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Conjugated Polyelectrolytes Based on Poly(para-Phenylene Ethynylene)

Permanent Link: http://ufdc.ufl.edu/UFE0022803/00001

Material Information

Title: Conjugated Polyelectrolytes Based on Poly(para-Phenylene Ethynylene) Fluorescent and Chemiluminescent Sensors
Physical Description: 1 online resource (205 p.)
Language: english
Creator: Liu, Yan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: assay, chemiluminescence, enzyme, fluorescence, sensors
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Our study focused on design and development of fluorescent and chemiluminescent biosensors based on functionalized poly(para-phenylene ethynylene)s (PPEs). By taking advantage of the amplified signal response of conjugated polyelectrolytes (CPEs) to aggregation and/or the presence of charged quenchers, the developed PPE-based optical biosensors afford high sensitivity and good specificity. They also allows for rapid, convenient and continuous determination of enzyme activity and/or inhibition. First, a PPE derivative, biphenyl poly(phenylene ethynylene) sulfonate (BpPPE-SO3-) is used to develop a fluorescence turn-off assay for phospholipase C (PLC) based on the reversible interaction between BpPPE-SO3- and phosphatidylcholine, which is regarded as the natural substrate of PLC. The PLC assay operates with phospholipid substrate concentrations in the micromolar range and the analytical detection limit for PLC is < 1 nM. The real-time fluorescence intensity from the CPE can be converted to substrate concentration by using an ex-situ calibration curve, allowing kinetic parameters of PLC to be determined. PLC activation by Ca2+ and inhibition by EDTA and fluoride ion are demonstrated using the optimized sensor. Second, another PPE derivative, poly(phenylene ethynylene) carboxylate (PPE-CO2-) is used to develop a fluorescent assay for alkaline phosphase (ALP) based on the fluorescence recovery of Cu2+-quenched PPE-CO2- by pyrophosphate (PPi), the natural substrate of ALP. Using the PPE-CO2--Cu2+ system as the signal transducer, a real-time fluorescence turn-off assay for ALP operates with PPi in the micromolar range, and it offers straight-forward and rapid detection of ALP activity with the enzyme present in the nanomolar concentration range with detection limit of 5 nM, operating either in an end-point or real-time format. Kinetic and product inhibition parameters are derived. Third, addition of adenosine 5?-triphosphate disodium (ATP) into a solution of PPE-CO2- and Cu2+ also recovers the Cu2+-quenched fluorescence of PPE-CO2- to a notable higher extent compared with the introduction of adenosine 5?-diphosphate sodium (ADP) and adenosine 5?-monophosphate sodium (AMP) under the same concentration level. Using ATP as substrate or product, a PPE-CO2--based real-time fluorescence assay to detect the catalytic activity of adenylate kinase (ADK) in an equilibrium transphosphorylation is designed and developed based on the different response of adenosine phosphates to quenched fluorescence of polymer. The ADK assay shares the same advantage of the ALP assay and allows for calculation of ADK catalyzed reaction rates as well as kinetic, activation and inhibition parameters. Last, the first chemiluminescence system for CPE was developed based on the peroxyoxalate chemiluminescence reaction. The chemiluminescence systems consists of bis(2,4,6-trichlorophenyl) oxalate (TCPO) as aryl oxalate, imidazole as catalyst, H2O2 as oxidant, and two PPEs as fluorophores. The effects on chemiluminescence signals of concentrations of oxalate, catalyst, oxidant and fluorophores as well as solvent composition are studied and the amplified chemiluminescence quenching property of CPE is found. The chemiluminescence of CPE is successfully applied as a biosensor platform by qualitatively and quantitatively detecting activities of two enzymes, peptidase and ALP.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Yan Liu.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Schanze, Kirk S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022803:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022803/00001

Material Information

Title: Conjugated Polyelectrolytes Based on Poly(para-Phenylene Ethynylene) Fluorescent and Chemiluminescent Sensors
Physical Description: 1 online resource (205 p.)
Language: english
Creator: Liu, Yan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: assay, chemiluminescence, enzyme, fluorescence, sensors
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Our study focused on design and development of fluorescent and chemiluminescent biosensors based on functionalized poly(para-phenylene ethynylene)s (PPEs). By taking advantage of the amplified signal response of conjugated polyelectrolytes (CPEs) to aggregation and/or the presence of charged quenchers, the developed PPE-based optical biosensors afford high sensitivity and good specificity. They also allows for rapid, convenient and continuous determination of enzyme activity and/or inhibition. First, a PPE derivative, biphenyl poly(phenylene ethynylene) sulfonate (BpPPE-SO3-) is used to develop a fluorescence turn-off assay for phospholipase C (PLC) based on the reversible interaction between BpPPE-SO3- and phosphatidylcholine, which is regarded as the natural substrate of PLC. The PLC assay operates with phospholipid substrate concentrations in the micromolar range and the analytical detection limit for PLC is < 1 nM. The real-time fluorescence intensity from the CPE can be converted to substrate concentration by using an ex-situ calibration curve, allowing kinetic parameters of PLC to be determined. PLC activation by Ca2+ and inhibition by EDTA and fluoride ion are demonstrated using the optimized sensor. Second, another PPE derivative, poly(phenylene ethynylene) carboxylate (PPE-CO2-) is used to develop a fluorescent assay for alkaline phosphase (ALP) based on the fluorescence recovery of Cu2+-quenched PPE-CO2- by pyrophosphate (PPi), the natural substrate of ALP. Using the PPE-CO2--Cu2+ system as the signal transducer, a real-time fluorescence turn-off assay for ALP operates with PPi in the micromolar range, and it offers straight-forward and rapid detection of ALP activity with the enzyme present in the nanomolar concentration range with detection limit of 5 nM, operating either in an end-point or real-time format. Kinetic and product inhibition parameters are derived. Third, addition of adenosine 5?-triphosphate disodium (ATP) into a solution of PPE-CO2- and Cu2+ also recovers the Cu2+-quenched fluorescence of PPE-CO2- to a notable higher extent compared with the introduction of adenosine 5?-diphosphate sodium (ADP) and adenosine 5?-monophosphate sodium (AMP) under the same concentration level. Using ATP as substrate or product, a PPE-CO2--based real-time fluorescence assay to detect the catalytic activity of adenylate kinase (ADK) in an equilibrium transphosphorylation is designed and developed based on the different response of adenosine phosphates to quenched fluorescence of polymer. The ADK assay shares the same advantage of the ALP assay and allows for calculation of ADK catalyzed reaction rates as well as kinetic, activation and inhibition parameters. Last, the first chemiluminescence system for CPE was developed based on the peroxyoxalate chemiluminescence reaction. The chemiluminescence systems consists of bis(2,4,6-trichlorophenyl) oxalate (TCPO) as aryl oxalate, imidazole as catalyst, H2O2 as oxidant, and two PPEs as fluorophores. The effects on chemiluminescence signals of concentrations of oxalate, catalyst, oxidant and fluorophores as well as solvent composition are studied and the amplified chemiluminescence quenching property of CPE is found. The chemiluminescence of CPE is successfully applied as a biosensor platform by qualitatively and quantitatively detecting activities of two enzymes, peptidase and ALP.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Yan Liu.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Schanze, Kirk S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022803:00001


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1 CONJUGATED POLYELECTROLYTES BA SED ON POLY(PARA-PHENYLENE ETHYNYLENE): FLUORESCENT AND CHEMILUMINESCENT SENSORS By YAN LIU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Yan Liu

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3 To my parents

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4 ACKNOWLEDGMENTS It is rea lly hard to list all the people who sin cerely helped me during the past five years and I owe my gratitude to all of them who have made this dissertation possible. Because of them, my graduate experience has been one I will cherish forever. First of all, I would especially like to thank my supervisor, Dr. Kirk S. Schanze, for his support, advice and encouragement. He guided me to investigate the amazing area of sensor applications of conducting polymers. He also taug ht me to think independently, helped me to overcome difficulties, assisted me with paper writing, and supported me to develop other skills beyond chemistry. I am fortunate to have such a great supervisor whose enthusiasm for science and kindness to people set the norm for me in my future career. My deepest gratitude also goes to all the former and current members in the Schanze research group for their help and friendship. When I first joined the group, Dr. Mauricio Pinto and Dr. Chunyan Tan taught me how to use the in struments and help me to learn about our polymers as soon as possible. Dr. Hui Jiang is a photochemistry encycl opaedia and whenever I had questions about my research, he is there with solutions. I am deeply grateful to him for the long discussions in the past three years that he lped me to finish this dissertation. Dr. Kastu Ogawa has always been there to listen and give advi ce, especially with the first project. He also assisted me with writing my first paper. Dr. Xiaoyong Zhao discovered the wonderful pyrophosphate sensor, which provide s the basis for my second and third projects. I am also thankful to him as well as Jarret Vella who t ook their time to synthesize the polymers for me so that I could finish my work. Dr. John Peak re vised my oral report and presentation, and helped me a lot in scientific wring. Jonathan Sommer is a great labmate, his humor enabled me to be cheerful through tough research, and his friendship and encouragement has helped me to become more aclamated in a totally different country. I give my thanks to Dr. Kye-Young Kim, Julia

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5 Keller and Abigail Shelton for managing the orders for the group. Of course, I also want to thank many other group members, Emine Demir, Dr. Richard Farley, Lijuan Huang, Eunkyung Ji, Seoung-Ho Lee and Youngjun Li for their group wo rk which makes my research much easier. I would like to extend my appreciation to Dr. Weihong Tan, one of my committee members, for his kindness in writ ing a recommendation letter for me. I am also thankful to other committee members, Dr. Charles Cao, Dr. Valeria Kleiman and Dr. Paul Holloway for their time and suggestions. Finally, I would like to express my heart-felt gratitude to my family. My parents are a constant source of support and they provide me with the best condition for education. Since I was very young, they have taught me how to face challenges, how to discover my potential, how to be responsible. I am also grateful to my twin brother who takes most of the responsibility to take care of my parents. Without him, I could not go abroad and continue my dream. I have to give a special mention for the support given by my boyfriend. None of this would have been possible without his love and encouragement which I will definitely give for him in his doctorate journey.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES .........................................................................................................................10LIST OF FIGURES .......................................................................................................................11LIST OF ABBREVIATIONS ........................................................................................................ 16ABSTRACT ...................................................................................................................... .............22CHAPTER 1 INTRODUCTION .................................................................................................................. 24Conjugated Polymers ........................................................................................................... ...24Conjugated Polyelectrolytes ...................................................................................................26Amplified Quenching of Conj ugated Polyelectrolytes ...........................................................27Fluorescence Quenching ................................................................................................. 27Stern-Volmer Equation .................................................................................................... 29Amplified Quenching in Neutral CPs: Molecular Wire Effect ....................................... 31Amplified Quenching in CPEs: Combinati on of Ion-Pair Complex Formation and Molecular Wire Effect ................................................................................................. 33Stern-Volmer Plots in Amplified Quenching of CPEs: Sphere-of-Action ......................35Aggregation of Conjugated Polyelectrolytes ..........................................................................37Existence of Aggregation: Solvent De pendent Optical Properties of CPEs ................... 37Effect of Aggregation on Am plified Quenching of CPEs ...............................................38Interaction of Surfactants with CPEs : Tuning the Conformation and Optical Properties .....................................................................................................................40Optical Sensor Application of Conjugated Polyelectrolytes ..................................................43Sensing Mechanisms .......................................................................................................44Superquenching mechanism .....................................................................................44Light harvesting mechanism via fluores cence resonance energy transfer ............... 45Conformation change mechanism ............................................................................ 47Small Ion Sensing with CPEs ..........................................................................................48Small Biomolecule Sensing with CPEs ........................................................................... 50Protein Sensing with CPEs ..............................................................................................52Indirect detection of enzymes .................................................................................. 52Direct detection of proteins ......................................................................................57DNA Sensing with CPEs ................................................................................................. 58DNA sensing based on conf ormation transition ...................................................... 58DNA sensing based on FRET .................................................................................. 61DNA sensing based on cova lent bioconjugation ......................................................64Challenges of CPE-based Optical Sensors: Non-specific Interactions of CPEs ............. 66Biosensors Using CPEs in Biological Environments ...................................................... 68

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7 Peroxyoxalate Chemiluminescence and Its Sensor Application ............................................ 69Chemiluminescence ......................................................................................................... 69Peroxyoxalate Chemiluminescence ................................................................................. 71Sensor Application of Peroxyoxalte Chemiluminescence .............................................. 74Scope of Our Study .................................................................................................................762 PHOSPHOLIPASE C ASSAY USING BIPHENYL POLY(PHENYLENE ETHYNYLENE) SULFONATE ............................................................................................78Introduction .................................................................................................................. ...........78Results and Discussion ........................................................................................................ ...80Overview of PLC Turn-off Assay ................................................................................... 80Effect of 10CPC on Fluorescence of BpPPE-SO3 ..........................................................82PLC Turn-off Assay ........................................................................................................ 87Determination of PLC Ca talyzed 10CPC Hydrolysis Kinetic Parameters ...................... 91Inhibition of the PLC Catalysis .......................................................................................92Specificity of the PLC Turn-off Assay ............................................................................ 93Discussion. ................................................................................................................... ....95Experimental .................................................................................................................. .........97Materials ..................................................................................................................... .....97Instrumentation ............................................................................................................... .97General Methods .............................................................................................................98Solution preparation ................................................................................................. 98Fluorescence turn-off assay procedure ..................................................................... 98Calculation of initial rate of reaction ( v0) ................................................................. 99Calculation of kinetic parameters ........................................................................... 100Synthetic Procedures of BpPPE-SO3 ............................................................................1003 ALKALINE PHOSPHATASE USING POLY(PHENYLENE ETHYNYLENE) CARBOXYLATE ................................................................................................................. 102Introduction .................................................................................................................. .........102Results and Discussion ........................................................................................................ .106Overview of ALP Turn-off Assay ................................................................................. 106Fluorescence Quenching of PPE-CO2 by Cu2+ .............................................................108Fluorescence Recovery of PPE-CO2 -/Cu2+ by PPi ........................................................109ALP Turn-off Assay ...................................................................................................... 113Determination of ALP Catalyzed PPi Hydrolysis Kinetic Parameters .........................117Inhibition of the ALP Catalysis .....................................................................................120Specificity of the ALP Turn-off Assay ......................................................................... 121Discussion .................................................................................................................... ..123Experimental .................................................................................................................. .......125Materials ..................................................................................................................... ...125Instrumentation .............................................................................................................. 125General Methods ...........................................................................................................126Fluorescence turn-off assay procedure ................................................................... 126Calculation of initial rate of reaction ( v0) ............................................................... 127

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8 Calculation of kinetic an d inhibition param eters ................................................... 127Synthetic Procedures of PPE-CO2 ................................................................................128PPE-CO2C12H25 ......................................................................................................128PPE-CO2 ................................................................................................................1294 ADENYLATE KINASE ASSAY USING POLY(PHENYLENE ETHYNYLENE) CARBOXYLATE ................................................................................................................. 130Introduction .................................................................................................................. .........130Results and Discussion ........................................................................................................ .133Overview of ADK Turn-off Assay ................................................................................ 133Fluorescence Recovery by ATP, ADP and AMP .......................................................... 135Calibration Curves of ADK Turn-off Assay ................................................................. 138ADK Turn-off Assay ..................................................................................................... 139Effect of Mg2+ on ADK Activity in Turn-off Assay ..................................................... 144Determination of ADK Catalyzed ATP Tr ansphosphorylation Kinetic Parameters ..... 145Inhibition of the ADK Catalysis in Turn-off Assay ......................................................147Specificity of the ADK Turn-off Assay ........................................................................ 148ADK Turn-on Assay ...................................................................................................... 151Discussion .................................................................................................................... ..152Experimental .................................................................................................................. .......153Materials ..................................................................................................................... ...153Instrumentation .............................................................................................................. 153General Methods ...........................................................................................................154Fluorescence assay procedure ................................................................................ 154Calculation of initial rate of reaction ( v0) ............................................................... 155Calculation of kinetic parameters ........................................................................... 1555 CHEMILUMINESCENT CONJUG ATE POLYELECTROLYTES ...................................156Introduction .................................................................................................................. .........156Results and Discussion ........................................................................................................ .158Photophysical Property of CPEs ................................................................................... 158Chemiluminescence of CPEs ........................................................................................ 159Optimization of Chemiluminescence Signal ................................................................. 161Quenching Study ...........................................................................................................167Peptidase Chemiluminescence Turn-on Assay with BpPPE-SO3 ................................169Alkaline Phosphatase Chemilumines cence Turn-off Assay with PPE-CO2 ................173Discussion .................................................................................................................... ..176Experimental .................................................................................................................. .......177Materials ..................................................................................................................... ...177General Methods ...........................................................................................................178Chemiluminescence measurements ........................................................................ 178Quenching behavior ...............................................................................................179Peptidase chemiluminescence assay with BpPPE-SO3 .........................................179Alkaline phosphatase chemilumi nescence assay with PPE-CO2 ..........................180

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9 6 CONCLUSION .................................................................................................................... .181Fluorescence PLC Assay Based on Conformation Change ..................................................181Fluorescent ALP and ADK Assays Based on Superquenching ............................................ 182Chemiluminescent Conjugated Polyelectrolytes .................................................................. 184Outlook of CPE-based Sensor Application .......................................................................... 185APPENDIX A FLUORESCENCE INTENSITY CORRE CTION FOR PHOTOB LEACHING OF CPES .....................................................................................................................................186B LINEAR FITTED VS. SIGMOIDAL FITTED CALIBRATION PLOTS .......................... 188LIST OF REFERENCES .............................................................................................................192BIOGRAPHICAL SKETCH .......................................................................................................205

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10 LIST OF TABLES Table page 5-1 Comparison of Stern-Volmer quenching of chemiluminescence and photolum inescence of BpPPE-SO3 by K-pNA. .............................................................. 171A-1 Comparison of initial rates of reaction derived by two patterns. ..................................... 187B-1 Parameters and correlation coefficient fo r sigmoidal fitted calibration plots in PLC and ALP Assay ................................................................................................................ 188

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11 LIST OF FIGURES Figure page 1-1 Molecular structures of some commonly seen CPs ........................................................... 251-2 Molecular structures of some common CPEs ....................................................................271-3 Generation of fluorescence and m echanism of fluorescence quenching ........................... 281-4 Molecular structure of MV2+ and the CPs studied by Swagers group .............................. 311-5 Molecular wire effect expressed by conjugated polymers ................................................. 321-6 Absorption and fluorescence of 1.7 10-5 M PPV-SO3 in water with and without 100 nM MV2+ .....................................................................................................................341-7 Molecular structure of PPE-SO3 and its model compound PE-SO3 used by Schanzes group .................................................................................................................351-8 The SV plots of 1 10-5 M MBL-PPV with MV2+ and liner static SV fittings .................361-9 Absorption and fluorescence spectra of PPE-SO3 in methanol, methanol:water (1:1) and water ............................................................................................................................371-10 Molecular structure of PPE-CO2 used by Schanzes group .............................................. 381-11 Quenching of 10 M PPE-CO2 emission by MV2+ in water ( ) and in methanol with 0 M ( ), 2.5 M ( ), 5.0 M ( ), 7.5 M ( ), or 10.0 M ( ) CaCl2 ..............391-12 The emission spectra of 2 10-5 M PPV-SO3 in water: (A) PPV-SO3 alone and (B) in the presence 2 10-6 M DTA .........................................................................................401-13 Effect of DTA on quenching of PPV-SO3 by MV2+ and TNT ......................................... 421-14 Detection of avidin using CPE via the superquenching mechanism ................................. 451-15 Molecular struct ure of cationic CPE 3 used by Heegers group in the DNA sensor .........461-16 The PNA-C*/ 3 assay to detect a comp lementary ssDNA sequence ..................................461-17 Molecular structures of cationic PT deriva tives used in CPE-based sensor application via conformation change mechanism ................................................................................. 471-18 Specific detection of human -thrombin by use of ssDNA thrombin aptamer and cationic 4 ............................................................................................................................481-19 Structure of carboxylated PPE 7 and pictures taken under a hand-held UV light to show the fluorescence under different situations ............................................................... 49

