, energy-level

Ei

'

W, is degeneration numbers of the

Wv is the degeneration numbers of energy-level E v. We can

determine the probability distribution of the microscopic states by the Lagrange

~ a, g, I = 0 where al is Lagrange uncertainty factor. Thus we ~rther obtain: uncertain factor method of

Ln(m. + W. -\)/ m.)+

(n, / W, ), i.e., the numbers of localizors with higher energy is more than in the lower energy-levels or the numbers of localizors in each microscopic state in the higher energy-levels is more than in the lower energy-levels. This shows that the bio-entropy of the living system decreases, if the energy, material and information acquired from the environments are not to increase kinetic energy of thermal motion of the localizors, but to make these localizors transit to higher energy-levels from the lower energy levels, or to make the particle numbers with higher energy be more than one in the lower energy levels which forms a reversed distribution of particle numbers. This represents a degree of self-organization, it has the features of high energy and low entropy, and is formed naturally in the living system after absorbing energy, material and information from the environment. Substituting now Eq. (3) into Eq. (5), we obtain:

418

Pang X-F & Cao X-Y

This shows that the change of bio-entropy is negative, only if an appropriate energy, material and information are absorbed from the environments, e.g., the condition:

a a a dad a d a [ - - - + - - - ( - ) + - ( - ) - - ( - ) ] g ,>0 anj ani amv dt a· dt a· dt a . .

~

~

~

is satisfied. Thus the bio-self- organization can be spontaneously formed. 7 or enhanced, and it is also a kind of dissipation structure. 7 because the above condition of n quanta in ith state converting to n quanta in the jth state after absorbing n photons is just the condition of form of dissipation structure formed in a non-equilibrium state. Meanwhile, the bio-self-organization has the features of higher energy and lower entropy. From Eq. (6) we know that if Ll S '",,,,,,metD"''' bb10~O~

N ...... '"

£:X"'.O

6'" 1.

",ROCNO

_ "'"c"e"";'''9

p.~"'''''''' :32,,"6&

:aD'-I'J.,l300l;;i ...

.~::g It' h

,

F

7

Fig. 2.

'"

. o

,

ppon

IH NMR spectra of f3-CD and fJ-CD and cuprous iodide-pyridine.

Synthesis of a Novel Fluorescence Probe of (3-CD and Cuprous Iodide Pyridine

423

To study the structure of the inclusion complex, IH NMR experiment was performed (Fig. 2). According to the shift of the IH (below), it was shown that an inclusion complex of f3-CD and cuprous iodide pyridine was formed. Fig. 2 illustrates the alteration of the fluorescence spectrum of the inclusion complex of f3-CD and cuprous iodide-pyridine upon addition of different concentrations of methane. The fluorescence maximum excitation and emission wavelengths of the inclusion complex of f3-CD and cuprous iodide-pyridine were at 282 nm and 365 nm, respectively. Addition of different concentrations of methane caused a noticeable decrease in fluorescence intensity. The maximum emission wavelength produced a small red shift from 365 nm to 367 nm and the corresponding excitation wavelength was slightly red shifted from 282 nm to 286 nm. The marked fluorescence quenching and the bathochromic displacement proved that there was interaction between the methane and this inclusion complex.

260

280

300

320

340

360

380

400

420

440

460

v.eveiength Inm

Fig. 3.

Fluorescence spectral changes of inclusion complex f3-CD and cuprous iodide-pyridine bubbling in various concentrations of methane.

ACKNOWLEDGEMENTS We appreciated the support of the key Funding of the National Natural Science of China (No: 50534100) and Shanxi Province graduate student innovative plan (No: 07010700). REFERENCES 1. Wenz G. Cyclodextrins as building blocks for supramolecular structures and functional units. Angew Chern Int Ed 1994;33:803-22. 2. Harada A. Cylodextrin based molecular machines. Acc Chern Res 2001;34: 456-64. 3. Liu Y, Li L, Zhang H-Y, Zhao Y-L, Wu X .. Bis(pseudopolyrotaxane) s possessing copper ions formed by different polymer chains and bis(~-cyclodextrin)s bridged with 2,2' -bipyridine-4,4' -dicarboxyether. J

424

4. 5.

6.

Qiao Jet al.