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12 1-20 Qualitative interpretation of the Hg2+-induced agglutination of the 7-papain complex .... 491-21 Interaction between p-BV2+ and sugar at near neutral pH ................................................. 511-22 Titration curves agai nst sugar for the PPE-SO3 -/ p-BV2+ system ....................................... 511-23 Changes in the color of solution of ca tionic PT derivative in water induced by the addition of equimolar amounts of various anions .............................................................. 521-24 Structures of quencher substrates us ed in protease assay by Schanzes group .................. 541-25 Mechanism of the turn-on and tu rn-off CPE-based sensors for protease activity................................................................................................................................541-26 General scheme for the kina se and phosphatase assays ..................................................... 561-27 Fluorescence spectral cha nges of MBL-PPV upon titration of different concentration of cyt c ................................................................................................................................571-28 Photographs and UV-visible absorpti on spectra changes observed in the PT 4-based DNA sensor ........................................................................................................................591-29 Formation of a planar 4/ssDNA duplex and a helical 4/dsDNA triplex ............................ 601-30 Proposed signal amplification detecti on mechanism based on the conformational change of 4 upon forming triplex with dsDNA and energy transfer for DNA detection ..................................................................................................................... ........611-31 Detection of DNA by two process FRET a nd relative orientation of three optical components: CPE 6, FL and EB ........................................................................................ 621-32 Molecular structure of cationic CPE-based copolymer 8 used by Bazans group ............. 631-33 Detection of DNA by PPE-SO3 labeled molecular beacon ............................................... 641-34 Molecular structure of PPE 9 used by Kims group in the DNA sensor ........................... 651-35 The PPE 9-oligonucleotide bioconjugation to form PPE-DNA to detect target DNA based on FRET and form PPE-DNA beacon ..................................................................... 651-36 Sensor for anti-DNP antibody ............................................................................................681-37 Visualization of mu tant and mannose-binding E.coli strains after incubation with mannosylated CPE ............................................................................................................. 691-38 Luminol chemiluminescence reaction. .............................................................................. 711-39 The CIEEL mechanism for the pero xyoxalate chemiluminescence reaction .................... 72

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13 1-40 Structures of co mm only used oxalates .............................................................................. 741-41 The time course for GOD reaction detected by TCPO chemiluminescence when GOD is ( ) 5; ( ) 20; ( ) 30; ( ) 40 units in the reaction mixture ..................................752-1 Results of radiometric as say for PLC based on BHC12PC ............................................... 792-2 Structures of polymer, BpPPE-SO3 and substrate, 10CPC, and mechanism and illustration of PLC turn-off assay ......................................................................................812-3 Normalized absorption of 1 M BpPPE-SO3 before and after addition of 8 M 10CPC in water at 25 C .................................................................................................... 832-4 Fluorescence changes upon titra tion of 10CPC into BpPPE-SO3 water solution ............. 842-5 Fluorescence changes upon titration of 10CPC into BpPPE-SO3buffer solution ........... 862-6 Fluorescence spectroscopic changes observed in the PLC turn-off assay ......................... 872-7 Effect of concentr ation of activator, [Ca2+] on the initial rate of hydrolysis of 10CPC by PLC ...............................................................................................................................882-8 Changes in fluorescence emission intens ity during the PLC turn-off assay as a function of reaction time for various concentr ations of PLC and dependence of initial rate of reaction on PLC concentration ............................................................................... 892-9 Dependence of initial rates of reaction ( v0) on substrate concentration [10CPC] ............. 922-10 Inhibition of PLC turn-off assay ........................................................................................932-11 Hydrolysis of phospholipid at di fferent positions catalyzed by PLA2, PLC and PLD ......942-12 Specificity of PLC turn-off assay ...................................................................................... 952-13 Reduced viscosity vs. concentration for BpPPE-SO3 in 0.1 M LiBr/DMSO at 20 C ... 1013-1 Structures of literature reported ALP substrates ..............................................................1033-2 Reaction progress curves for ALP during an electrochemical immunoassay .................. 1043-3 Recovery response of Cu2+-quenched fluorescence of PPE-CO2 by PPi and 11 control anions ...................................................................................................................1053-4 Mechanism and illustration of ALP turn-off assay ..........................................................1073-5 Fluorescence spectroscopic ch anges observed up on titration of Cu2+ into a solution of PPE-CO2 -..........................................................................................................................1083-6 Fluorescence changes upon titration of PPi into solution of PPE-CO2 and Cu2+ ...........110

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14 3-7 Logarithm of fluorescence r ecov ery efficiencyof PPE-CO2 -/Cu2+ solution titrated with PPi at different [Cu2+] .............................................................................................. 1113-8 Calibration curve for PPE-CO2 fluorescence recovery indu ced by addition of PPi ....... 1123-9 Fluorescence changes observed in the ALP turn-off assay ............................................. 1153-10 Decrease of [PPi] during the realtime ALP turn-off assays with varying concentration of ALP .......................................................................................................1163-11 Dependence of initial rate of enzy matic reaction on ALP concentration ........................ 1173-12 Natural logarithm of concentration of PPi as a function of incu bation time for various ALP concentrations ..........................................................................................................1183-13 Concentrations of hydrolyzed product, Pi, as a function of time at various initial PPi concentrations ................................................................................................................ ..1193-14 Inhibition of ALP turn-off assay by Pi ............................................................................1203-15 Changes in fluorescence emission intensity at 525 nm as a function of incubation time after addition of ALP and control proteins .............................................................. 1223-16 Specificity of ALP turn-off assay .................................................................................... 1234-1 Bioluminescent assay for ADK ....................................................................................... 1314-2 Mechanism of coupled enzyme assays for ADK ............................................................. 1324-3 Mechanism of ADK turn-off and turn-on assay ..............................................................1344-4 Comparison of fluorescence intensity incr ease at 525 nm upon titration of ATP, ADP and AMP, respectively .....................................................................................................1364-5 Calibration curves, logarithm of fluorescen ce recovery efficiencies as a function of [ATP], at different concentration of Mg2+. ......................................................................1394-6 Fluorescence changes observed in the ADK turn-off assay. ........................................... 1414-7 Decrease of [ATP] during first 60 sec of the real-time ADK turn-off assays with varying concentration of ADK ......................................................................................... 1424-8 Dependence of initial rate of reaction ( v0) on ADK concentration .................................. 1434-9 Effect of concentration of Mg2+ on initial rate of reaction at different concentrations of ATP. .............................................................................................................................1454-10 Dependence of initial rates of reaction ( v0) on substrate concentration [MgATP] .......... 146

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15 4-11 Inhibition of ADK turn-off assay by Ag+. ....................................................................... 1474-12 Specificity of ADK turn-off assay ...................................................................................1494-13 Changes in fluorescence emission intensity at 525 nm as a function of incubation time after addition of ADK and BSA .............................................................................. 1504-14 Change of fluorescence intensit y observed during ADK turn-on assay .......................... 1515-1 Three steps in peroxyoxalate chemiluminescence ........................................................... 1565-2 Normalized absorption a nd fluorescence spectra of 1 M BpPPE-SO3 in methanol and water ..........................................................................................................................1585-3 The chemiluminescence signal profiles ........................................................................... 1605-4 Effect of H2O2 concentration on chemiluminescence intensity and duration time .......... 1625-5 Effect of ImH concentration on chem iluminescence intensity and duration time ........... 1635-6 Effect of concentration of TC PO on chemiluminescence intensity ................................. 1645-7 Effect of concen tration of BpPPE-SO3 on chemiluminescence intensity. ...................... 1655-8 Effect of solvent composition on chem iluminescence intensity and duration time ......... 1665-9 SV plots of chemiluminescence a nd photoluminescence quenching of CPE .................. 1685-10 Mechanism of chemiluminescen ce turn-on assay for peptidase ...................................... 1705-11 Change of chemiluminescence intensity obs erved in the turn-on assay for peptidase ....1725-12 Mechanism of chemiluminescence turn-off assay for ALP ............................................. 1735-13 Changes of chemiluminescence intensity observed in the turn-off assay for ALP ......... 1755-14 Structures of oxamide reagents. .......................................................................................177B-1 Sigmoidal fitted calibration plots ..................................................................................... 189B-2 Comparsion of relative erro r at different substrate concen trations from linear fitted and sigmoidal fitted calibraton plots ................................................................................191

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16 LIST OF ABBREVIATIONS ADK Adenylate Kinase ADP Adenosine 5 -diphosphate sodium ALP Alkaline phosphase AMP Adenosine 5 -monophosphate sodium ATP Adenosine 5 -triphosphate disodium AVI Avidin BpPPE-SO3 Biphenyl poly(phenylene ethynylene) sulfonate BSA Bovine serum albumin BT 2,1,3-Benzothiadizole BTZP Benzothiazolyl-5-phosphate CaM Protein calmodulin CE Capillary electrophoresis CIEEL Chemically initiated elec tron-exchange luminescence CNC Charge neutral complex CP Conjugated polymer 10CPC 1,2-Didecanoylsn -glycero-3-phosphocholine CPE Conjugated polyelectrolyte cyt c Cytochrome c DAG Diacylglycerol, 1,2-didecanoylsn -glycerol DMPC Dimyristoylphosphatidylcholine DMPG Dimyristoyl phosphatidyl glycerol DNP Dinitrophenol DNP-BS1-Sulfobutoxy-2, 4-dinitrobenzene DNPO Bis-(2,4,dinitrophenyl) oxalate

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17 DNT 2,4-Dinitrotoluene DOTAP 1,2-Dioleoyl-3-trimethyl-ammonium propane dsDNA Double-stranded DNA DTA Dodecyltrimethylammonium bromide EB Ethidium bromide EDTA Ethylenediamine tetraace tic acid, disodium salt dihydrate ELISA Enzyme-linked immunosorbent assay F Fluorophore F* Excited fluorophore CL Chemiluminescence quantum yield FcEtOPO3Na2 Sodium salt of ferrocene ethyl phosphate ester FET Field-effect transistor ex Efficiency of production of the excited species FIA Flow injection analysis FL Fluorescein L Luminescence quantum yield FQ Non-fluorescent complex betw een the fluorophore and quencher FRET Fluorescence resonance energy transfer GOx Glucose oxidase HEPES 4-(2-Hydroxyethyl)-1-pip erazineethanesulfonic acid HRP Peroxidase from horseradish HTS High-throughput screening I Inhibitor I Fluorescence intensity I0 Initial fluorescence intensity at t = 0 of the enzyme assay

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18 I0c Initial corrected fluorescence intensity at t = 0 of the enzyme assay Ib0 Fluorescence intensity of a blank solu tion at time = 0 of the a bloank assay Ibt Fluorescence intensity of blank so lution at time t of a blank assay ICL Chemiluminescence intensity IgG Immunoglobulin Ii ( or I0) Initial fluorescence intensity ImH Imidazole Ip Fluorescence intensity of pure polymer Iq Quenched fluorescence intensity Ir Recovered fluorescence intensity It Fluorescence intensity at time t of the enzyme assay Itc Corrected fluorescence intensity at time t of the enzyme assay Ka Association constant Kapp Apparent equilibrium constant kcat Catalytic constant or turnover number kcat/ Km Specificity constant that determines the relative rate of reaction at low substrate concentration KD Quenching constant in the dynamic quenching process Ki Inhibition constant Km Michaelis-Menten constant, the substrate concentration at which the rate of the enzyme reaction is half of the maximum rate of enzymatic reaction KpNA L-lysine p-nitroanilide dihydrobromide, a cat ionic peptide labeled with a p-nitroanilide unit kq Bimolecular quenching constant KS Quenching constant in th e static quenching process KSV Stern-Volmer constant

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19 LC Liquid chromatography LEC Light emitting electrochemical cell LED Light-emitting diodes MBL-PPV Poly[5-methoxy-2-(4-sulfobutoxy)-1,4-phenylenevinylene] 4-MUP 4-Methylumbellyferyl phosphate MV2+ Methyl viologen, N,N -dimethyl-4,4 -bipyridinium M.W. Molecular weight ODI 1, 1 -Oxalyldiimidazole PA Polyacetylene PAE Poly(aryle ne ethynylene) PANI Polyanaline PBS Phosphate buffer solution p-BV2+ Boronic acid-fuctionalized benzyl viologen PDA Polydiacetylene PDMAE Poly(N,N-dimethylamino-ethylene iodide) PEP Peptidase PF Polyfluorene Pi Phosphate PLA2 Phospholipase A2 PLC Phospholipase C PLD Phospholipase D pNA P -nitroanilide PNA Peptide nucleic acid PNA-C* A peptide nucleic aci d strand labeled at the 5 end with a chromophore pNPP P -nitrophenylphosphate

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20 PPE Poly( para-phenylene ethynylene) PPE-CO2 Poly(phenylene ethynylene) carboxylate PPE-SO3 Poly(phenylene ethynylene) sulfonate PPi Pyrophosphate PPP Poly( para-phenylene) PPP-NEt3 + Poly( para-phenylene) triethylammonium PPV Poly( para-phenylene vinylene) PPV-SO3 Poly( para-phenylene vinylene) sulfonate PPy Polypyrrole PT Polythiophene Q Quencher QTL Quencher-tether-ligand S0 Ground singlet electronic state S1 Singlet excited electronic state S/B Sample and blank ratio SDC Sodium deoxycholate SNP Single nucleotide polymorphism ssDNA Single-stranded DNA SV Stern-Volmer Fluorescence lifetime m easured without quencher 0 Fluorescence lifetime m easured with quencher TCP Trichlorophenol TCPO Bis(2,4,6-trichlorophenyl) oxalate TNP 2,4,6-Trinitrophenol TNT 2,4,6-Trinitrotoluene

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21 Tris Tris(hydroxymethyl)aminomethane v0 Initial rate of enzymatic reaction Vmax Maximum rate of enzymatic reaction

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22 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CONJUGATED POLYELECTROLYTE BA SED ON POLY(PARA-PHENYLENE ETHYNYLENE): FLUORESCENCE AN D CHEMILUMINESCENT SENSORS Yan Liu December 2008 Chair: Kirk S. Schanze Major: Chemistry Our study focused on design and developmen t of fluorescent and chemiluminescent biosensors based on functionalized poly(para-phenylene ethynylene)s (PPEs). By taking advantage of the amplified signal response of conj ugated polyelectrolytes (CPEs) to aggregation and/or the presence of charged quenchers, the de veloped PPE-based optical biosensors afford high sensitivity and good specifi city. They also allows for rapid, convenient and continuous determination of enzyme activity and/or inhibition. First, a PPE derivative, biphenyl poly(phenylene ethynylene) sulfonate (BpPPE-SO3 -) is used to develop a fluorescence turn-off assay fo r phospholipase C (PLC) based on the reversible interaction between BpPPE-SO3 and phosphatidylcholine, which is regarded as the natural substrate of PLC. The PLC assay operates with phospholipid substrate concentrations in the micromolar range and the analytical detec tion limit for PLC is < 1 nM. The real-time fluorescence intensity from the CPE can be conve rted to substrate conc entration by using an exsitu calibration curve, allowing kinetic parameters of PLC to be determined. PLC activation by Ca2+ and inhibition by EDTA and fluoride ion ar e demonstrated using the optimized sensor. Second, another PPE derivative, poly( phenylene ethynylene) carboxylate (PPE-CO2 -) is used to develop a fluorescent assay for alkaline phosphase (ALP) based on the fluorescence recovery of Cu2+-quenched PPE-CO2 by pyrophosphate (PPi), the natural substrate of ALP.

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23 Using the PPE-CO2 --Cu2+ system as the signal transducer, a real-time fluorescence turn-off assay for ALP operates with PPi in the micromolar range, and it offers straight-forward and rapid detection of ALP activity with the enzyme pres ent in the nanomolar concentration range with detection limit of 5 nM, operating either in an en d-point or real-time format. Kinetic and product inhibition parameters are derived. Third, addition of adenosine 5 -triphosphate disodium (ATP ) into a solution of PPE-CO2 and Cu2+ also recovers the Cu2+-quenched fluorescence of PPE-CO2 to a notable higher extent compared with the introduction of adenosine 5 -diphosphate sodium (ADP) and adenosine 5 monophosphate sodium (AMP) under the same concen tration level. Using ATP as substrate or product, a PPE-CO2 --based real-time fluorescence assay to detect the catalytic activity of adenylate kinase (ADK) in an equilibrium transp hosphorylation is designed and developed based on the different response of adenosine phosphates to quenched fluorescence of polymer. The ADK assay shares the same advantage of the ALP assay and allows for calculation of ADK catalyzed reaction rates as well as kine tic, activation and inhibition parameters. Last, the first chemiluminescence system for CPE was developed based on the peroxyoxalate chemiluminescence reaction. The chemiluminescence systems consists of bis(2,4,6-trichlorophenyl) oxalate (TCPO) as aryl oxalate, imidazole as catalyst, H2O2 as oxidant, and two PPEs as fluorophores. The effects on chem iluminescence signals of concentrations of oxalate, catalyst, oxidant and fluorophores as well as solvent composition are studied and the amplified chemiluminescence quenching property of CPE is found. The chemiluminescence of CPE is successfully applied as a biosensor platform by qualitatively and quantitatively detecting activities of two enzyme s, peptidase and ALP.