MacromoI2002;35:9934-8. Ma shikun, Wang Jinlin, Li Aixiu, et al. Synthesis and crystal structure of P-CD and paradioxy benzene. J.Chinese Science Bulletin 2000;45:1383-1386. Liu Y, Zhao YL, Zhang HY, et al. Polymeric rotaxane constructed from the inclusion complex of p-cyclodextrin and 4,4-dipyridine by coordination with nickel ions. Angew Chern Int Ed 2003;42:3260-3. Song L, Meng QJ, You X. Cyclodextrin and inclusion compounds. J Inorg Chern 1997;13:368-74.

PHOSPHORESCENCE PROPERTIES OF 2-BROMOQUINOLINE-3BORONIC ACID IN SODIUM DEOXYCHOLATE AND ITS POTENTIAL APPLICA TION IN RECOGNITION OF CARBOHYDRATES QJ SHEN,I WS ZOU,I WJ JIN,1,2· Y WANG 1• 1School a/Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, P.R. China 2College a/Chemistry, Beijing Normal University, Beijing 100875, P.R.China *Email: [email protected]

INTRODUCTION Recently, a considerable effort has been devoted towards aromatic boronic acids because of their remarkable luminescence properties which can be altered when they bind to carbohydrates. I,2 The signal measured in such research was usually fluorescence rather than phosphorescence, because the latter is easily quenched by oxygen. However, phosphorimetry has its own advantages, such as large Stokes shift, high signal-to-noise ratio and long lifetime, which make these signals easy to separate from background signal and thus become a potential tool for analysis of biological samples. Here we report a new approach using 3-bromoquinoline as a phosphorescent sensing molecule for the detection of carbohydrates in sodium deoxycholate (NaDC) solution. NaDC can form aggregates in aqueous solution and provide a rigid hydrophobic environment that can protect a phosphor from quenching by external molecules such as oxygen. Although 5-quinolineboronic acid and 8quinolineboronic acid have been reported to be used as fluorescent agents for the detection of carbohydrates3,4 there is still little development of phosphorescencebased sensors for carbohydrates. MATERIALS AND METHODS Materials and apparatus. The 2-bromoquinoline-3-boronic acid (BrQBA) (97%), D-glucose (99%) and D-mannose (99%) were purchased from Alfa Aesar. NaDC (+99%), D-fructose (99%) and D-galactose (99%) are products of Acros Organics. A Cary Eclipse luminescence spectrometer (Varian Company) was employed to obtain room temperature phosphorescence (RTP) spectra and measure lifetime. Intensity and lifetime measurement of BrQBA. IOO!J.L BrQBA ethanol stock solution of 5.0 mmoIlL and an appropriate volume of NaDC solution were transferred into a 10 mL comparison tube, and mixed and finally diluted to 10 mL. The excitation and emission slits were both set at 20 nm. The delay time and gate time were set at 0.10 and 5.0 ms for intensity measurement, and both at 0.1 ms for lifetime measurement. 425

426

Shen QJ et al.

RESUL TS AND DISCUSSION Room temperature phosphorescence (RTP) spectrum and lifetime of BrQBA in NaDC. BrQBA gave a weak fluorescence and no phosphorescence in aqueous solution because of the quenching by dissolved oxygen. However, a strong RTP signal was observed in a NaDC solution without any deoxygenation, with emission at 500 nm. ~~--------------------~ '0;;

~

~

0 ~asured value - - Fitted decay curve

~

~

0·~0---::1;0~~20~~3~0~~40~~~5'0 Lifetime/ms

10

20

30

40

50

Lifetime/ms

Fig. 1. RTP decay curve (left) and analysis of residual (right), [BrQBA] =5.0xI0- 5 mollL, [NaDC] =4.0 mmollL. This is because NaDC in aqueous solution formed micelle complexes that provide a near rigid and hydrophobic structure,5 that are favorable not only for the stabilization of the triplet states of BrQBA, but also preventing from the quenching of oxygen. As 6 shown in Fig. I, phosphorescence decay showed as diexponential decay model, with a long lifetime of 6.99 ms and a short lifetime of 1.39 ms. The fractional contribution of long-lived component was> 73.5%, suggesting that the majority of BrQBA molecules were well protected by NaDC aggregates. Effect of NaDC concentration on RTP intensity. The RTP intensities of BrQBA increased at first upon increase of NaDC concentration and reached a maximum at 4.0 mmollL. Then, RTP intensity fell sharply to zero when the NaDC concentration was greater than 6.0 mmollL. This result suggests that it is essential to maintain the NaDC concentration so as to obtain optimal RTP emission. The reason is that NaDC in aqueous solution exists as a variety of clusters with different sizes and is prone to form a dimer in the lower concentration range, 5,7,8 which can tightly capture individual phosphors as a "sandwiched" structure that isolates the phosphor from quenchers such as molecular oxygen. RTP change with incubation time. When the NaDC concentration was fixed at 4.0 mmollL, the RTP intensity of BrQBA increased with incubation time, accompanying by an increase in the fractional contribution of the long-lived species up to 95%. This is because the equilibrium between BrQBA and NaDC is reached slowly at room temperature. However, we found that heating accelerated this process and also produced the stronger RTP intensity.

Phosphorescence Properties of2-Bromoquinoline-3-Boronic Acid 427 1.7r-T"99%)(O.lOS g in SO mL dichloromethane) was diluted to prepare working solutions just before use. Mineral salts medium (MSM) comprised - (NH4)2S04, 1000mg; Na2HP04, 800mg; K2HP0 4, 200mg; MgS0 4 '7H 20, 200mg; CaCh'2H 20, 100mg; FeCI 3 'H 20, Smg; CNH4)6M07024'H20,1 mg; 1000ml ofMilli-Q water; pH=7]. 2.682g DL-malic acid CAR, Shantou Xilong Chemical, China), 4.203g citric acid monohydrate CAR, Sinopharm Chemical Reagent, China) and 1.94 mL butylic acid CAR, Shantou Xilong chemical, China) were dissolved in 100 mL MSM solution respectively to 441

442

Wei XY et al.

prepare as stock solutions, of which the concentration were all 200 mmol/L. Phenanthrene-degrading microorganisms were from School of Life Science, Xiamen University. Fluorescence was measured using a Cary Ellipse spectrofluorimeter (Varian, USA)( ex and em slits 5.0 nm). UV and visible absorption spectra obtained on an Ultrospec 2100 pro UV-Visible spectrophotometer (Amersham Bioscience) (xenon lamps as light source). 600 249.06,495.33(1

164.00,484.813

347.07,377.026

150

300 350 Wavelength (urn)

400

Fig. 1. Excitation (a) and emission (b) spectra of phenanthrene in Milli-Q water

W ...... d.'ngd!. (run)

Fig. 2. Excitation (a) and emission (b) spectra of phenanthrene in MSM solution

Fluorescence method for phenanthrene. Fig. I and 2 show that the excitation and emission fluorescence spectra of phenanthrene in Milli-Q water and MSM solution are similar, maximal excitation and emission peaks at 249.06 and 364.00 nm respectively, and signal of phenanthrene was determined at this wavelength. Serial concentrations of phenanthrene prepared in MSM were used to determine the fluorescence signa!.3 The experimental results shown that in the range of 60 Ilg/L to 1000 IlglL, a good relationship between the concentration (x) and fluorescence signal (y) value of phenanthrene in MSM was obtained (y=1351.6x (r2 =0.9978). The detection limit was 44.9 Ilg phenanthrene/L (B + 3SD), with a relative standard deviation of less than 1.42% Cn = 11). The fluorescence signal blank was measured throughout the experiment; the average value was 1.23 (n = II), which was much lower than the fluorescence signal of phenanthrene. Fluorescence spectra of phenanthrene in LMWOA solution Control experiments showed that malic acid, citric acid and butylic acid had little effect on the spectral characteristics of phenanthrene or its fluorimetric determination. Measurement of phenanthrene biodegradation by fluorescence method. 50.0 mL MSM were added to each incubation flask; the final concentration of phenanthrene was 1000 j.l giL. After sterilization at 121"C for 15 min, the bacterial strain was inoculated into individual incubation flasks and incubated on a rotary shaking incubator at 25 'C and 150 rpm in the dark. The biodegradation rate of phenanthrene was monitored directly by the fluorescence method without solvent extraction. Quantification of LMWOA by UV-VIS method. A series concentration of malic acid, citric acid and butylic acid were prepared in MSM respectively and their absorption signals were determined all at 205 nm 9 to obtained absorption calibration

Effects of LMWOA on Biodegradation of Phenanthrene Studied by Fluorimetry

443

standard curves. All of the standard curves for these three LMWOA have good linear relationship in experiment demanded concentration range. RESULTS AND DISCUSSION Effects of different LMWOA on the degradation of phenanthrene. Biodegradation of phenanthrene in MSM solution with or without malic acid, citric acid and butylic acid (LMWOA 1600 ilmol/L) are shown in Fig. 3. 800

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