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24 CHAPTER 1 INTRODUCTION Conjugated Polymers Until abou t 40 years ago, all carbon based polym ers were rigidly regarded as insulators. However, an era of conducting polymers started in 1967 from an experimental mistake in Hideki Shirakawas lab of the University of Tsukuba in Japan.1 A bizarre-looking and semiconducting polyacetylene (PA, Figure 1-1) was obtained by accidental addition of 1000 times excess of the catalyst. With the cooperation of Alan MacDia rmid of the University of Pennsylvania at Philadelphia and Alan Heeger of the University of California at Santa Barbara, the conductivity of PA was increased by 10 milli on times in 1977 upon doping with electron acceptors or electron donors.2 This breakthrough in discovery of conjugat ed polymers (CPs) created an entirely new field of chemistry and eventually resulted in th e award to three scientists of the 2000 Chemistry Nobel Prize.3 Ever since, CPs with delocalized -electron systems have attracted an overwhelming interest in laboratories around the gl obe due to their versa tile optical, electrical, and magnetic properties. Figure 1-1 shows struct ures of a variety of other CPs commonly investigated, including poly( para-phenylene) (PPP),4 poly( para -phenylene vinylene) (PPV),5 poly( para -phenylene ethynylene) (PPE),6 polythiophene (PT),7 polypyrrole (PPy),8 polyanaline (PANI)9 and polyfluorene (PF).10 Most CPs are prepared via a palladium-catalyzed cross-linking polymerization which offers the benefits of mild reaction condition, and wide functional group and solvents compatibility.11 Conjugated polymers have become an important class of materials in a wide variety of applications, including light-emitting diodes (LEDs),5,12 light emitting electrochemical cells (LECs),13 plastic lasers,14 solar cells,15 and field-effect transistors (FETs).16,17 One recent area of interest in CPs is their use in chemi cal or biological sensor applications.18,19 In particular, the

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25 chemical structures of CPs offer several advantag es in sensor applications, especially increased sensitivity. The delocalized electronic structur e of CPs enables them to exhibit efficient absorption and strong emission, and produce amplified signal changes up on interacting with various analytes. The increased sensitivity (amplifi cation) is also derived from their efficient coupling between optoelectronic segments20,21 and rapid transport of electronic excitations.19 Based on different types of observe d signals, CP-based sensors have been formulated in a variety of schemes, such as conductometric met hod (electrical conductiv ity as the signal),22 potentiometric method (chemical potential as the signal),23 colorimetric method (absorption characteristic as the signal),24 and fluorometric method (fluor escence characteristic as the signal).19 Figure 1-1. Molecular structures of some commonly seen CPs. Poly( para-phenylene ethynylene)s (PPEs, Figure 11) are representative poly(arylene ethynylene) (PAE) compounds in the CP family and characteristic of having benzene rings as the aromatic groups which are polymerized via triple bonds. Because of the 180 bond angle between the phenyl carbon and sp1 carbon, the low-molecular-we ight PPEs display a rod-like structure and offer promising app lications such as nonlinear optics,25 liquid-crystal displays26 and molecular wires to bridge nanoelectrodes.27 Compared with PPVs, the first group of CP compounds were used as a platform for fluor escence-based sensors for biological targets,28 PPE

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26 exhibit extended electronic structure and higher quantum yield. Due to their optimal photophysical characteristics, PPEs have been explor ed as an important class of CPs for sensing and serve as potential transducers.29-31 Conjugated Polyelectrolytes Solubility in aqueous m edia is essential for CP s sensing ability to in teract with analytes, especially in the biological e nvironment. Water-soluble CPs are achieved by incorporating ionic functionality as pendant groups on the conjugated backbone; th e resulting polymers are known as conjugated polyelectrolytes (CPEs). Commonly used ionic side groups include sulfonate (SO3 -), carboxylate (-CO2 -), phosphonate (-PO3 2-) and alkyl ammonium (-NR3 +).11 Some examples of CPEs are shown in Figure 1-2. CPEs integrate the intrinsic electronic and optical properties of the organic -conjugated backbone and the unique charge interaction ability of polyelectrolytes, which also makes this amphi philic material as a perfect platform for development of chemoand bio-sensors. In additi on to their water solubility, the charged nature of CPE facilitates the ability to control the dist ance and the strength of interaction between ionic partners. For example, most CPE-based sensor approaches developed hitherto depend on the electrostatic interaction between the probes (e.g., CPE) and the ta rget ionic species, such as metal ions, anions, polyelectro lytes, proteins and DNA.19 Due to their unique structural properties, CPEs also provide other advantages in various applications. For example, CPEs can be processed into films from water or other polar solvents (e.g., methanol), which are regarded as being superior to organic solvents in terms of green chemistry. In addition, because of their intrinsi c amphiphilic nature, CPEs are capable of selfassembly into supramolecular structures such as colloids and polyelectrolyte layer-by-layer films, which could provid e functional materials with supra-molecular order.32

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27 O O n NEt3 NEt3 O O n CO2 n S O O SO3 nPPP-NEt3 +PPV-SO3 -PPE-PO3 2-PT-CO2 PO3 2 PO3 2 Figure 1-2. Molecular structur es of some common CPEs. Amplified Quenching of Conjugated Polyelectrolytes Fluorescence Quenching One of m ost exciting properties of CPEs which accounts for their high sensitivity in the sensor application is so-called amplified quenc hing or superquenching effect. That is, a number of fluorescent CPEs exhibit very hi gh sensitivity to oppositely charged molecular quenchers anticipated to quench fluor escence by electron or energy transfer.28 Before exploring this effect, it is important to understand the basic process, mechanisms and expression of fluorescence quenching. A simplified Jablonski diagram33 shown in Figure 1-3A illustrates that upon absorption of photons, a fluorophore (F) is excited to singlet excited electronic state (S1) and form a excited fluorophore (F*). The fluorescence is generated when F* relaxes to ground singlet electronic state (S0) via photon emission. However, F* could return to S0 by various competing pathways such as non-radiative relaxation and phosphores cence. Fluorescence quenching is another important process competing with fluorescence, in which the fluorescence intensity and/or

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28 lifetime is decreased th rough interaction of F (o r F*) with a second mol ecule called quencher (Q). These interactions include excited-state interaction, molecula r rearrangement, energy transfer, charge transfer, ground-state complex formation and dynamic collision. Figure 1-3. Generation of fluorescence and mechanism of fluorescence quenching. A) Simplified Jablonski diagram. B) Dynamic quenching mechanism. C) Static quenching mechanism. Generally, fluorescence quenching can occu r by two different mechanisms, dynamic quenching and static quenching.33 Dynamic quenching, also ca lled collisional quenching, depends on the diffusion of the quencher. As shown in Figure 1-3B, dynamic quenching occurs when the F* is deactivated upon a diffusi ve encounter of Q and return to S0 without emission of a photon, while Q is not chemically altered in th e process. Figure 1-3C illustrates the static quenching which happens as a result of forma tion of a stable non-fluorescent complex (FQ) between the F and Q. When this complex absorb s light, it immediately re turns to the ground state

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29 without emitting fluorescence. The static quenchi ng occurs in the ground state and its efficiency is related to the association constant ( Ka) for formation of FQ. There are several ways to distinguis h between dynamic and static quenching.33 The first method is to determine the dependence of th e fluorescence lifetime on quencher concentration. In the case of dynamic quenching, the additional deactivation pathway shortens the observed lifetime of the fluorescence. In contrast, in st atic quenching, the lifetim e doesnt change since those Fs that are not complexed, and hence are able to emit after excitation, will have normal excited state properties. Second, the temperature effect on the quenching efficiency could be carefully examined to distinguish between these two mechanisms. Dynamic quenching efficiency is expected to increase with increase temperature due to larger diffusion coefficients at higher temperature, while increased temperature is likely to result in decreased stability of the complexes, and thus reduce the static quenching efficiency. Third, note that the complex, FQ, typically has a different absorption spectrum from the fluorophore, therefore, the presence of an absorption change is diagnostic of a static quenc hing mechanism. However, it is important to recognize that dynamic and static processes ar e concurrently present in many systems. Stern-Volmer Equation33 Both dynamic quenching and static quenchi ng are described by Stern-Volmer (SV) equation which is given by 0 SV1[Q] I K I (1-1) where I0 and I are the fluorescence intensity observed in the absence and presence of quencher, respectively, [Q] is the quencher concentration, and KSV is the SV quenching constant. In the dynamic quenching, the fluorescence intensity is quenched to same extent as the fluorescence lifetime. As a result, the SV equation can be written as

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30 00 q0 D1[Q]1[Q] I kK I (1-2) where 0 and are the fluorescence lifetime measured w ith and without quencher, respectively, kq is the bimolecular quenching constant, and KD is used to represent KSV in the dynamic quenching process. The SV quenching constant is given by kq0. In the case of pure diffusioncontrolled quenching, kq cannot exceed the diffusion rate constant ( ca. 1010 M-1s-1). The typical fluorescence lifetime is around 10-8 s,33 which indicates the upper limit of KD to be around 100 M-1 (i.e. 1010 M-1s-1 10-8 s = 100 M-1). Thus a moderate to large observed SV quenching constant infers the presence of static quenching process, in whic h the SV equation is written as 0 S1[Q] I K I (1-3) where KS is represent KSV in the static quenching process a nd equal to association constant ( Ka) between the fluorophore and the quencher. The lifetime of the fluorophore in the static quenching process is not affected by th e presence of Q and the ratio of 0 to is 1. A plot of I0/ I versus [Q] is known as a Stern-Volmer (SV) plot, which yields a straight line with a slope equal to KSV in both pure dynamic and static quenching process. However, in many instances, the SV plot displays a non-linear, upward curvature, wh ich indicates the fluorophore is quenched by both collisions and complex form ation with the same quencher. In such circumstances, a modification of original SV equation yields 0 DS(1[Q])(1[Q]) I KK I (1-4) This form of SV equation combines dynamic and static quenching e ffect on the fluorescence intensity and displays as a s econd-order in [Q]. The dynamic portion of the observed quenching can be determined by lifetime quenching as shown in Equation 1-2.

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31 Amplified Quenching in Neutral CPs: Molecular Wire Effect The concept of amplified quenching in CP s was first described by Swager and coworkers in 1995.34,35 They observed that the fluorescence of a series of neutral, organic soluble PPEs was quenched by methyl viologen salt (MV2+ or N,N -dimethyl-4,4 -bipyridinium, Figure 1-4) with very high efficiency and ascribed this signal enhancement to the molecular wire effect. Figure 1-4. Molecular structure of MV2+ and the CPs studied by Swagers group. Swager and co-workers studied a fluor escent single cyclophane receptor compound 1 and a PPE-type polymer 2 which has a cyclophane group on each repeat unit (Figure 1-4). While MV2+ binds to the cyclophane unit, the fluorescence of both compounds is quenched by MV2+ via an electron transfer mechanism. The static quenching process was confirmed by comparing the intensity quenching and lifetime quenching. However, compared to model 1, 2 with many receptors wired in series exhibits a 67-fold enhancement in quenching efficiency in terms of KSV value which is reached about 105 M-1. Swager attributed this greatly enhanced fluorescence quenching of PPE to extended electronic communication and exciton transport on the conjugated polymer chain, which was termed the molecular wire effect.36 As shown in Figure 1-5, upon photoexcitation, excited

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32 states consisting of elec tron-hole pairs, also known as excito ns, can be generated randomly and migrate along the polymer chain. Each exciton passes multiple cyclophane receptors until it encounters a MV2+ bound receptor, where the excitation is quenched. This energy migration is a collective property rather than a property of discrete units of the polymer. As a result of extremely rapid exciton diffusion and energy mi gration along the polymer backbone, a single MV2+ occupied on the receptor site is able to qu ench many repeat units in the polymer chain. Therefore, the quenching response of polymer 2 to MV2+ is greatly amplified. The large KSV value obtained from SV plot is interpreted as the product of association constant of MV2+ to the cyclophane receptor ( Ka) and an amplification factor, which is the number of receptors visited by the exciton. The known value of Ka allows the calculation of the true amplification factor of 67 as mentioned earlier. + + eeET Quencher h receptor analyte Molecular Wire Receptor Assembly Figure 1-5. Schematic illustration of the m olecular wire effect expressed by conjugated polymers. Reprinted with permission from Zhou et al .35 Swager found that multiple factors including po lymer structure delocalization length and fluorescence efficiency w ould influence the amplified quenching effect.35 In addition, the quenching efficiency increases steadily with in creasing polymer molecula r weight and plateaus after the molecular weight reaches 65,000. The molecular weight dependent feature of quenching suggests that in the higher molecu lar weight samples, the excitons are not able to visit the entire

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33 length of polymer due to its smaller diffusion length in limited lifetime. Moreover, the wired in series design confines the exciton to walk al ong the polymer chain one-dimensionally and visit a limited number of receptors. In order to maximize the number of receptors that an exciton can reach throughout its lifetime and increase the quenching efficiency, Swager and co-workers further designed a two-dimensional film that incorporates a rigid pe ntiptycene-derived PPE, 37,38 and a three-dimensional film composed of multiple layers of different PPEs.39,40 In the multipledimensional PPE films, the intermolecular energy transfer is greatly enhanced due to the prevention of -stacking of the polymer backbone and the rapid diffusion of exciton in 3dimensions. In particular, the two-dimensional film provides cavities to allow the diffusion of small organic compounds within the solid state f ilm, especially the electron-deficient analytes. This system was used to detect nitroaromatic compounds such as 2,4,6-trinitrotoluene (TNT) and 2,4-dinitrotoluene (DNT) within tens of seconds Because of its high sensitivity, a commercial explosive detector called Fido based on this technology was introduced by the ICx Technologies, Inc. in 2005. Amplified Quenching in CPEs: Combination of Ion-Pair Complex Formation and Molecular Wire Effect The amplified quenching effect of water-solu ble and charged CPEs, which is regarded as the basis of their sensitive sensory application, was first observed by Chen and Whitten in 1999.28 In their work, an anionic derivativ e of poly (phenylen e vinylene), PPV-SO3 (structure shown in Figure 1-2) is que nched very efficiently by MV2+ via photoinduced electron transfer. As shown in Figure 1-6, addition of very low concentrations of MV2+ to a dilute water solution of PPE-SO3 induces a noticeable red-shift in the ab sorption spectrum and a dramatic quenching of the polymers fluorescence. The KSV constant was reported to be ~1.7 107 M-1, which is a million-fold amplified relative the quenching of isolated stilbene in dilute solutions. Because the

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34 binding constant of neutral stilbene to MV2+ is apparently smaller than that of anionic polymer to MV2+, the real amplification factor could not be es timated. However, from the concentrations of polymer and quencher, it was conf irmed that one molecule of MV2+ effectively quenches about 1,000 repeat units of PPV-SO3 which is approximately the length of a polymer chain. PPV-SO3 PPV-SO3 -/MV2+ Abs Flu Figure 1-6. Absorption (left) a nd fluorescence (right) of 1.7 10-5 M PPV-SO3 in water with and without 100 nM MV2+. Reprinted with permission from Chen et al .28 After the work of Chen and Whitten, it was disc overed that similar or even more amplified quenching occurs for other CPEs with various quenchers,29,30,41-44 which suggests that the amplified quenching effect is a general property for CPEs. For example, Schanze et al observed that an anionic derivative of PPE, PPE-SO3 (Figure 1-7), which has a higher fluorescence quantum yield than PPV-SO3 exhibits amplified quenching by MV2+ in both water and methanol.30 The KSV value was reported in the magnitude of 107 M-1 which is four orders of magnitude greater than that of the small molecule analog, PE-SO3 (Figure 1-7). In comparison to neutral CPs studied by Swag er, charged CPEs exhibit more amplified quenching efficiency in terms of KSV constant. This is due to the formation of the strong association complex between the oppositely charged polyelectrolyte and quencher via a

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35 combination of Coulombic and hydrophobic inter actions. Therefore, the amplified quenching effect in CPEs is attributed to a combination of two effects: the formation of ion-pair complex and the ultrafast exciton migration along the pol ymer chain (molecular wire effect) to the quencher trap site. As a result, any factor affecting these two effects could influence the amplified quenching effect of CPEs and thus chan ge the SV constant. It has been demonstrated that these factors include polymer chain length,42,45 polymer aggregation,45,46 quencher properties (charge,47 hydrophobicity48 and size46), solution conditions (pH,29,49 ionic strength49,50 and buffer concentration50), and additives (metal ions,51 surfactants,52-54 polyelectrolytes55,56 and proteins57,58). O O SO3 SO3 n PPE-SO3 -PE-SO3 O O SO3 SO3 Figure 1-7. Molecula r structure of PPE-SO3 and its model compound PE-SO3 used by Schanzes group. Stern-Volmer Plots in Amplified Qu enching of CPEs: Sphere-of-Action A typical SV plot for CPE quenching by an oppositely charged quencher ion is shown in Figure 1-8, where butoxy-substitu ted PPV, MBL-PPV is quenched by MV2+.50 The SV plot exhibits a linear correlation at low quencher co ncentration range, and upw ard curvature (inset, Figure 1-8) at higher quencher concentration. The study of effect of buffer on quenching indicates a static quenching mechanism for MBLPPV at low quencher concentration, and the static SV equation (Equation 1-3) pe rfectly accounts for the linear co rrelation of SV plot at this regime. However, with increase of the concen tration of quencher, the superlinear behavior cannot be explained by Equation 1-3.

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36 Figure 1-8. The SV plots of 1 10-5 M MBL-PPV with MV2+ and liner static SV fittings. Inset: the same plot extended to higher MV2+ concentration. Reprinted with permission from Wang et al .50 Bazan and Heeger applied a s phere-of-action quenching model to explain the superlinear quenching at high quencher concentration.47,50 In the theory of sphere-o f-action, there is a type of apparent static quenching of fluorophores at nearly unit efficiency when a quencher is within the quenchi ng sphere. As MV2+ concentration increases, the local concentration of the quencher is enhanced greatly due to the tendency toward complex formation, and the average of MV2+ molecules separation is only about 800 C onsidering the large size of the conjugated polymer in aqueous solution, there is always a quencher ion within a ch arge transfer distance from one of the polymer chain. In the ot her word, there is always one or more MV2+ molecules within the sphere-of-action of the polymer. As a result, the quenching in this regime increases superlinearly with the quencher concentration. In addition to the sphere-of action quenching model, recent research showed that the inter-chain exciton migrati on induced by the polymer aggregation also accounts for the superlinear SV plot.30,51 In order to better understand the ag gregation of CPEs as well as its effect on amplified quenching, we will discuss it in more detail below. O O n SO3 Li MBL-PPV

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37 Aggregation of Conjugated Polyelectrolytes Existence of Aggregation: Solvent Dependent Optical Properties of CPEs As pointed out earlier, the amphiphilic characteristic of CPEs facilitates their self-assembly into supramolecular structures, wh ich could strongly influence their optical properties. In a series of reports, Schanze et al have carefully studied the solvent dependent optical properties of PPESO3 (structure shown in Figure 1-7), which prov ides the evidence for the existence of CPE aggregation.30,44 This work has been extende d by Waldeck and co-workers.59 Figure 1-9. Absorption (left) and fluorescence (right) spectra of PPE-SO3 in methanol, methanol:water (1:1) and water. Arrows show the direction of change with increasing water content. Reprinted with permission from Tan et al .30 As shown in Figure 1-9, th e absorption band of PPE-SO3 progressively red-shifts and narrows with increasing of volume fraction of water. In addition, the most pronounced change is seen in the fluorescence spectrum. In methanol, PPE-SO3 features a sharp, narrow and strong structured florescence band at 450 nm with relati vely small Stokes shift. In contrast, as the fraction of water increases, a new broad, red-shifted and weaker band appears at 550 nm. The fluorescence properties of PPE-SO3 in methanol are very similar to those exhibited by neutral PPEs dissolved in good solvents such as CHCl3 or THF, where the polymer exists as in an unaggregated monomeric state.60 Therefore, it suggests that PPE-SO3 also exists in a molecularly-dissolved state in its good solv ent, methanol. While, the changed fluorescence of

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38 the polymer in water indicates the change of polymer conformation, because the characteristics of fluorescence spectrum are very similar to those observed from conjugated polymer (and oligomer) aggregates.61-63 In addition, the decrease in the quantum yield indicates that this aggregate emission is dominated by an inter-chain excimer-like state, which has lower radiative rate than the intra-chain exciton.64 From this work, Schanze et al concluded that PPE-SO3 aggregates in water via the inter-chain hydrophobic in teractions and leads to a broad, red-shifted and weaker emission due to stacking between two or more polymer chains. Effect of Aggregation on Amplified Quenching of CPEs Early studies by Schanze et al demonstrated that quencher ions could induce aggregation of CPEs and the resulting a ggregation had a pronounced e ffect on amplified quenching.30,43 For example, compared with the KSV constant of PPE-SO3 quenching by MV2+ in methanol, a larger KSV constant was obtained in wate r, which indicated that the que nching was further amplified by aggregates. Recently, Schanze et al further confirmed this correlation by studying the aggregation of anothe r anionic PPE, PPE-CO2 (Figure 1-10) induced by Ca2+, as well as its effect on amplified quenching of the polymer by MV2+.51 Figure 1-10. Molecula r structure of PPE-CO2 used by Schanzes group. In this study, the divalent cation Ca2+ was shown to induce th e aggregation of PPE-CO2 by ionic bridging between polymer chains and polyvalent quencher i ons. As shown in Figure 1-11, MV2+ quenches the fluorescence of PPE-CO2 more efficiently in water than in methanol in which the polymer is considered not aggregat ed at low polymer concentration. However,

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39 addition of different equivalents of Ca2+ into methanol solution of PPE-CO2 leads to a significant increase of quenching efficiency and a decrease of the linear range of SV plots until the quenching efficiency reaches the level in water. Furthermore, the superlinear quenching is observed immediately upon addition of a very small amount of MV2+ in the presence of 1 equivalent of Ca2+ per PPE-CO2 repeat unit. This finding clearl y demonstrates that the quencherinduced aggregating of CPEs facilitates the inter-chain ex citon delocalization among the individual CPE chains in the polymer aggregates which is regarded as another important reason to explain superlinear SV behavior. This expl anation also challenges the sphere-of-action quenching model described earlier which is more reasonable with small molecules or unaggregated polymers. Schanze et al suggested that under the situation with polyvalent quencher ions which induce the aggregation of CPEs, it is more appropriate to use a model which incorporates the possibilities of three-dimensional exciton diffu sion within a polymer aggregate. This idea is consistent with the previously mentioned work of Swager in which a three-dimensional film of neutral CP was cons tructed in order to enhance the intermolecular energy transfer.39,40 Figure 1-11. Quenching of 10 M PPE-CO2 emission by MV2+ in water () and in methanol with 0 M (), 2.5 M (), 5.0 M (), 7.5 M (), or 10.0 M () CaCl2. Reprinted with permission from Jiang et al .51

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40 Interaction of Surfactants with CPEs: Tuni ng the Conformation and Optical Properties Since polymer aggregation plays an important ro le in determining the optical properties of CPEs, any factor that influences the polymer aggregation is e xpected to affect the optical properties including the absorption, emission a nd emission quenching behavior. Inspired by the study that polyelectrolytes can form stable complexes with oppositely charged amphiphilic molecules (surfactants),65,66 Whitten et al. first demonstrated that CPEs were capable of forming complexes with oppositely charge surfactants via Coulombic attraction, which resulted in dramatic and tunable changes in both geometric conformation a nd optical properties of CPEs.53 Figure 1-12. The emission spectra of 2 10-5 M PPV-SO3 in water: (A) PPV-SO3 alone and (B) in the presence 2 10-6 M DTA. Inset: Normalized absorption and emission spectra of PPV-SO3 in water. Reprinted w ith permission from Chen et al .53 Specifically, Whitten et al monitored the optical response of PPV-SO3 to the addition of 10 mol% of the surfactant dodecyltrimethylamm onium bromide (DTA). As shown in Figure 112, compared with the emission of PPV-SO3 in water, the fluorescence after addition of DTA shows a well-defined vibrational structure with a noticeable blue -shift and dramatic intensity enhancement. Meanwhile, the absorption spectrum is narrowed and red-shifted. Note that the fluorescence spectrum of polymer alone in wate r undoubtedly displays the aggregate state emission; however, the fluorescence spectrum of th e polymer/surfactant complex is very similar

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41 to the monomeric state emission exhibited by pur e polymer in good solvent. Consequently, the authors concluded that the polymer underw ent a conformation change after forming a complex with the surfactant, i.e., the polymer backbone became more ordered and extended due to its hydrophobic interactions with the surf actant tails, which thereby disrupted the aggregation of polymer chains. The more homogeneous conformation of the polymer in the PPV-SO3 -/DTA complex was also confirmed by the structured ab sorption spectrum. In a ddition, a pure aqueous solution of PPV-SO3 exhibits excitation wavelength-depend ent fluorescence; however, this siteselective fluorescence is diminished in the presence of DTA. Whitten et al also examined the surfactant effects on the amplified quenching behavior of CPEs and found that the surfactan t with opposite charges readily m odified the susceptibility of the polymer to quenching by different quenchers.52 As shown in Figure 1-13, the KSV constant of PPV-SO3 for the charged quencher, MV2+, is depressed by more than 2 orders of magnitude in the presence of 10 mol% of DTA relative to the polymer repeat unit. In contrast, the KSV constant for neutral quencher, 2,4,6-trinitrotoluene (TNT), is enhanced nearly 10-fold with same amount of surfactant. The decrease of the quenching sensitivity to MV2+ is believed to be due to two effects: 1) the cationic su rfactant competes with MV2+ for binding with the anionic polymer, thus decreasing the association consta nt between the polymer and MV2+; 2) The disruption of aggregation of polymer by surfactan t dramatically decreases or ev en eliminates the inter-chain interaction, and thereby reduces the amplified quenching extent. However, in the case of TNT, formation of complex between polymer and su rfactant affords a loca l hydrophobic environment near the polymer backbone, which facilitates the solubilization of neutral TNT molecules in this local environment and increases its association constant with the polymer. As a result, the quenching efficiency for TNT is impr oved with the aid of surfactant.

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42 Figure 1-13. Effect of DTA on quenching of 1.5 10-5 M PPV-SO3 by MV2+ and TNT. A) SV plot for PPV-SO3 without DTA. B) SV plot for PPV-SO3 with 1.5 10-6 M DTA. Reprinted with permission from Chen et al .52 Building on the work of Whitten et al ., Dalvi-Malhotra and Chen demonstrated the similar impact on the fluorescence quantum yield and the quenching sensitivity of PPV-SO3 by another surfactant 1,2-diole oyl-3-trimethyl-ammonium propane (DOTAP).54 Furthermore, they synthesized a viologen-based quencher functionalized with a long, hydrophobic alkyl chain which was proved to be a more effective quencher than MV2+ in the presence of DOTAP. The higher sensitivity to alkyl chain-f unctionalized quencher is attributed to the formation of bilayers between DOTAP and PPV-SO3 backbone which facilitates the interaction of aliphatically functionalized-quenc her with polymer. It is important to note that the conformation and optical modification upon addition of surfactant is reversible by removing the surfactant. In addition to surfactants, some other charged additives exclusive from commonly used quenchers, such as proteins58,67 and other polyelectrolytes56 (i.e., oppositely charge CPE55), can modify the optical properties of CPEs via changing the geometric conformation of the pol ymer. The ability to tune the optical and electronic properties of CPEs by manipulating their conforma tion clearly provides another platform for sensor applicatio ns of this powerful material.

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43 Optical Sensor Application of Conjugated Polyelectrolytes Over the past few years, CPEs have been stud ied extensively as optical sensors for various analytes,19 including small ions or biomolecules, proteins and nucleic acids. The unique structural and optical properties of CPEs provide several adva ntages over the routine sensor methods. First, the multiple-charged structure of CPEs affords the water solubility which is essential for carrying out biologi cal assays in aqueous media. And the amphiphilic structure property of CPEs provides a plat form to interface with the an alytes through electrostatic or hydrophobic interactions. Second, the CPE-base d optical sensors afford a homogeneous approach which is less labor intensive and less time consuming compared with heterogeneous assay such as enzyme-linked immunosorbent assay (ELISA). As a result, the CPE-based assay is simpler, faster, and readily adapted to a fl uorescence-based high-thr oughput screening (HTS) format.68 Third, and most importantly, signal amplif ication is achieved based on sensitive and collective response of CPEs to external stimuli including a very small amount of quenchers (amplified quenching effect) and minor changes in aggregation or conformation. Therefore, the CPE-based optical sensors show su perior sensitivity with typical detection limits in nanomolar concentration range69 or even in zeptomolar range.70 CPE-based optical sensors have been realized in two detection schemes, colorimetric and fluorometric. Colorimetric detection is based on a change in absorption wavelength of CPEs; while the fluorometric assay affords inherent high sensitivity as well as versatility in detection of various signals, which include th e changes in intensity, wavelength and lifetime. In most of the CPE-based sensors that have been developed, th e fluorescence can be either enhanced (turn-on approach) or quenched (turn-off approach) upon direct or indirect in teraction with targets. Both the turn-on and turn-off approach es are realized by three mechanisms, i.e., superquenching mechanism, light harvesting mechanism via fluor escence resonance energy transfer (FRET) and

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44 conformation change mechanism. It is important to point out that the th ree mechanisms are not exclusive, that is, some already developed CPE-ba sed sensors utilize more than one or even all of them in one assay. Here, we will first describe the three sensing mechanisms, and then discuss the sensor application of CP Es for different analytes. Sensing Mechanisms Superquenching mechanism Superquenching or amplified quenching has been explored as a basis for sensitive chemical or biological sensing. In the superquenching strategy, a quenche r-tether-ligand (QTL) complex is synthesized by connecting a quenche r to a biologically interes ting ligand. The superquenching behavior of CPE is modulated by addition of analytes which is capable to binding with the ligands. In 1999, Chen and Whitten published the first example of a CPE-based biosensor based on this mechanism.28 As shown in Figure 1-14, a biotin-f unctionalized viologen quencher serves as the QTL complex and it quenches the fluorescence of PPV-SO3 via charge transfer. Addition of avidin into the solution of the polymer and quencher results in a fluorescence turn-on response. The process from quenching to unquenchi ng is attributed to binding of the biotinfunctionalized viologen by avidin thus producing a sterically large avid in-bound quencher and preventing the close association of quencher with polymer. By em ploying different ligands in the QTL complex, Whitten and co-workers also deve loped sensors for cholera toxin and hepatitis C.19 However, further studies by Bazan and co workers on the same polymer, quencher and avidin systems have indicated that the increase of the fluorescence is at least partially due to the formation of the cationic avidin-anionic polym er complex by non-specific interaction and this complex shields the polymer from the quencher.58 We will discuss the non-specific interaction in more detail below.

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45 Figure 1-14. Detection of avidin using CPE vi a the superquenching mechanism. Reprinted with permission from Chen et al .28 Light harvesting mechanism via fl uorescence resonance energy transfer Fluorescence resonance energy transfer (FR ET) occurs via a long range dipole-dipole interaction between the electroni c excited states of two dye molecules in which excitation is transferred from a donor molecule to an accepto r molecule without emission of a photon. The efficiency of FRET strongly depe nds on the distance between the donor and acceptor, as well as the overlap between the emission spectrum of the donor and the absorp tion spectrum of the acceptor. When FRET is used in a sensor scheme the detection signal co uld be the enhancement or diminution of the fluorescence of the donor or acceptor. CPEs are excellent donor candidates due to their high extinction coe fficients, high fluorescence quantum yields and efficient exciton migration, which result in the amplif ied fluorescence from energy acceptor. As a specific example of FRET, we describe the detection scheme that was applied to DNA detection by Gaylord, Bazan and Heeger.71 More detail on their wo rk is given in a later section. The DNA assay contains three ingredients, namely, a cationic poly(fluorineco phenylene) 3 (Figure 1-15), a probe peptide nucleic acid (PNA) strand labeled at the 5 end with a chromophore (C*, fluorescein) having spectral overlap with 3, and the target DNA strand. As shown in Figure 1-16, the neural PNA -C* displays no electrostatic interaction with cationic 3, and the average distance between them is too large for effectiv e FRET. Introduction of complementary ssDNA into the solution of 3/PNA-C* indicated by the left arrow results in the

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46 formation of PNA-DNA comple x which features a net nega tive charge and favorable electrostatic interaction with 3. As a result, the distance between DNA-PNA-C* and 3 is greatly decreased, thereby allows for effective FRET to take place followed by the detection of the presence of complementary ssDNA from a turn-on fluorescence signal from fluorescein. On the contrary, the addition of noncomplementr y ssDNA indicated by the right arrow doesnt induce the hybridization w ith PNA-C*, thus resulting in little FRET. The detection limit of this FRET-based DNA assay for the complementary strand was demonstrated to be 10 pM and this superior sensitivity was attributed to the inhe rent optical signal amplification of CPE. n N N 3 Figure 1-15. Molecular st ructure of cationic CPE 3 used by Heegers group in the DNA sensor. Figure 1-16. Schematic representation of the PNA-C*/3 assay to detect a complementary ssDNA sequence. Reprinted with permission from Gaylord et al.71

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47 Conformation change mechanism The conformation change of the conjugate d backbone upon complexation with different analytes results in the chromic changes, both in absorbance and fluorescence of CPE. One of the advantages of conformation change-based sensor a pproach over the other two mechanisms is that it depends on nonvalent interactions and therefore eliminates the need of covalent labeling of probe molecules. Several cationic poly(thiophene) (PT) derivatives as shown in Figure 1-17 are the most commonly used CPEs in this mechanism. It was first employed to detect DNA hybridization70,72 and then used to detect a specific protein.73 Figure 1-17. Molecular structures of cationic PT derivatives used in CPE-based sensor application via conforma tion change mechanism. Leclerc and coworkers have develo ped an assay to detect a human -thrombin by utilizing 4 and a ssDNA thrombin aptamer.73 As shown in Path A of Figure 1-18, a compact unimolecular quadruplex structure is formed by binding aptamer to specific protein, thrombin. The added 4 wraps around this structure, exists in a less aggregated conformation and shows blue-shifted absorption and enhanced fluorescence compared with those displayed in Path B where nonspecific protein is used. Unde r the condition of path B, the free apatmer complexes with 4 and forms a weakly emissive duplex. Therefore, the specific protein is distinguished by monitoring the conformation perturbation-induc ed optical signal which is governed by aptamer structure. By using this mechanism, the authors could detect human -thrombin with a detection limit of 10

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48 pM. In addition, Nilsson and co-workers ha ve utilized the same strategy and reported a 5-based assay to detect the protein calmodulin (CaM) and CaM-Calcineurin interaction74 as well as a 6based sensor to detect amyloid fibril formation.75 Figure 1-18. Schematic illustration of the specific detection of human -thrombin by use of ssDNA thrombin aptamer and cationic 4. Reprinted with permission from Ho et al .73 Small Ion Sensing with CPEs Small ions, including protons, metal ions, me tal complexes and inorganic anions are known to interact with oppositely charged CPEs and influence th eir optical properties via the mechanisms of superquenching,76 energy transfer41 and conformation perturbation.73 In 2005, Bunz and co-workers reported a sens itive lead sensor which was based on the Pb2+-induced fluorescence quenching of a carboxylated PPE 7 (Figure 1-19).77 The high sensitivity of this lead sensor was attributed to the combination of multivalent effect between 7 and Hg2+, as well as the exciton migration (amplified quenching effect) along the polymer chain. More recently, the same group employed the same polymer and developed a selective sensor for Hg2+.78 The assay was based on formation of an electrostatic complex between 7 and papain, a cationic cysteine protease. As shown in Figure 1-19, the 7-papain complex displays the selective fluorescence quenching response only to Hg2+ over 9 other control metal ions (i.e., Zn2+, Cd2+,

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49 Pb2+, Fe2+, Ni2+, Co2+, Cu2+, Ca2+ and Mg2+). The authors proposed an agglutination mechanism to explain this selectivity as shown in Figure 120. Papain with free thio l groups is known to bind Hg2+. In the 7-papain complex, the protein chains are strongly cross-linked by the anionic polymer, forming a supramolecular structure which is more sensitive towards agglutination than either 7 or papain alone. As a result, a weak emissi ve precipitation and a non-fluorescent solution were observed after adding Hg2+ to the 7-papain complex solution. Figure 1-19. Structure of carboxylated PPE 7 and pictures taken under a hand-held UV light to show the fluorescence under different situations: A) 7-papain complex (7, 5 M; papain, 5 M). B) All 10 metals added to 7-papain complex (each metal, 0.4 mM). C) Same as B without Hg2+. Reprinted with permission from Kim et al .78 Figure 1-20. Qualitative interpretation of the Hg2+-induced agglutination of the 7-papain complex. A) 7 alone. B) Electrostatic complex of 7 and papain. C) The addition of Hg2+ to 7-papain complex leads to its precip itation by cross-linki ng of the papain molecules through Hg2+. Reprinted with permission from Kim et al .78

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50 In 2000, Schanze and co-workers report ed an efficient sensor for Ru(phen )3 4(phen = 4,7bis(4-sulfophenyl)-1,10-phena nthroline) and Fe(CN)6 4-.41 Both of these two ions act as the direct quenchers for PPP-NEt3 + (structure shown in Figure 1-2) via energy transfer from the cationic polymer to the metal-to-ligand charge transfer state of the ions. The KSV values were reported around 108 M-1 and SV plot displayed the typical upward curvature because of the quencher induced aggregation.51 In addition to the ion sensors desc ribed above, CPE-based assays have also been developed to detect the other ions, such as H+ (pH sensor),79 K+,73,80 Eu2+,76 Cu2+,76 Ru(bpy)2(dppz)2+ (bpy = 2,2 -bypyridine; dppz = dipyrido[3,2-a:2 3 -c]phenazine),81 and multicationic amines.82 Small Biomolecule Sensing with CPEs The use of CPEs in the biosensor area is one of the most important applications of CPEs and has been studied intensively over the past years. Followed by the pi oneering avidin assay invented by Whitten et al in 1999,28 several CPE-based biosensors ha ve been reported to detect small biomolecules, including saccharides,83,84 adenosine triphosphate (ATP),85 H2O2 84and antioxidants.86 In 2002, Schanze and co-workers reported a sensitive fluorescent sensor for saccharides based on amplified quenching of PPE-SO3 (structure shown in Figure 1-7) by a boronic acidfuctionalized benzyl viologen, p -BV2+ (Figure 1-21).83 The cationic p -BV2+ can strongly associate with anionic PPE-SO3 and quenches its fluorescence ( KSV = 2.8 107 M-1) with comparable quenching efficiency as MV2+. However, as the reaction shown in Figure 1-21, the added sugar reacts with p -BV2+ in neutral medium to generate a bisboronate derivative, which is overall charge neutral. Therefore, upon addi tion of sugars into the solution of PPE-SO3 -/ p -BV2+, the carbohydrate-bound p -BV2+ cant complex with PPE-SO3 and the quenched fluorescence is recovered, which is regarded as a turn-on response to added sugars. As shown in Figure 1-22, as

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51 the concentration of the sugars ( D -fructose, D -galactose or D -glucose) increases, the amplified quenching is eliminated and a sign ificant increase in the fluorescence intensity is observed. The sensory system affords a 50-fold incr ease in intensity upon addition of 10 mM D -fructose. In addition, the sugar sensor also shows selectivity towards D -fructose over the other two saccharides, which is believed to arise from more stable association between D -fructose and p BV2+. Figure 1-21. Interaction between p -BV2+ and sugar at near neutral pH. Figure 1-22. Titration curves against sugar for the PPE-SO3 (2.5 10-6 M)/ p -BV2+ (8 10-7 M) system, measured in PBS (6 mM) pH 7.4. Reprinted with permission from DiCesare et al .83 More recently, Shinkai and co-w orkers reported a cationic PT -based selective sensor for ATP.85 As shown in Figure 1-23, the sensory system displays a red shift in absorption spectrum or a color change of the pol ymer solution from yellow to pink-red upon introduction of ATP. The authors proposed that the a dded ATP formed an electrostatic complex with PT derivative and the complex increased the planarity of the polymer and therefore induced the -stacked

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52 aggregation. The smaller response to the inorganic phosphates (e.g., HPO4 2-) suggests that the adenine base structure-induced hydrophobic effect is an important factor in the aggregation. Note that adenosine diphosphate (ADP) and ade nosine monophosphate (AMP) show observable difference in the color change from ATP, which is due to their weaker electrostatic attraction towards PT derivative because of less negative charges. The different responses to adenosine phosphates was adopted by Zhu and Bazan to de velop a PT-based colorimetric assay for hexokinase, an important enzyme used in glucose metabolism.24 Figure 1-23. Changes in the color of solution of 1.0 10-4 M cationic PT derivative in water induced by the addition of equimolar amount s of various anions. Reprinted with permission from Li et al .85 Protein Sensing with CPEs Protein sensing is another important research interest in the CPE-based optical sensor application and has undergone an ex tensive growth in recent years. Protein sensing consists of two segments, indirect detection of various en zymes and direct detect ion of other proteins. Indirect detection of enzymes The research interest in the field of enzyme sensing with CPEs started from the detection of three important enzymes including protease,87-89 kinase90 and phosphatases.90 Generally, a quencher-labeled or fluorophore-la beled substrate is applied as a probe. In the presence of enzymes, the linker between the substrate and quencher/fluorophore is cleaved and the fluorescence of CPE is either turned on or turned off.

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53 A protease is an enzyme that conducts hydrolysis that is, begins protein catabolism by hydrolysis of the peptide bonds that link am ino acids together in the polypeptide chain. In 2004, Pinto and Schanze reported fluorescent turn-on and turn-off assays for protease utilizing two anionic CPE s, PPE-SO3 (structure shown in Figure 1-7) and PPE-CO2 (structure shown in Figure 1-10), respectively.87 In the turn-on approach, Kp NA (structure shown in Figure 1-24), a cationic peptide (the protease substrate) labeled with a p -nitroanilide (p NA) unit (the fluorescence quencher), was used in conjunction with the anionic PPE-SO3 -. As the turn-on mechanism shown in Figure 1-25, the cat ionic peptide ion-pairs with PPE-SO3 and quenches the polymer fluorescence. Introduction of the protease to a solution of the quencher/substrate and polymer induces hydrolysis of the Kp NA and formation of a charge neutral p NA moiety, which loses the ability to associate with PPE-SO3 and quench the fluorescence. As a result, the fluorescence intensity increases concomitantly wi th peptide hydrolysis. The turn-on assay was demonstrated to detect the activity of as low as 2.7 nM thrombin within 100 s and provide reliable measurement of kinetic data. While, in the turn-off approach developed by the Pinto and Schanze, a colorless and nonemissive bis-argi nine derivative of Rhodamine-110, Rho-Arg2 (Figure 1-24), was used as the substrate for pe ptidase. As shown in Figure 1-25, the cationic substrate is capable of electrostatic binding PPE-CO2 -, but it doesnt quench the fluorescence of the polymer. However, when Rho-Arg2 is hydrolyzed by added protea se, the liberated emissive mono-arginine derivative, Rho-Arg (Figure 1-24), could strongly quench the fluorescence of PPE-CO2 via FRET due to a good spectra overlap. Th erefore, the decreased fluorescence was observed accompanying the hydrolysis of Rho-Arg2 by protease. The turn-off sensor was reported to successfully detect as low as 3,5 nM papain and display a 10 -fold signal enhancement compared to a pure rhodamine alone.

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54 Figure 1-24. Structures of que ncher substrates used in prot ease assay by Schanzes group. Figure 1-25. Mechanism of the turn-on and turn-off CPE-based sensors for protease activity. Reprinted with permission from Pinto et al .87 Simultaneously with Schanzes work, Whitten and co-workers reported a CPE-based protease sensor in 2004.88 In their study, they utilized CPE-coated microspheres functionalized with biotin-binding protein, as we ll as a polypeptide substrate labe led with biotin at one end and quencher at the other end. Sim ilar to Schanzes turn-on protea se assay, the quencher labeled substrate strongly quenches the florescence of CPE via an association betw een biotin and biotin binding protein. However, when the CPE-coated microspheres are added into the solution of

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55 substrate in the presence of pr otease, the fluorescence is unquen ched, because the substrate is hydrolyzed by protease and incapable of associating with polymer. More recently, Swager and co-workers developed another protease assay which was based on the concept of molecular beacon.89 The unique aspect of this protease sensor is that the polypeptide is covalently attached to the CPE, and the resulting peptide conjugated CPE serves as both the enzyme substrate and the fluorescent reporter. Kinase and phosphatase contro l the phosphorylation and dephos phorylation of proteins, so they play an important role in the regulation of cellular metabolism, growth, differentiation, and proliferation. In 2004, Whitten and co-workers published an anionic PPE-based fluorescent assay for kinase and phosphatase.90 Similar to their work in the protease sensor, microspheres are coated with the PPE derivative containi ng both sulfonate and carboxylate groups. The carboxylate groups are complexed with Ga3+ ions, which are known to have affinity towards phosphorylated peptides. As shown in Figure 1-26, a rhodamine-labeled peptide, designed in a QTL format, is used as the substrate for th e kinase. Upon the phosphorylation by kinase, the phosphorylated peptide associates with Ga3+-functionalized and PPE-c oated microspheres via Ga3+-phosphate binding, quenches of fluorescence of PPE via energy transfer to rhodamine and generates a turn-off signal response. Meanwhile a fluorescence turn-on assay to detect the dephosphorylation by phosphatase wa s also designed via the same strategy. In addition to the direct assay, Whitten and co-workers reported another competitive assay to detect the kinasemediated phosphorylation, in which an unlabeled peptide substrate and a dye-labeled tracer phosphopeptide were used.90 Upon phosphorylation, the phosphoryl ated label-free substrate displaces the tracer phosphopeptid e on the surface of polymer-coa ted microspheres and leads to recovery of the fluorescence whic h is initially quenched by tracer.

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56 Figure 1-26. General scheme for the kinase a nd phosphatase assays. Reprinted with permission from Rininsland et al .90 Phospholipase A2 (PLA2) is another enzyme detected by the CPE-based sensor. As an upstream regulator of many in flammatory processes, PLA2 specifically catalyzes the hydrolysis of sn -2 acyl bond of phospholipid to release a free fatty acid and a lysophospholipids. In 1999, Charych and co-workers published a colori m etric one-step assay to detect PLA2 activity and inhibition by utilizing vesicles co mprised of polydiacetylene (PDA) and dimyristoylphosphatidylcholine (DMPC).91 Inspired by this work, Whitten and co-workers recently developed a fluorescent turn-off assay for PLA2 based on a so-called frustrated superquenching mechanism.92 Using the layer by layer method of assembly, a cationic microsphere-supported CPE is coated by dimyri stoyl phosphatidyl glycerol (DMPG), which protects CPE from attack of quencher and serves as substrate for PLA2. Upon hydrolysis of DMPG induced by addition of PLA2, the CPE is exposed to the quencher and the fluorescence is quenched efficiently. A similar FRET-based turn -on assay with a dye-labeled DMPG was also designed using the same strategy. This homogeneous CPE-based assay is s uperior to previously developed PLA2 assay in terms of using natural s ubstrates without any modification. Additionally, more recently, Zhu et al have reported the CPE-based optical assays for sensing activity and inhibition behavior of other enzymes, such as acetylcholinesterase93 and hexokinase.24

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57 Direct detection of proteins Direct detection of proteins is referred to specifically monitoring the optical signal changes induced by direct interaction between CPEs and proteins vi a either superquenching or conformation perturbation. Genera lly, it doesnt require the involve ment of other molecules such as ligands and quenchers. We have already given the examples of direct protein sensing based on conformation perturbation in the previous confor mation change mechanism section, therefore, we will herein focus on direct detection of proteins based on superquenching strategy. In 2002, Heeger and co-workers reported th e direct detection of the heme-containing protein, cytochrome c (cyt c), which was used as a direct fluorescence quencher with high quenching efficiency.94 Cyt c associates with an anionic CP E, MBL-PPV (structure shown in Figure 1-8) driven by electro static attraction, and quenches the fluorescence with a KSV of 3.2 108 M-1 (Figure 1-27). The authors attrib uted the superquenching effect to electron transfer from the excited polymer to the Fe3+ center in cyt c. In 2006, Huang and co -workers published the fluorescence quenching of a cationi c CPE by an anionic iron-contai ning protein, rubredoxin via the same mechanism.95 Figure 1-27. Fluorescence spectral changes of 1.0 10-6 M MBL-PPV upon titration of different concentration of cyt c: from top to bottom: 0, 0.25, 1, 2.5, 5, and 9.4 nM. Reprinted with permission from Fan et al .94

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58 However, a non-specific quenching effect on the fluorescence of MBL-PPV by lysozyme, a positively charged protein that lacks a charge tr ansfer center, was also observed by Heeger and co-workers.94 The quenching effect is due to the fl uorescence self-quenchi ng caused by proteininduced aggregation of anionic polymers. The ex istence of non-specific in teraction restricts the application of CPE in di rect protein detection. More recently, Bunz and Rotello have c onstructed an array-ba sed protein sensor comprising six functionalized PPE polymer s to identify and quantify 17 proteins.96 Characteristic with various charges and molecular scales, th ese PPEs interact with each protein based on different mechanisms, and show distinct fluor escence response in the presence of a given protein. The method of linear disc riminant analysis was applied to differentiate the patterns of 17 proteins at nanomolar to micromolar concentratio ns. Finally, an identification accuracy of 97% was reported. DNA Sensing with CPEs DNA sensing with CPEs has been studied ex tensively by several gr oups. Generally, the developed DNA sensors are based on three mech anisms by using different cationic CPEs, i.e., conformation transition from cationic PT deri vatives, light harvesti ng by FRET from cationic poly(fluorineco -phenylene)s, and covalent bioconjugation of PPEs with DNA. DNA sensing based on conformation transition In 2002, the group of Leclerc developed the first CPE-based DNA sens or by applying the cationic PT derivative, 4 (structure shown in Figure 1-17).72 CPE 4 exists in a random-coil conformation with a twisted conj ugated backbone in aqueous soluti on, whereas transmits to an aggregated and planar form with 143 nm red-shifted absorption in th e solid state. As a result, its conformation is very sensitive to changes in the environment. Addition of one equivalent of 20base segment of ssDNA leads the color of an aqueous solution of 4 changed from yellow ( max =

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59 397 nm, Figure 1-28Aa and Ba) to red ( max = 527 nm, Figure 1-28Ab a nd Bb) within 5 min of incubation. As shown in Figure 1-29, the color ch ange is due to the formation of a so-called duplex between 4 and the oligonucleotide probe driven by electrostatic attraction. The duplex forces the polymer to adapt a highly conjugate d and planar conformation from its original random-coil form, which accounts for the red-sh ifted absorption. After several hours of incubation, precipitation was obser ved from duplex solution. Howeve r, if another one equivalent of complementary target ssDNA is added into th e duplex solution at high temperature before precipitation, the color again becomes yellow ( max = 421 nm, Figure 1-28Ac and Bc) after 5 min. This change is caused by the formation of triplex between 4 and hybridized oligonucleotides, with the charac teristics of right-handed helica l orientation of the polymer backbone and electrostatic bi nding between polymer and ds DNA (Figure 1-29). The less conjugated and nonplanar conformation of polymer results in the color change in the triplex solution. Figure 1-28. Photographs and UV-visible absorption spectra changes observed in the PT 4-based DNA sensor. A) Photographs of 7.9 10-5 M solution of a) 4 alone, b) 4/ssDNA duplex, c) 4/dsDNA triplex, d) 4/ssDNA plus a complementary target with a two-base mismatch, and e) 4/ssDNA plus a complementary target with a one-base mismatch after five minutes of mixing at 55 C. B) UV-visible absorption spectra corresponding to photograph A. Reprinted with permission from Ho et al .72

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60 In order to verify the selectivity of this 4-based ssDNA sensor, Leclerc and co-workers tested the sensor response to target ssDNA cont aining two or even one mismatched nucleotides. Both of them exhibit distinct absorption chan ges from the perfect matched target (Figure 128Ad, Bd, Ae, Be), which are caused by incomplete hybridization. In addition to concomitant colorimetric change with the DNA assay, the auth ors also observed the associated fluorescence change which was demonstrated to detect ssDNA with a 7 orders of magnitude improvement over colorimetric assay.72 Figure 1-29. Scheme description of the formation of a planar 4/ssDNA duplex and a helical 4/dsDNA triplex. Reprinted with permission from Ho et al .72 Followed by this work, Leclercs group furt her improved their PT conformation changebased DNA assay in terms of sensitivity and need for elevated temperature. For example, they increased the sensitivity of the DNA assay to the zeptomole level by specially designing a tailored detection platform consisting of a stable light-emitting diode (LED) source and simple interference filters.70 In addition, they also developed an ambient temperature compatible DNA assay by addition of formamide as a denaturant.97 Later in 2005, Lecl erc and co-workers incorporated their assay with FRET technique and developed an even more sensitive DNA sensor. In this approach, the oligonucleotide probe is labeled with a fluorophore (e.g., Alexa Fluor 546), and forms a le ss emissive duplex with 4. As shown in Figure 1-30, when this duplex

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61 hybridizes with its complementary target oligonucleotide, the triplex is form ed concomitant with FRET from polymer to the fluorophore acceptor. Capable of distinguishing target DNA from noncomplementary or mismatched DNA, this turn-on strategy is able to detect as low as 3 zM target DNA. Figure 1-30. Schematic description of the pr oposed signal amplification detection mechanism based on the conformational change of 4 upon forming triplex with dsDNA and energy transfer for DNA detection. Reprinted with permission from Ho et al .98 DNA sensing based on FRET The group of Bazan pioneered the developmen t of DNA sensors using FRET of CPE. The FRET based sensing strategy was initially applied to probe RNA-RNA assemblies99 and RNAprotein interactions.100 Then Bazan and co-workers expanded it to DNA sensing. We have described the first DNA sensor they developed in the section of light harvesting mechanism via FRET (Figure 1-16). Using the same cationic poly(fluorineco -phenylene) derivative, 3 (structure shown in Figure 1-15), they furt her improved this DNA assay by replacing the expensive neutral probe peptide nucleic acid with ssDNA probe strand.101 In spite of the existence of an electrostatic attraction between cationic 3 and the ssDNA probe, the FRET

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62 appears to be more efficient upon hybridizati on with complementary target ssDNA. The less efficient FRET in the case of the noncomplementary sequence arises b ecause the nonhybridized strand intereferes with the interaction of the ssDNA probe st rand and the polymer. Additionally, the good selectivity of this assay depends on the stronger electrostatic attraction between 3 and hybridized dsDNA (ssDNA probe + complementary ta rget ssDNA) compared with that between 3 and the ssDNA (ssDNA probe or noncomplementary ssDNA). The authors emphasized the importance of both electrostatic interaction and hydrophobic in teraction in this DNA assay.102 In addition to the detection of ssDNA, the same strategy was demonstrated to be a feasible detection method for dsDNA without the need fo r the thermal denaturing steps which are typically required for DNA hybridization probe assays.103 Figure 1-31. Schematic representation of A) DNA detection by twoprocess FRET and B) relative orientations of th ree optical components: CPE 6, FL and EB. Reprinted with permission from Xu et al .104 In order to attenuate the non-specific interaction which limits the specificity of the DNA assay, Bazan and co-workers applied a two-st ep energy transfer process by incorporating fluorescein (FL) as an intermediate FRET gate and ethidium bromide (EB) as the second energy acceptor as shown in Figure 1-31A.104 EB is known to intercalate only between the stacked bases of dsDNA, therefore, there is no FRET in the presence of non-complementary ssDNA. This is because the non-complement ary ssDNA could not hybridize with probe DNA and form dsDNA. Fluorescein is re quired in this assay due to the nearly orthogonal arrangement

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63 of transition dipole moments betw een polymer and EB as shown in Figure 1-31B. More recently, Bazan and co-workers successfully applied th is two-step FRET and inercalator-based DNA sensing strategy to detect the quadruplex-to-dupl ex transition of guanine -rich oligonucleotides.105 In 2006, the group of Bazan repo rted a different DNA assay whic h was capable of accurate determination of DNA concentration.106 This assay employed a series of carefully designed copolymers 8 (Figure 1-32), which contains different percentage of 2,1,3-benzothiadizole (BT) unit per polymer chain.107 The fluorescence of 8 is characteristic with changing from blue (from CPE unit) to green (from BT unit) emission as polymer concentration increases, which is believed to arise from formation of the aggregation and efficient interchain energy transfer to the lower energy BT sites. Addition of DNA to 8 results in polymer aggregation as well as the fluorescence shift from blue to green light. And the relative in tensities of blue and green emission quantitatively measure the amount of DNA that is bound with the polymer. More recently, 8 was used to incorporate the strand-s pecific DNA detection into DNA chips and microarrays by the same group.108 Figure 1-32. Molecular structure of cationic CPE-based copolymer 8 used by Bazans group. In addition to the detection of short strands of oligonucleotides, Bazan and co-workers have combined their PNA/ssDNA (Figure 1-16) a ssay with S1 nuclease enzyme to detect the single nucleotide polymorphism (SNP ) which contains 249 base pairs.109 The function of S1 nuclease in this assay is to digest all unbound ssDNA after PNA/ssDNA annealing and thereby

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64 increase the selectivity. This assay is capable of distinguishing the normal, wild-type human DNA sequences from those sequences containing a si ngle base mutation. It is encouraging to see its potential ability in the clin ical application such as iden tification of neurodegenerative diseases. DNA sensing based on covalent bioconjugation Tan and Schanze reported the first self -signaling DNA sensor based on covalent conjugation by synthesizing a molecula r beacon with the anionic PPE-SO3 (structure shown in Figure 1-7) chain as the fluorophore.110 Figure 1-33 illustrates the mechanism of DNA detection by the PPE-SO3 --labeled molecular beacon. In the absen ce of the target DNA, the quencher is close to PPE-SO3 due to self-complementary folding of the DNA and amplified quenching of the fluorescence of the polymer. Upon addition of complementary DNA, a more stable duplex is formed, thus disrupting the single-stranded hair pin structure, separating the polymer from the quencher and restoring the fluorescence. Compared to a traditional molecular beacon, this new design increases the assay sensit ivity by taking advantage of amp lified quenching effect of CPE. However, the downside of this DNA assay is the limited solubility of the DNA-tagged PPE-SO3 -. Therefore, it requires the addition of surfactants into the assay solution to increase the solubility of the molecular beacon, alt hough surfactants have the potentia l to interfere with assay. Figure 1-33. Schematic representa tion of DNA detection by PPE-SO3 labeled molecular beacon. Reprinted with permission from Yang et al .110

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65 Figure 1-34. Molecula r structure of PPE 9 used by Kims group in the DNA sensor. Figure 1-35. The PPE 9-oligonucleotide bioconjuga tion to form PPE-DNA (top) to detect target DNA based on FRET and form PPE-DNA beacon (bottom), demonstrating self-signal amplifying label free detection. Re printed with permission from Lee et al .111 Based on the work of Tan and Schanze, Kim an d co-workers reported a label-free and selfsignal amplifying molecular DNA sensor using a sulfonated PPE, 9 (Figure 1-34), in 2007. PPE 9 contains carboxylic acid groups on both ends of the polymer chain, which are capable of coupling to amino-functionalized oligonucleotides.111 The water solubility of 9 is greatly increased because of the bulky ethylene oxide side chains whic h prohibit the polymer chains

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66 from aggregating by shielding the hydrophobic polymer backbone. As shown in Figure 1-35, the authors designed two DNA sensing schemes using 9. The first scheme depends on the direct bioconjugation of 9 with a ssDNA probe via carbodiimide chemistry and detection of the complementary dye-labeled ssDNA based on FRET. Similar to Tan and Schanzes work, the second scheme is based on formation of molecular beacon containing 9, and it eliminates the need of surfactants because of th e increased water solubility of 9. Challenges of CPE-based Optical Sensor s: Non-specific Interactions of CPEs In the foregoing text, we have mentioned se veral times the non-specific interactions of CPEs, which are regarded as a significant ch allenge associated with the optical sensor application of CPEs. In the limited numbers of sensors that are free of impact by non-specific interaction, CPEs are usually f unctionalized with groups that c ould specifically and covalently interact with target analytes (e.g. protease sensor developed Swager et al89). However, these sensor schemes require special synthesis a nd lack versatility. On the contrary, the unfuntionalized CPE-based sensor schemes feature the advantages of being more versatile and cost efficient, but they rely on electrostatic or hydrophobic interacti ons between CPEs and targets, which leads to a strong susceptibility to the presence of other species in the sensing environment. Interfering species includes char ged small molecules (ions and surfactants), branched molecules (dendrimers), charged biomelucules (carbohydrates, nucleic acids and proteins) and other polyelectrolyt es. They associate with CPEs driven by the same interaction forces as the targets, quench/unquenc h the fluorescence and/or perturb the conformation/aggregation of CPE, and thereby generating fals e positive or false negative signals. Compared with the mechanisms of super quenching and conformation change, CPE-based sensors via FRET mechanism are less vulne rable to non-specific interactions.

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67 Several groups have intens ely studied non-specific intera ctions between CPEs and proteins.57,58,112-115 For example, Heeger and co-workers57 observed that the fluorescence of MBL-PPV (structure shown in Fi gure 1-8) was enhanced upon addi tion of 150 nM of an anionic protein, mouse immunoglobulin (IgG). Furtherm ore, the quenched fl uorescence by the viologen type quencher which is labeled by an antibody fragment specific to mouse IgG could be recovered by mouse IgG as well as non-specific an tibodies to even as high as 150% of original fluorescence intensity. In order to minimize the in fluence of non-specific interaction, a charge neutral complex (CNC) consisting of MBL-PPV and a cationic satura ted polyelectrolyte poly(N,N-dimethylamino-ethylene iodide) (PDMAE) in a 1:1 ra tio was prepared by Heeger, et al .57 CNC is readily quenched by both cationic and anionic quenchers and is less sensitive to non-specific interactions because of two factors. First, the ability of CNC to associate with charged species is attenuated because it is ch arge neutral. Second, CNC is more rigid upon complexation and thereby becomes less suscepti ble to conformation changes induced by other species. Heeger et al successfully applied CNC to detect as low as 300 nM anti-dinitrophenol (DNP) IgG.57 As shown in Figure 1-36, the quenche d fluorescence of CNC by 1-sulfobutoxy-2, 4-dinitrobenzene (DNP-BS-) is selectively recovered by anti-DNP antibody. Non-specific match of quenchers and antibodies (DNP-BSand control antibody, Rat IgG; 2,4,6-trinitrophenol (TNP) and anti-DNP antibody) results in no response in the fluorescence recovery. This method is demonstrated to be a great improvement to minimize the non-specific interaction of CPEs. In addition to prepare CNC, other methods were also used to decrease the susceptibility of CPE-based sensors to non-specific interactions. For example, CPEs were coated on the microspheres88,90 or grafted on the surface of colloids116 to confine the aggregation extent and restrict the conformation perturba tion induced by interfering species in the sensing environment.

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68 Figure 1-36. Schematic of the sensor for an ti-DNP antibody. A) Fluor escence from CNC is quenched by DNP-BSand recovered on the specif ic binding between DNP-BSand anti-DNP antibody. B) Fluorescen ce from CNC is quenched by DNP-BSand not recovered on addition of non-specific an tibodyrat IgG. C) Fluorescence from CNC is quenched by TNP and not recovered on addition of non-specific anti-DNP antibody. Reprinted with permission from Wang et al .57 Biosensors Using CPEs in Biological Environments Due to the existence of non-specific interac tion, very few of the developed CPE-based assays were applied to more complex systems, such as blood and urine, which contain a mixture of influencing species. However, CPEs have been demonstrated to be useful materials in the in vivo detection of bacteria,117-119 virus,117 amyloid deposits120 and cellular structures (i.e. chromatin and cytoplasmatic vesicles)121 by a staining process. We will give the examples about the bacteria detection. In 2004, Disney and co-workers prepared an anionic carbohydrate mannose-functionalized PPE and applied it as a biosensor for the mannose binding lectin concanavalin and the E. coli bacteria which contain mannose receptors.118 As shown in Figure 1-37, the specific binding between PPE and bacteria controls the staining of the bacteria and efficiently distinguishes mannose-binding E. coli strains from mutant bacteria which lack mannose receptors. In addition, Whitten and co-workers demonstrated that anot her cationic CPE showed light-induced biocidal

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69 activity against Gram-negative bacteria ( E. coli ) and Gram-positive bacteria spores ( Bacillus anthracis ).119,122 The authors attributed the biocidal activit y to the coating or even penetration of the CPE into the cell wall of the bacteria, as well as the generation of singlet oxygen upon photoexcitation. Most recently, th e group of Bunz constructed PPE-b ased microplates to detect and identify three different strains of E. coli in minutes.123 In their study, an anionic PPE is bound to cationic gold nanoparticles which efficien tly quench the fluorescence of the polymer. In the presence of the anionic bacteria, the fluorescence is restored differentially by three distinct strains of E.coli, which have different ability to re place the PPE on the gold nanoparticles. Figure 1-37. Visualization of mutant (left) and mannose-binding (right) E.coli strains after incubation with mannosylated CPE. Reprinted with permission from Disney et al .118 Peroxyoxalate Chemiluminescence and Its Sensor Application Chemiluminescence Since the phenomena of chemiluminescence wa s first observed in 1888 when green light was emitted from lophine oxidized by oxygen,124 the exploration of chemiluminescence has undergone an extensive growth. Chemilumine scence is defined as the production of electromagnetic radiation (ultraviolet, visible or infrared) observed when a chemical reaction yields an electronically exc ited intermediate or product, wh ich either luminesces (direct chemiluminescence) or donates its energy to an other molecule responsible for the emission (indirect or sensitized chemiluminescence).125 The initial interest of chemiluminescence

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70 reactions was focused on the gas-phase reactions with sulfur or nitrogen compounds, but more recently, liquid-phase chemiluminescence reactions have become more important due to their versatility. Furthermore, by taking the advant age of coupling chemiluminescence with other efficient injection or separation techniques (i .e., flow injection analysis (FIA), liquid chromatography (LC), capillary elec trophoresis (CE), and immunoassays),126 chemiluminescence technique has become more us eful in various disciplines, including the clinical,127 pharmaceutical,128 biomedical,129 food130 and environmental131 analysis. Compared with photoluminescence, chemilumine scence is superior in terms of simpler instrumentation without need for excitation light source. Because of the resulting absence of the noise caused by light scattering, background emi ssion and source instability, chemiluminescence is generally regarded as a dark-field technique. Moreover, th e avoidance of background noise leads to improved detection limit and signalto-noise. Therefore, chemiluminescence is considered more sensitive than photoluminescen ce. However, some deficiencies in applying chemiluminescence include shorter lifetime as we ll as time-dependent and environment-sensitive signal. In addition, the lack of selectivity in the presence of multiple chemiluminescence capable analytes is another limita tion of chemiluminescence. The chemiluminescence intensity is affected by two factors: the efficiency of generating molecules in the excited state (chemilumine scence quantum yield) and the rate of the chemiluminescence reaction.132 The expression of this dependence is given as CLCLdA I dt (1-7) where ICL is the chemiluminescence emission intensity, CL is the chemiluminescence quantum yield, and (dA / dt ) is the rate at which the chemilumi nescence precursor A is consumed. Generally, the reaction rate can be im proved by adding catalyst. The value of CL ranges from

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71 0.001 to 0.1, but even systems with much lower CL can be used in analysis due to the absence of background emission. The value of CL is defined as CLexL (1-8) where ex is the efficiency of production of the excited species (the fraction of the chemiluminescence precursor A that produces an excited molecule) and L is the luminescence efficiency of the luminescent species. Peroxyoxalate Chemiluminescence As seen in the definition of chemilumines cence, two types of chemiluminescence (direct chemiluminescence and sensitized chemilumine scence) are assigned based on the different species emitting the light. In direct chemilumine scence, the product of the reaction emits the light. The best known example of direct chemilu minescence precursor is luminol, which can be oxidized in alkaline media to produce excited 3-aminophthalate ion as shown in Figure 1-38.132 This reaction is catalyzed by various metal ions, such as Fe2+, Cu2+ and Co2+. The excited ion returns to ground state by emitting chemilumines cence, and the chemiluminescence spectrum is identical with the fluorescence spectrum of 3-aminophthalate. Figure 1-38. Scheme of luminol chemiluminescence reaction. The second chemiluminescence approach, sens itized chemiluminescence, involves the excitation of a fluorophore via energy transfer from the intermediate of chemiluminescence reaction, and the chemiluminescence emission from the excited fluorephore. Peroxyoxalate

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72 chemiluminescence is one of the most efficien t and versatile sensit ized chemiluminescence processes available today.133 It consists of a reaction between an aryl oxalate and an oxidant, typically hydrogen peroxide, to form a high-energy intermediate, which is capable of exciting a large number of fluorophores and the resulting ch emiluminescence emission is identical to the fluorescence of the fluorophore. Due to the complex and rapid side reactions as well as unstable intermediates, the mechanism for peroxyoxa late chemiluminescence reaction and the identification for intermediate are still under controversy.134-137 However, it is generally believed that the reaction follows a chemically init iated electron-exchange luminescence (CIEEL) mechanism with 1,2-dioxetanedione as one of the key intermediates.132 As the general mechanism of peroxyoxalate chemiluminescence r eaction shown in Figure 139, after the oxalate is oxidized, the generated high-energy 1,2-dioxetanedione forms a charge complex with fluorophore, accepting one electron by the intermediate The electron is tran sferred back to the fluorophore raising it to an exci ted state and liberating emission characteristic typical for the fluorophore nature. Figure 1-39. The CIEEL mechanism for the peroxyoxalate chemiluminescence reaction. Compared with the direct chemiluminescence system utilizing luminol, peroxyoxalate chemiluminescence has a number of advantages. Firs t, it is more efficient and sensitive due to relatively higher CL, and it is not susceptible to metal ion catalysis or effects of oxygen. Second,

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73 in addition to the detection of the oxidant which is the most applications of direct chemiluminescence reaction, peroxyoxalate chemiluminescence can also detect other fluorophore-related species. Thus peroxyoxalate chemiluminescence has wider application scope. Third, different from the alkaline requ irement of direct chemiluminescence reaction, peroxyoxalate chemiluminescence reac tion can be carried out at pH 7, the optimal pH for most biological process. Therefore, peroxyoxalate chemiluminescence is suitable for the biosensor application. Nevertheless, there are several limitations with peroxyoxalate chemiluminescence systems that are not found in the direct chemiluminescence system. For example, the peroxyoxalate chemiluminescence reagents (oxalate s) are insoluble in wa ter. Moreover, they become unstable due to hydrolysis upon contact with water, and the hydrolyzed products lose their ability as chemiluminescence sensitizer. Consequently, organic solutions, such as acetonitrile or acetone, are requir ed in the peroxyoxalate chemiluminescence system. However, the aqueous peroxyoxalate chemiluminescence was achieved by combining it with FIA and delivering oxalate solution via a separate flow line.138 The idea of coupling FIA was first introduced by Rule and Seitz,139 and since then has been used widely. It affords rapid and reproducible mixing as well as reliable emi ssion measurement, and allows for sample throughput. The commonly used oxalates (peroxyoxalate chemiluminescence reagents) include bis(2,4,6-trichlorophenyl) oxalate (TCPO), bis-(2 ,4,dinitrophenyl) oxalate (DNPO) and 1, 1 oxalyldiimidazole (ODI) (Figure 1-40). The reactivities of peroxyoxalate chemiluminescence reagents are pH-dependent, and TC PO demonstrates the greatest intensity at neutral pH (~7.5).140 TCPO and DNPO feature higher stability a nd solubility over ODI, but they are less reactive.141As a result, a weak base is generally empl oyed as a catalyst to increase the reaction

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74 rate in TCPO chemiluminescence or DNPO chem iluminescence system. Compared with other amine bases, imidazole (ImH) was reported to be th e most efficient catalyst and have the greatest effect on improvement of peroxyoxalate chemiluminescence yield.142,143 Figure 1-40. Structures of commonly used oxalates. Sensor Application of Peroxyoxalte Chemiluminescence In the past three decades, peroxyoxalate chem iluminescence reaction has been proven to be a useful, selective and se nsitive detection method fo r sensing various species.144 It has been used to directly detect H2O2, fluorophores (e.g., polycyclic aromat ic hydrocarbons) or compounds that are fluorescent derivatized usi ng dansyl chloride (e.g., ami no acids, carboxylic acids and aliphatic amines). Moreover, it has also been appl ied as a sensitive sensor to indirectly determine the substrates following their conversion to H2O2 by enzymatic reactions. In the latter application, a combination of peroxyoxalate chemiluminescence and FIA/LC is generally applied. By immobilizing an appropriate enzy me which is usually substrate-corresponding oxidase, this method has accurately detected a number of substrat es including glucose,138 uric acid,145 amino acids,146 glutamic acid,147 choline (or choline-contaning phospholipids)148 and polyamines.149 Taking the detection of glucose as an example, we will discuss in more detail below. By using immobilized glucose oxidase to ca talyze the oxidation of glucose and produce H2O2, Williams et al first introduced the idea of de tecting enzymatically-generated H2O2 by peroxyoxalate chemiluminescence in 1976.150 Base on this work, Nakashima and co-workers

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75 utilized the same pair of substrates and en zyme and developed a TCPO chemiluminescencebased glucose assay with 10 nM fluorophore.151 A linear calibration grap h was obtained up to 1.5 -4 M of glucose solution and the detection lim it was reported to be 10 nM at the ratio of chemiluminescence intensity for sample and blank (S/B) of 3. Figure 1-41 illustrates the chemiluminescence signal as a function of reactio n time at different concentration of glucose oxidase. Nakashima and co-workers later combin ed this assay with FIA and measured the glucose or uric acid in serum.145 More recently, Irgum and co-workers have developed an aqueous-compatible ODI chemiluminescence system to detect glucose and acetylcholine/choline by employing immobilized enzyme reactor and coupling ODI chemiluminescence with FIA and LC.138 Little effect on the chemiluminescence reacti on was observed by the aqueous solution that was used to dissolve glucose and delivered to th e reaction cell through a separate analyte flow line. Favorable detection limits were obtained, i. e. 3 nM for glucose and 50 nM for acetylcholine and choline either in aqueous buffer solution or in urine sample. The low detection limits are believed to arise from the inherent sensitivity of peroxyoxalate chemiluminescence technique because of eliminating the light source and background noise. Figure 1-41. The time course for GOD reactio n detected by TCPO chemiluminescence when GOD is ( ) 5; ( ) 20; ( ) 30; ( ) 40 units in the reaction mixture. Reprinted with permission from Nakashima et al .151

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76 Scope of Our Study The aim of this research is to investigate optical sensor applica tions of functionalized poly( para-phenylene ethynylene) (PPE ), design and develop PPE-based fluorescent and chemiluminescent assays for various enzymes. Chapter 2 4 describes real-time fluorescent assays for phospholipase C (PLC), alkaline phosphase (ALP) and adenylate kinase (ADK), resp ectively using two PPE derivatives, biphenyl poly(phenylene ethynylene) sulfonate (BpPPE-SO3 -) and poly(phenylene ethynylene) carboxylate (PPE-CO2 -). These assays were designed based on different sensor mechanisms. For example, the PLC assay relied on the confor mation and aggregation changes of BpPPE-SO3 induced by the PLC substrate, phospholipid. Whereas the ALP and ADK a ssays were based on superquenching-unquenching cycles of the fluorescence of PPE-CO2 which resulted from to the availability of quenchers in the proximity of polymer. The PPE-based homogeneous enzyme assay provided a rapid, convenient and continuous method to accurately detect the kinetic activity of the enzymes and evaluate the performan ce of the activators and i nhibitors. In addition, by applying micromolar range of substrates, the assays were found to be very sensitive and selective over the non-specific control proteins. In Chapter 5, in order to expand the PPE-bas ed optical sensor to the chemiluminescence field, the first chemiluminescence system for a water soluble CPE was developed based on the imidazole-catalyzed TCPO chemiluminescence reaction. The effects on chemiluminescence signals of concentrations of oxalate, catalyst, oxidant and fluorophore as well as solvent composition (water and acetonitrile) were demonstrated to be consistent with the kinetics of peroxyoxalate chemiluminescence reaction. Studies of chemiluminescence quenching of BpPPESO3 and PPE-CO2 by small opposite charged molecules displayed amplified chemiluminescence quenching with comparable quenching efficiency of photoluminescence.

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77 The chemiluminescence of CPE was successfully a pplied as a biosensor to qualitatively and quantitatively detect the activity of peptidase and ALP.

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78 CHAPTER 2 PHOSPHOLIPASE C ASSAY USING BIPH ENYL POLY(PHENYLENE ETHYNYLENE) SULFONATE Introduction Phospholipids are naturally occu rring amphiphiles which serve as the major component of biological membranes. Phospholipase C (PLC) catal yzes the hydrolysis of the phosphate ester in a phospholipid selectively at the glycerol side yielding a diacylglycerol (DAG) and a phosphatecontaining head group.152 The ability to quantitatively m onitor PLC catalytic activity and inhibition is important as DAGs play a critical role in cell function and the signal transduction cascade in mammalian systems.153-156 Several PLC assays have been developed based on turbidimetric,157,158 pH-stat titration,159 radiometric,160,161 and continuous fluorometric162-171 methods. The turbidimetric and titration assays suffer from low sensitivity and the inherent disadvantage that larg e quantities of enzyme and substrate are required. Radiom etric assay attains the lowest detection limit for PLC that has been reported. For example, most recently, De lfino and co-workers developed a subnanogram radiometric assay for PLC based on a longchain radioiodinatabl e phosphatidylchline, BHC12PC.160 As shown in Figure 2-1A, BHC12PC ha s a 4-hydroxyphenyl group attached at the end of the fatty acyl chain located in position sn -2, which enables this phospholipid to be radioiodinated. Figure 2-1B illustrates the linea r dependence of the enzymatic activity on the PLC mass. It is clear that the assa y is very sensitive with ability to detect as low as 0.05 ng PLC. However, in the radiometric assays, 3H-,172 14C-,173 32P-174 or 125I-160 labeled phospholipids are required as substrates making the approach expensive, laborious and time-consuming. Fluorometric assays based on th e determination of choline165 or inorganic phosphate164 offer high sensitivity and are continuous. Although they ar e advantageous when carrying out studies on

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79 enzyme kinetics,163 most of the fluorometric assays requi re synthetic fluorogenic substrates, which results in low catalytic turnover of enzymes and reduced reaction rates.165,166 Figure 2-1. Results of radiom etric assay for PLC based on BH C12PC. A) Structure of the radioiodinatable long-chain phosphatidylchloine, BHC12P C. B) Dependence of the measured activity with the amount of PL C. Inset shows extended ranges for the assays. Reprinted with permission from Caramelo et al .160 In this chapter, we introduce a CPE-based sensitive and specific fluorescent turn-off assay for PLC with natural phospholipid as substrate. The earlier studies of CPE-surfactant complexes provide the basis for th e study presented herein,52,53 where we explore the interactions between an anionic CPE and phospholipids. We demonstrat e that phospholipids inte ract strongly with CPEs, eliciting significant change s in the fluorescence properties of the polymer. The effects are reversible and consequently the CPE-lipid comple x provides a platform for the development of a

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80 fluorescence turn-off assay for the lipase enzyme PL C. In particular, the fluorescence of the CPE is enhanced and blue shifted upon complexation with phosphatidylcholin e. Incubation of the polymer-phospholipid complex with PLC results in a decrease of the fluorescence which is due to the enzyme catalyzed hydrolysis of the phospholipid. The PLC enzyme assay conditions are optimized, and the effects of th e addition of an activator (Ca2+) and several inhibitors of PLC are studied. The assay is calibrated for substrate concentration, allowing the determination of the catalytic kinetic parameters, Km and Vmax. Results and Discussion Overview of PLC Turn-off Assay The structure of the conjugated polyelect rolyte used in the assay, BpPPE-SO3 -, is shown in Figure 2-2A. The synthesis and char acterization of this polymer is described in the experimental section. Previous studies on structura lly-similar anionic PPE-type CPEs, PPE-SO3 (structure shown in Figure 1-7), have shown that in water these polymers exist as aggregates. The characteristic feature of the aggregated state of CPEs is that the fluorescence emission appears as a broad, structureless band that is Stokes shifted significantly from the absorption band. Like the previously studied CPEs, in water BpPPE-SO3 exists in an aggregated state, as is clearly signaled by the broad, structurel ess fluorescence band that is Stoke s shifted from the absorption band ( max flr = 503 nm and max abs = 419 nm). The phosphatidylcholine, 1,2-didecanoylsn glycero-3-phosphocholine (10CPC) was chosen as the natural subs trate for PLC. As shown in Figure 2-2A, PLC catalyzes the hydrolysis of 10CPC to generate DAG and phosphorycholine. The cartoon shown in Figure 2-2B illustrates the mechanism of the PLC turn-off assay. Although 10CPC is a zwitterion, electrostatic (ion-dipole) and hydrophobic forces induce the formation of a polymer-lipid comple x between the phospholipid and BpPPE-SO3 -. As a result, the backbone of BpPPE-SO3 is more extended and aggregatio n of the polymer is reduced. The

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81 lipid-induced changes in polymer conformation and aggregation state are signaled by a blue-shift and enhancement in the polymers fluorescence intensity. Figure 2-2. A) Structur es of polymer, BpPPE-SO3 and substrate, 10CPC, and reaction scheme for hydrolysis of 10CPC by PLC. B) Mech anism and illustration of PLC turn-off assay. Photographs of solutions illuminated with near UV light illustrate the polymer fluorescence under the different condition of the assay. Reprinted with permission from Liu et al .175 Introduction of PLC to the polymer-lipid comp lex induces hydrolysis of 10CPC. The PLC catalyzes hydrolysis of the zwitterionic head group from the hydrophobic tail, disrupting the amphiphilic structure of the lipid which is a key factor allowing 10CPC to complex with the

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82 polymer. One of the 10CPC hydrolysis products, DAG, is hydrophobic and charge neutral while the second product, phosphorylcholin e, has a net negative charge. Apparently neither of these species causes the spectra changes of the polymer associated with electrostatic interaction and induced modified aggregati on state. As a result, the fluorescence of BpPPE-SO3 reverts to its original (aggregated) state, in which the spectrum is red shifted and less intense. As shown by the photograph in Figure 2-2B, the changes in BpPPE-SO3 fluorescence that accompany the assay are easily observed by eye. After additi on of 10CPC to the solution of BpPPE-SO3 the fluorescence is considerably bright er (compare middle vial with le ft vial), and the subsequent addition of PLC causes the fluorescence intensity to decrease again (right vial). The 10CPC/PLC assay is rendered quantita tive by applying a calibration, which relates the BpPPE-SO3 fluorescence intensity to 10CPC concentrati on (see Figure 2-5B and Equation 2-1 in experimental section). This allows one to dete rmine the enzyme reaction kinetics from changes in polymer fluorescence intensity. Effect of 10CPC on Fluorescence of BpPPE-SO3 A series of titrations was carried out to quantify the effects 10CPC and polymer concentration on the polymers absorption and fluorescence. U pon addition of 10CPC to an aqueous BpPPE-SO3 solution at room temperature, the ab sorption of the polymer blue shifts by only 2 nm without a significant change in the shape of the absorption band (Figure 2-3). However, as shown in Figure 2-4A, the fluorescence spectrum changes dramatically. In particular, addition of 10CPC (0 15 M) to a solution of BpPPE-SO3 (c = 1 M) induces a blue shift of the fluorescence maximum from 503 nm to 436 nm combined with a 50-fold increase in the fluorescence in tensity at 436 nm. Th e significant change of the fluorescence intensity suggests that 10CPC inhibits the aggreg ation of the polymer as has been reported for the addition of other surfactants to CPEs.53

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83 Figure 2-3. Normalized absorption of 1 M BpPPE-SO3 before () and after ( ) addition of 8 M 10CPC in water at 25 C. Repr inted with permission from Liu et al .175 Figure 2-4B illustrates the plots of the relative fluorescence intensity at 436 nm as a function of concentration of added 10CPC at different BpPPE-SO3 concentrations. For each plot, the fluorescence intensity increases gradually at low 10C PC concentration. After the concentration of 10CPC reaches a certain point, the intensity incr eases sharply and linearly until reaching a plateau, at which point further addition of 10CPC causes little additional change in the intensity. (The plot for 5 M BpPPE-SO3 in Figure 2-4B only shows the effect of added 10CPC below the concentration range where the sharp in crease is observed.) Note that as the BpPPESO3 concentration increases, more lipid is need ed to induce the fluorescence change. This is consistent with the involvement of a polyme r-lipid complex in the observed fluorescence intensity enhancement. Importantly, over the concentration range in which the fluorescence intensity increases sharply, the relati ve increase in fluorescence intensity (I / Ip) is nearly linearly proportional to 10CPC concentration. This sugge sts that is should be possible to create a calibration curve which relates fluorescence intensity to 10CPC concentration.

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84 Figure 2-4. Fluorescence changes upon titration of 10CPC into BpPPE-SO3 -. A) Fluorescence spectroscopic changes of a solution of 1 M BpPPE-SO3 in water observed upon addition of 10CPC at 25 C, ex = 400 nm. B) Fluorescence intensity increase at 436 nm upon titration of 10CPC at diffe rent concentration of BpPPE-SO3 in water, ex = 400 nm. Reprinted with permission from Liu et al .175

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85 Prior to developing a calibrati on allowing one to follow the 10 CPC substrate concentration during PLC hydrolysis, a study was carried out to determine the solution conditions under which the PLC activity was optimized, and yet the poly mers response to 10CPC was still acceptable. This survey demonstrated that the optimum so lution conditions include the use of Tris-HCl buffer (c = 50 mM, pH 7.4) along with added Ca2+ as an activator for the PLC.152 Figure 2-5A shows the effect of 10CPC on the fluorescence of 15 M BpPPE-SO3 in 50 mM Tris-HCl with 2 mM Ca2+ at 37 C. Note that under thes e solution conditions upon addition of 10CPC the fluorescence of BpPPE-SO3 is blue shifted and enhanced to a lesser extent in comparison to the change in fluorescence for the polymer seen in pure water (see Figure 2-4A). This difference arises because Ca2+ induces aggregation of BpPPE-SO3 -,11,51 and this effect partially offsets the ability of 10CPC to influence the extent of BpPPE-SO3 -aggregation. Despite the fact that under these solution conditions more 10CPC is needed to elicit a strong fluorescence response, at [BpPPE-SO3 -] = 15 M, a 10-fold fluorescence enhancement is observed upon addition of 100 M of 10CPC. In particular, Figure 2-5B illustrates the increase in fluorescence intensity at 460 nm ( ex = 400 nm) as a function of added 10C PC. This plot features a similar trend as that shown in Fi gure 2-4B, with the BpPPE-SO3 exhibiting a significan t and nearly liner intensity increase until reachi ng a plateau for [10CPC] > 250 M. The inset in Figure 2-5B shows a calibration plot of the rela tive fluorescence intensity increase ( I / Ip) as a function of [10CPC]. The plot features a good linear rela tionship over the concentration range 0 100 M with a y-intercept of 1, a nd consequently the [10CPC] is directly proportional to ( I / Ip 1) at any time during a PLC hydrolysis reactio n. This calibration is used to derive Equation 2-1 which is used to determine [10CPC]t during the PLC assay (see experimental section).

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86 Figure 2-5. Fluorescence change s upon titration of 10C PC into BpPPE-SO3buffer solution. A) Fluorescence spectroscopic changes for a solution of BpPPE-SO3 (15 M) observed upon addition of 10CPC in 50 mM Tr is-HCl (pH 7.4) with 2 mM Ca2+ at 37 C, ex = 400 nm. B) Fluorescence intensity incr ease at 460 nm upon titration of 10CPC, ex = 400 nm. Inset: Calibration plot of relati ve fluorescence intensity increase as a function of 10CPC concentration. Reprinted with permission from Liu et al .175

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87 PLC Turn-off Assay In order to demonstrate the feasibility of using BpPPE-SO3 as the basis for a PLC turn-off assay, we initially examined the effect of added 10CPC and PLC on the polymers fluorescence intensity in water without added buffer or Ca2+. The results of this ini tial experiment are shown in Figure 2-6. In particular, the initial fluorescence from a solution BpPPE-SO3 (c = 1 M) is blue-shifted and signifi cantly enhanced in intensity by addition of 10 M 10CPC (change indicated by arrow 1). After introduction of PLC (c = 2.3 nM) to the solution, hydrolysis of 10CPC causes a red shift of fl uorescence band and a decrease in intensity with increasing incubation time (indicated by arrow 2). The BpPPE-SO3 -fluorescence decreases ~50% within one minute even in the unbuffererd solution and without added Ca2+ activator, which demonstrates the potential for the PLC turn-off assay. Figure 2-6. Fluorescence spectroscopic change s observed in the PLC turn-off assay. () Initial fluorescence of 1.0 M BpPPE-SO3 in water at 25 C; ( ) fluorescence after step (1): addition of 10 M 10CPC; fluorescence intensity as a function of time after step (2) : addition of 2.3 nM PLC and incubate for 1 ( ), 5 ( ), 20 ( ) and 45 () min, ex = 400 nm. Reprinted with permission from Liu et al .175

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88 In order to optimize the conditions fo r PLC assay, the effect of the Ca2+ activator was investigated when assays were conducted in 50 mM Tris-HCl buffer (pH 7.4). Calcium ion serves as an activator for PLC because Ca2+ interacts with the substr ate providing charge density which enhances binding of th e enzyme with the substrate.176 The dependence of the initial enzyme catalyzed rate ( v0) on [Ca2+] is shown in Figure 2-7 (see calculation of initial rate of reaction in experimental secti on). The experiment was conduct ed with a solution containing BpPPE-SO3 (c = 15 M), 10CPC (cinitial = 80 M) and PLC (c = 20 nM). Note that v0 increases almost linearly until it reaches a maximum rate when [Ca2+] = 2 mM and at higher concentrations v0 decreases. The observed dependence of v0 on [Ca2+] is similar to that observed in previous kinetic st udies of PLC activity.168,177 Figure 2-7. Effect of con centration of activator, [Ca2+] on the initial rate of hydrolysis ( v0) of 10CPC by PLC. Experiment conditions: 15 M BpPPE-SO3 -, 80 M initial 10CPC and 20 nM PLC in 50 mM Tris-HCl (pH 7.4) at 37 C, ex = 400 nm. Reprinted with permission from Liu et al .175

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89 Figure 2-8. A) Changes in fluorescence emission intensity ( Itc) at 460 nm during the PLC turnoff assay as a function of reaction time for various concentrations of PLC: 0 (), 5 ( ), 10 ( ), 25 ( ) and 50 () nM. Itc was corrected for photobleaching by blank fluorescence intensity Ibt (see appendix). Experiment conditions: 15 M BpPPE-SO3 and 30 M initial 10CPC in 50 mM Tris-HCl (pH 7.4) with 2 mM Ca2+ at 37 C, ex = 400 nm. B) Dependence of initial rate of reaction ( v0) on PLC concentration. Reprinted with permission from Liu et al .175

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90 In a series of investigations, we examined the kinetics of the PLC catalyzed hydrolysis of 10CPC at varying PLC concentration. These real -time kinetic assays were carried out with a solution containing BpPPE-SO3 (c = 15 M) and 10CPC (cinitial = 30 M) in Tris-HCl buffer (50 mM, pH 7.4) in the presence of 2 mM Ca2+ at 37 C. Figure 2-8A illustrates plots of the BpPPESO3 fluorescence intensity monitore d at 460 nm as a function of time for solutions containing PLC at concentrations ranging fr om 0 50 nM. Note that the ra te of decrease in the BpPPE-SO3 fluorescence intensity increases as the concentrat ion of PLC increases. Figure 2-8B illustrates a plot of v0 as a function of PLC concentration for the ra nge 0 75 nM, and it is evident from this presentation that the initial catalyzed reaction ra te varies linearly with enzyme concentration. The analytical detection limit for PLC is 0.5 nM (22 ng-mL-1, 6.6 10-4 unit-mL-1, one unit is defined as the amount of enzyme hydrolyzing 1.0 mole of 10CPC per min at pH 7.4 at 37 C) and was obtained from the calibration curve in Fi gure 2-8B. This sensitivity is comparable to values obtained by using different assays.178,179 An interesting observation is that the BpPPE-SO3 -/10CPC/PLC solution becomes turbid as the reaction proceeds when solutions containing 35 M 10CPC undergo PLC-catalyzed hydrolysis. Under these conditions, at longer reaction time a green ish-yellow precipitate forms and the fluorescence of the polymer decreases shar ply. This observation suggests that the product diacylglyceride (DAG) produced by PLC-catalyzed hydrolysis of 10CPC co-precipitates with BpPPE-SO3 and this effect interferes with the sensor response. In order to prove that DAG is the origin of the precipitation, its effect on the assay was investigated. Thus, various concentrations of DAG were added to a solution containing BpPPE-SO3 and 10CPC (c = 15 M and 50 M, respectively). It was found that DAG dissolves with a negligible effect on the fluorescence intensity when added at lo w concentration (c < 10 M). However, with increasing DAG

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91 concentration, the fluorescence intensity gradua lly decreases followed by a co-precipitation of the DAG/polymer complex at higher concentration of DAG (c > 30 M). The precipitation results in a significant decrease in the fluorescen ce intensity. Therefore quantitative analyses, including the calculations for initial rate of r eaction and determination of kinetic parameters, were conducted at DAG concentration less than 5 M. Determination of PLC Catalyzed 10CPC Hydrolysis Kinetic Parameters The BpPPE-SO3 based fluorescence assay was used to determine the kinetic parameters ( Km and Vmax) for the PLC catalyzed hydrolysis of 10CPC The kinetics experiments were carried out using solutions containing BpPPE-SO3 (c = 15 M), PLC (c = 20 nM), Ca2+ (c = 2 mM) and Tris-HCl buffer (50 mM, pH 7.4) at 37 C. The in itial concentration of 10CPC was varied from 0 90 M, which is the range corres ponding to the linear range in the calibration curve shown in Figure 2-5B (inset), and the v0 values were obtaine d and plotted as a function of [10CPC] as shown in Figure 2-9. Using a non-linear regression routine, the plot of v0 vs. [10CPC] was fitted with the Michaelis-Menten equation (see Equation 2-2 in the experimental section). The fit yields values for Km (Michaelis-Menten constant) and Vmax (maximum rate of enzymatic reaction) of 28 3 M and 19 0.7 mol-min-1-mg-1, respectively. Unfortunately, there are no other reports of the values for Km and Vmax with 10CPC as substrate. However, using natural egg lecithin (a mixture of several different phospholipid s including phosphatidylcholine), Km was reported to be 16 or 28 M and Vmax was 68 mol-min-1-mg-1 by monitoring the hydrolysis kinetics by phosphorus determination or by fluorometric method w ith dye-labeled lec ithin as substrate.168,180 This comparison shows that the Km value obtained from the BpPPE-SO3 --based fluorescence turn-off assay is in good agreement with values obtained using other assays; however the Vmax value is

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92 smaller. It is likely that the lower Vmax arises due to complex form ation between 10CPC and the polymer, which effectively decreases the binding of the lipid to PLC. Figure 2-9. Dependence of initial rates of reaction ( v0) on substrate concentration [10CPC] ranging from 0 and 90 M. Vmax and Km values were derived by nonlinear regression of these data. 15 M BpPPE-SO3 and 20 nM PLC in 50 mM Tris-HCl (pH 7.4) with 2 mM Ca2+ at 37 C, ex = 400 nm. Reprinted with permission from Liu et al .175 Inhibition of the PLC Catalysis In order to further demonstrate that the obs erved fluorescence intensity decrease that is induced by addition of PLC to the BpPPE-SO3 -/10CPC solution arises due to PLC catalyzed hydrolysis of the 10CPC, we tested the effect of known PLC inhibitors on the fluorescence assay. Two reported inhibitors for PLC from Clostridium perfringens are fluoride ion181,182 and EDTA.166,183 Both of these species were tested as i nhibitors in assays conducted under the same conditions used for the kinetics studies descri bed above using 10CPC at a concentration of 50 M. As shown in Figure 2-10, both fluoride ion and EDTA effectively inhibit the PLC activity. While the overall inhibition increases with incr easing inhibitor concentration, the inhibition efficiency is largest at low inhibitor concentration. Nonetheless, the inhibition experiments

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93 provide very strong evid ence that the assay is effectivel y reporting on the PLC catalyzed hydrolysis reaction. Figure 2-10. Inhibition of PLC tu rn-off assay. Plot illustrates the initial rate of reaction (v0) versus inhibitor concentration: F(), EDTA (). Experiment conditions: 15 M BpPPE-SO3 -, 50 M initial 10CPC and 20 nM PLC in 50 mM Tris-HCl (pH 7.4) with 2 mM Ca2+ at 37 C, ex = 400 nm. Reprinted with permission from Liu et al .175 Specificity of the PLC Turn-off Assay Given that an effective biosensor should exhib it specificity for the target enzyme, it is of interest to examine the response of the BpPPE-SO3 -/10CPC based PLC assa y to other proteins. In order to accomplish this objective, five proteins were selected, including phospholipase A2 (PLA2), phospholipase D (PLD), peptidases (PEP, a commercial mixture of protease), bovine serum albumin (BSA) and avidin (AVI). Three of these enzymes, PLC, PLA2 and PLD belong to the group of phospholipases. As shown in Figure 2-11, PLA2 catalyzes the hydrol ysis of an acyl ester bond exclusively at the 2-acyl position in the glycerophospholipid affording a free fatty acid and a lysophospholipid.184 The lysophospholipid ha s a surfactant-like st ructure, containing a single hydrophobic carbon chain and a polar head-group. Thus, like 10CPC, the

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94 lysophospholipid produced by PLA2 hydrolysis of 10CPC is expect ed to complex strongly with the polymer preventing aggregation and quenching of the fluorescence. On the other hand, PLD catalyzes hydrolysis of the phosphate ester bond on the choline side to form choline and phosphatidic acid (Figure 2-11).185 Although phosphatidic acid al so has a surfactant-like structure, its overall negativ e charge might prevent it from interacting with BpPPE-SO3 -. In addition, it is reported that 20 100 mM of Ca2+ is required for maximum activity of PLD, along with a pH of 5.6.186 Therefore PLD might not achieve its full activity in the optimal conditions for PLC turn-off assay (2 mM Ca2+ and pH 7.4). Figure 2-11. Hydrolysis of phospholipid at different positions catalyzed by PLA2, PLC and PLD. Reprinted with permission from Liu et al .175 The assays to study the specificity were carried out with the same conditions used for the kinetics studies described above, with 50 M 10CPC. In each case PLC was not added, but an aliquot containing the other protei n added in to achieve a final concentration of 20 nM (except for PEP, which was added to ach ieve a concentration of 0.86 g-mL-1). Figure 2-12 compares the fluorescence intensity changes (460 nm) observe d 2 min after addition of the protein aliquot to the BpPPE-SO3 -/10CPC/buffer solution. As expect ed, the assay with PLC exhibits ca. 70% decrease in fluorescence intensity, while the solu tions containing the control proteins exhibit less than an 8% decrease in intensity. Th is result demonstrates that the BpPPE-SO3 assay is specific for PLC. However, note th at a small, but significant decr ease in fluorescence intensity was observed upon addition of PLA2, PLD and PEP to the BpPPE-SO3 -/10CPC/buffer solution,

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95 suggesting the existence of a non-specific intera ction between the proteins and the BpPPE-SO3 -. Although such interac tion exists, a negligible decrease of fluorescence intensity (< 0.5%) is observed after 2 min incubation of a blank PLC assay (20 nM PLC) in which no substrate, 10CPC, is added. Therefore, the non-specific effect s are very small in comp arison to the effect of specific enzymatic hydrolysis of 10CPC by PLC. Thus, it is safe to conclude that the overall interaction between PLC and polymer/phosphatidylc holine complex consists mainly of specific enzymatic activity as well as very small contribution from non-specific interaction. Figure 2-12. Specificity of PLC turn-off assay. Changes in fluorescence emission intensity at 460 nm following 2 min incubation with PL C and other five control proteins. Experiment conditions: 15 M BpPPE-SO3 -, 50 M initial 10CPC and 20 nM PLC or control proteins (except for 0.86g/mL-1 PEP) in 50 mM Tris-HCl (pH 7.4) with 2 mM Ca2+ at 37 C, ex = 400 nm. Reprinted with permission from Liu et al .175 Discussion. The assay described herein affords the ability to monitor PLC activity rapidly and in realtime. In addition the method is qui te sensitive, both with respect to the amount of enzyme and substrate required (< 1 nM and 5 M, respectively). The method can also be readily adapted to a fluorescence-based high -throughput screening (HTS) assay format.68

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96 A comprehensive literature survey reveals that many assays for monitoring the enzymatic activity of PLC from Clostridium perfringens have been previously reported.69,158-160,162,163,168,171 The previously reported assays are based on different detection methods including turbidimetric, pH-stat titration, radiometric and continuous fluorometric methods. Concerning the sensitivity, the best detection limit of 0.005 units-mL-1 for PLC from Clostridium perfringens was achieved by using an ELISA assay.178 A second assay that is based on the acid-soluble phosphorus method affords a detection limit of 70 ng-mL-1 for PLC (M.W. 43 kDa).179 The BpPPE-SO3 --based PLC assay that is described herein clea rly affords sensitivity that is comp arable to (or be tter than) that of the earlier reports. With respect to the amount of lipid substrate needed for the assay, substrate concentrations in the millimolar range ar e required in the previously reported PLC assays.166,168,171,178-180,182,183,187 By contrast, our method not only al lows an assay to be carried out at significantly lower initial substrate concentrati on, but also affords the ability to carry out realtime detection of the enzyme activity. In addi tion, surfactants such as sodium deoxycholate (SDC) have been frequently used in the presence of Ca2+ ions to disperse water insoluble phospholipids so as to enhance the rate of reaction and leading to an effective increase the PLC activity.152,166,179,183 However, the addition of a surfactant does not afford any improvement in PLC activity if a water soluble phospholipid is the substrate.166 The effect of SDC was also studied for the BpPPE-SO3 -/10CPC/PLC assay and it was found that the polymers fluorescence response was suppressed in the presence of the surfact ant. The likely explanation for this effect is that the surfactant influences the fluorescence intensity change by comp lexing with the polymer (just like 10CPC), eff ectively counteracting the effect induced by hydrolysis of 10CPC. Nonetheless, since the formation of the polymer/lip id complex facilitates the solubilization of the lipid in water, addition of a surfactant such as SDC is not required for our assay.

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97 While the BpPPE-SO3 -/10CPC/PLC assay is relatively easy to implement and has many advantages, nonetheless the method still has some disadvantages. For example, the sensitivity of the assay is affected by various experimental conditions, such as buffer concentration, temperature and/or polymer concentration. In ad dition, non-specific interactions with various proteins and other solutes (e.g., metal ions, lipids or surfactants, etc.) a nd the polymer (and lipid substrate) could interfere with the sensor response, especially if quantitative (kinetic) data is needed. As a result, when applying this assay w ith real biological samples, such as serum sample, it might need to purify the PLC from th e samples to get rid of effects of non-specific interactions induced by different salts, proteins or other biologi cal components. Finally, the PLC turn-off assay is limited by one of hydrolysis pr oducts, DAG, which leads to a precipitation of polymer at higher concentration (c > 30 M). Experimental Materials All substrates, enzymes and proteins were purchased from Sigma-Aldrich and used as received, unless otherwise noted. Calcium chlo ride and ethylenediamine tetraacetic acid, disodium salt dihydrate (EDTA) were purchased from Fisher Chemical. Sodium fluoride was obtained from Mallinckrodt Chemical Works and 1,2-didecanoylsn -glycerol (DAG), was obtained from Cayman Chemical. All solvents were obtained from Fisher and used without further purification. Water was distilled and pur ified by using a Millipor e purification system. Instrumentation UV-Visible absorption spectra were obtained on a Perkin Elmer Lambda 25 UV/VIS spectrophotometer, with a scan rate of 960 nm-min-1. Fluorescence spectra were recorded on a Jobin Yvon-SPEX Industries Fluor olog-3 Model FL3-21 spectrofl uorometer and corrected by using correction factors generate d in-house with a primary standard lamp. The 1-cm fluorescence

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98 cuvette was placed in a custom-built thermostatte d cell holder which was maintained at 37 C during the assay and was e quipped with a micro-submer sible magnetic stirrer. General Methods Solution preparation Buffer solutions (pH 7.4) were prepared with Tris base and hydrochloric acid. A concentrated aqueous solution of BpPPE-SO3 was diluted with buffer solution to a final concentration ranging from 0.1 M to 15 M. The stock solutions of substrates, enzymes and proteins were prepared immedi ately before their use in the fluorescence assay. The enzyme substrate, 1,2-didecanoyl-sn -glycero-3-phosphocholine (10CPC), was dissolved in methanol and adjusted to 20 mM as stock con centration. Phospholipase C from Clostridium perfringens (C. welchii) (PLC) was dissolved in 50 mM Tris-H Cl buffer solution and adjusted to 10 M as stock concentration, and the assays were conducted in the same buffer. Control enzymes and proteins include phospholipase A2 from bovine pancreas (PLA2), phospholipase D from Arachis hypogaea (peanut) (PLD), bovine seru m albumin (BSA), avidin from egg white (AVI), and peptidase from porcine intestinal mucosa (PEP). They were used in place of phospholipase C in control experiments. Calcium chloride (1.0 M), s odium fluoride (0.2 M) a nd EDTA (0.2 M) were dissolved in water as stock solu tions. Sodium deoxycholate (SDC), was dissolved in methanol as 0.2 M stock solution. Fluorescence turn-off assay procedure The PLC enzyme assays were carried out at 37 C in a fluorescence cuvette with continuous stirring. For kinetics studies the fluorescen ce intensity was measured with excitation and emission wavelengths of 400 nm and 460 nm, respectively. A typical assay procedure was carried out as follows. First, a 2.0 mL aliquot of the BpPPE-SO3 solution was allowed to thermally equilibrate at 37 C and then the initial polymer fluorescence intensity ( Ip) at 460 nm

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99 was measured. The substrate (10CPC) was adde d to a second 2.0 mL aliquot of BpPPE-SO3 solution, this mixture was incubated for 15 min and then the solution was placed in the spectrophotometer and the fluorescence intensity ( Ibt) as a function of time was recorded as a blank. (Note that the blank signal Ibt decreased slightly with time due to photobleaching of the polymer, see appendix for more detail.) Another freshly prepared 2.0 mL aliquot of BpPPE-SO3 -/10CPC solution was incubated at 37 C, and then it was quickly pipetted into a cuvette containing a 4L aliquot of the enzyme solution, and the fluorescence intensity of the BpPPESO3 -/10CPC/PLC solution was monito red as a function of time (It). Subsequently, the sample fluorescence intensity It was corrected for photobleaching by using the blank intensity Ibt (see correction procedures and valida tion in appendix). The sample and blank fluorescence intensity ( Ibt and It, respectively) were measured using th e same conditions. After correction for photobleaching, the correct ed fluorescence intensity Itc at each time t was derived. For full wavelength-spectral scans of th e assays, the fluorescence intensity ( Ip, or It) vs. wavelength profiles were recorded with excita tion wavelength at 400 nm. Because fresh assay solution was used for spectral measurements at each time t, it was not necessary to correct for photobleaching. Calculation of initial rate of reaction ( v0) Itc was converted to substr ate concentration [10CPC]t as a function of time by using following Equation 2-1 which is de rived from the calibration plot of fluorescence intensity ratio ( I / Ip) vs. concentration of substr ate [10CPC] (Figure 2-5B). tc p t0 0c p1 [10CPC][10CPC] 1 I I I I (2-1)

PAGE 100

100 where, [10CPC]0 is the initial substrate concentration, [10CPC]t is the substrate concentration at time t, Ip is the fluorescence intensity of the polym er solution before addition of substrate, I0c is the initial corrected fluorescence intensity at t = 0, that is, the fluorescence intensity after addition of substrate but before the addition of enzyme and Itc is the corrected fluorescence intensity at time t after enzyme addition. A plot of [10CPC]t vs. time was then derived and the v0 was calculated from the slope of the plot by usin g data for the region where hydrolysis of 10CPC is less than 5 M. Calculation of kinetic parameters In the single-substrate enzymatic reactions, Michaelis-Menten equation188 is used to derive kinetic parameters and expressed as max0 0 m0[S] [S] V v K (2-2) where v0 is initial rate of reaction, [S]0 is the initial substrate concentration ([10CPC]0), Vmax is the maximum rate of enzymatic reac tion with saturated substrate and Km is Michaelis-Menten constant, that is, the substrate concentration at which the rate of the enzyme reaction is half Vmax. After a plot of v0 vs. [10CPC]0 was fitted with the MichaelisMenten equation using a non-linear regression routine, the value of kinetic parameters, Km and Vmax, were obtained. Synthetic Procedures of BpPPE-SO3 Sodium 3,3'-(2,5-diiodo-1,4-phenylene)bis(oxy) dipropane-1-sulfonate (0.975 g, 1.5 mmol) and 4,4'-diethynylbiphenyl (0.189 g, 1.5 mmol) were dissolved in a mixture of 30 mL of water, 30 ml of DMF and 10 ml of diisopropylamine at 55 C in a Schlenk flask. The resulting clear solution was deoxygenated by bubbling argon through the solution for 15 minutes. Then 52 mg of Pd(PPh3)4 and 10 mg of CuI were added under the pr otection of Argon. Upon the addition, the clear solution became turbid. The mixture was stirred under a positive pressure of Argon for 18

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101 hours. The viscous solution was poured into 700 mL of acetone, the polymer precipitated as yellow fibers. The polymer was re dissolved in 70 mL of DMSO and treated with 0.2 g NaCN. Then the resulting solution was filtrated through 2.5 m glass fiber filter and the filtrate was poured into another 700 mL of acetone. The polym er was collected by vacuum filtration and further purified by two repeated cycles of disso lution in DMSO and precipitation into a large volume of acetone. Finally, the polymer was dissolv ed in 80 mL of D.I. water and dialyzed against a large volume of water (Millipore Si mplicity water system) using a 12 KD MWCO regenerated cellulose membrane (F isher Scientific). After dialysis the solution was filter through a 2.5 m glass fiber membrane and the concentra tion of the aqueous solution was calibrated using gravimetric analysis and the polymer was stor ed in this format and diluted as appropriate for spectroscopic studies. 1H NMR (DMSO-d6, ppm from TMS, 80 C): 2.10 (t, 4H), 2.66-2.71 (m, 4H), 4.18 (br, t, 4H), 7.15 (s, 2H), 7.66-7.78 (br, 8H). FTIR ( max, KBr pellet): 2944, 2874, 2200, 1644, 1506, 1415, 1278, 1213, 1035, 953, 858, 823, 737, 667, 614, 529. [ ]DMSO, 20C = 1.39 dL/g (Figure 2-13) Figure 2-13. Reduced viscosity ( sp/c) vs. concentration (c) for BpPPE-SO3 in 0.1 M LiBr/DMSO at 20 C. Reprinte d with permission from Liu et al .175

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102 CHAPTER 3 ALKALINE PHOSPHATASE USING POLY(PHENYLENE ETHYNYLENE) CARBOXYLATE Introduction Alkaline phosphatase (ALP) is one of the mo st commonly assayed enzymes in clinical practice, because an abnormal level of serum alkaline phosphatase is an important index of several diseases such as bone disease (ri ckets, Pagets disease and osteomalacia),189 liver dysfunction (hepatitis and obstructive jaundice),190 breast and prostatic cancer,191 and diabetes.192 As a membrane bound enzyme found in a variety of tissues (intestine, liver, bone, kidney and placenta) of nearly all living organisms, ALP has broad substrate specificity and is capable of catalyzing hydrolysis or tran sphosphorylation of a wide variety of phosphate compounds in vitro .193 Among the many substrates for ALP, pyr ophosphate (PPi) is unique because it has a lower pH optimum compared to other substrates.193 Even though hydrolysis of PPi catalyzed by ALP is reported to be relatively slow,194,195 it is related to an impor tant role ALP plays during skeletal mineralization and vascular calcificat ion. ALP facilitates mineral precipitation and growth by hydrolyzing PPi, a poten t mineralization inhibitor, wh ile concomitantly producing phosphate (Pi) which is availabl e for deposition as hydroxyapatite.196,197 ALP also prevents excess vascular calcification by adjusting the le vel of PPi, the inhibitor of medial vascular calcification in vitro198 and in vivo ,199 through the same process. Therefore, a sensitive and continuous assay of ALP using PPi as substrate at physiological condition is of interest and offers a possibility to study the role of this enzyme in vivo A number of ALP assays that use different substrates are commercially available. The structure of some reported ALP subs trates are shown in Figure 3-1. P -nitrophenylphosphate ( pNPP) is a chromogenic substrate that serves as the basis for ALP colorimetric assays,200,201 while 4-methylumbellyferyl phosphate (4-MUP) is a substrate for a fl uorometric assay that

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103 operates at high pH.202,203 In addition, chemiluminescen ce assay using derivative of benzothiazolyl-5-phosphate (BTZP) as substrate,204 and electrochemical immunoassay using sodium salt of ferrocene ethyl phosphate ester (FcEtOPO3Na2) as substrate205 have also been used to determine the activity of ALP. Figure 32 illustrates the reaction progress curves in the electrochemical immunoassay, wh ich was employed for the indire ct detection of ALP down to 2 pM. Each of these systems offer high sensitivity and can be used in a continuous assay; however they are not able to assay the activity of ALP with PPi as substrate. The hydrolysis of PPi by ALP was first measured by a radiometric assay with 32P as tracer.206 But the high cost and hazard associated with the use of the radiolabeled substrate impedes its application. Currently the hydrolysis of PPi is typically assayed by c onverting the liberated Pi to chromogenic phosphomolybdate which is determined colorimetrically.207 However, this method is not very sensitive, it is time-consuming, and it cannot be used as a continuous (real-time) assay. To date, there has not been a report of a real-time assay for ALP using PPi as substrate. Figure 3-1. Structures of literature reported ALP substrates.

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104 Figure 3-2. Reaction progress curves for ALP at an electrode immersed in Tris buffer solution containing 10 M FcEtOPO3Na2 and different ALP concentrations from 3.7 pM to 230 pM. Reprinted with permission from Limoges et al .205 In this chapter, we describe a CPE-based continuous and sensitive fluorescent turn-off assay for ALP with natural PPi as substrate. This assay was designed based on a fluorescence turn-on sensor for PPi using PPE-CO2 -(structure shown in Figur e 1-10) as the signal transducer.208 In particular, the cupric ion (Cu2+) efficiently quenches the fluorescence of PPECO2 and the quenched fluorescence is recovered by addition of PPi. The fl uorescence recovery by PPi is based on the strong association of PPi with Cu2+. As a result, the polymer-metal complex is disrupted, leading to the recovery of the fluorescence intensity. Importantly, as shown in Figure 3-3, this fluorescence recovery response is very selective to PPi compared with 11 other control anions, in cluding inorganic Pi (H2PO4 and HPO4 2-) which is the only hydrolysis product of PPi catalyzed by ALP. Therefore, with th e introduction of ALP into a solution of PPE-CO2 -/Cu2+/PPi, PPi is hydrolyzed into Pi and the Cu2+ that is released quenches the

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105 fluorescence of PPE-CO2 -. By applying an ex-situ calibration, the decrease of the fluorescence intensity is quantitatively related to the decrease of [PPi] as a function of incubation time, thus realizing a real-time monitor of enzyme activity. The assay is carried out at physiological pH, provides flexibility of either end-point or re al-time measurement, allows determination of enzyme kinetic parameters and inhibition effici ency, and shows a relativ ely good specificity to ALP. By taking advantage of the intrinsic fl uorescence signal amplification of the CPE, a detection limit of ~5.0 nM of AL P is achieved, which is better than other ALP assays using PPi as substrate.206,207 Figure 3-3. Recovery response of Cu2+-quenched fluorescence of PPE-CO2 by PPi and 11 control anions. 5 M PPE-CO2 -/10 M Cu2+ with variousanions at 50 M concentration in 10 mM HEPES buffer at pH 7.5. (1, F-; 2, Br-; 3, I-; 4, HPO4 2-; 5, H2PO4 -; 6, P2O7 4-; 7, CH3CO2 -; 8, HSO4 -; 9, NO3 -; 10, HCO3 -; 11, SO4 2-; 12, CO3 2-). Inset shows a photograph of the sensor/ani on solutions illuminated with a UV-lamp. Reprinted with permission from Zhao et al .208

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106 Results and Discussion Overview of ALP Turn-off Assay The anionic CPE featuring car boxylate side groups, PPE-CO2 (structure shown in Figure 1-10) was selected as the signaling element in th e ALP assay. The synthesis and characterization of this polymer is described in the experimental section. In 10 mM Tr is-HCl buffer solution (pH 7.5), PPE-CO2 exhibits a broad fluores cence band with a maximum at 525 nm. On the basis of earlier work,30 it is clear from the appearance of the fluorescence that the polymer is aggregated in the aqueous buffer solution. The cartoon in Figure 3-4 illustrates the sequence and mechanism of the ALP turn-off assay, which is based on a fluorescence quench-unque nch-quench process. First, the fluorescence of PPE-CO2 is quenched by addition of an aliquot of the divalent ion Cu2+. The quenching likely occurs via a charge and/or energy transfer mechanism.209,210 Next, the fluorescence is recovered by addition of PPi to the PPE-CO2 -/Cu2+ solution. Fluorescence recovery occurs because PPi chelates Cu2+, effectively sequestering the ion so th at it is no longer complexed with the polymer. Finally, addition of ALP to the solution containing PPE-CO2 -/Cu2+/PPi initiates hydrolysis of PPi to Pi. The product, Pi, whic h exists as an equilibrium mixture of HPO4 2and H2PO4 in the buffered solution (pH 7.5), is unable to complex with Cu2+. Consequently the free Cu2+ again complexes with PPE-CO2 -, leading to quenching of the polymers fluorescence.208 As the ALP catalyzed hydrolysis reaction proceeds, th e amount of PPi available for association with Cu2+ decreases, and therefore the PPE-CO2 fluorescence intensity de creases with time. The photographs in Figure 3-4 clearly show the changes in PPE-CO2 fluorescence accompanying the assay. The substrate concentration, [PPi], is de termined as a function of time in the enzymatic reaction (see Equation 3-1 in e xperimental section), thereby a llowing kinetic investigation and inhibition study of ALP.

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107 Figure 3-4. Mechanism and illustration of AL P turn-off assay. Photographs of solutions illuminated with near UV light illustrate the polymer fluorescence under the different condition of the assay. Reprinted with permission from Liu et al .211 In the present study Tris-HCl buffer was used instead of HEPES, which was the buffer system used in a preliminary communication on the PPE-CO2 -/Cu2+/PPi ALP sensor.208 The TrisHCl buffer was selected for this study because pr eliminary results showed that ALP displays a 60-fold higher activity in Tris-HCl buffer compared to that ob tained in HEPES buffer under the same conditions. The higher ALP activity seen in Tris-HCl buffer is believed to arise because the Tris cation complexes and sequesters the Pi pr oduct which is a competitive inhibitor of ALP.193 We will describe the Pi inhi bition more thoroughly below. Magnesium ion, Mg2+, is reported to stimulate the monoesterase activity but almost completely inhibits PPi-ase activity of ALP.212 It was found that the addition of Mg2+ into solutions used in the present work does lead to a decrease of ALP activity even at very low concentration of Mg2+ (e.g. 10 M). Therefore, Mg2+ was not added to the solutions used in the work presented below.

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108 Fluorescence Quenching of PPE-CO2 by Cu2+ The interaction of carboxylate-substituted CPEs with metal ions has b een investigated in depth in a number of previous studies.51,77,213,214 In our earlier communica tion we reported that in HEPES buffer solution the fluorescence of PPE-CO2 is selectively quenched by Cu2+ compared to nine other divalent metal ions, including Ca2+, Mn2+ and Co2+.208 The fluorescence quenching mechanism is suggested to be singlet exciton qu enching via charge and/or energy-transfer when Cu2+ coordinates with carboxylate group(s) of the CPE chains.209,210 Figure 3-5. Fluorescence spectroscopic changes observed upon titration of Cu2+ into a solution of 3 M PPE-CO2 in 10 mM Tris-HCl buffer (pH 7.5) at 37 C, ex = 390 nm. Inset: Stern-Volmer plot of PPE-CO2 fluorescence quenching by Cu2+. Reprinted with permission from Liu et al .211 As shown in Figure 3-5, titration of Cu2+ (c = 0 300 M) into a Tris-HCl buffer (10 mM, pH 7.5) solution of 3 M PPE-CO2 results in quenching of the polymer fluorescence intensity. The Stern-Volmer plot ( Ii/ Iq vs. [Cu2+], Ii and Iq are the fluorescence intensities of PPE-CO2 before and after addition of Cu2+, respecitively) for the quenching da ta is shown in the inset; as

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109 seen for other CPE/quencher systems, the plot is nearly linear at low quenc her concentration, but it is curved upward at higher quencher con centrations. The Stern-Volmer constant ( KSV) derived from the linear region of the correlation is ca. 3.5 104 M-1, which is 30-fold lower than the value observed for Cu2+ quenching of PPE-CO2 in HEPES buffer ( KSV 106 M-1).208 The decreased quenching efficiency observed here is due to interaction of Tris base with Cu2+; apparently Tris is able to complex Cu2+ resulting in a shift of the equilibrium away from its binding with the polymer. However, the need to use Tris buffer as an acceptor for Pi to reduce its inhibition of the ALP activity leads us to co mpromise between higher ALP activity and lower quenching efficiency. Fluorescence Recovery of PPE-CO2 -/Cu2+ by PPi Recovery of the Cu2+-quenched fluorescence of PPE-CO2 by PPi occurs due to complex formation between PPi and the metal ion. The PPi-Cu2+ complex does not bind to PPE-CO2 -, and consequently the fluorescence from the polymer is recovered. This effect is demonstrated by a series of titrations in which PPi was added to Tris-HCl bu ffer solutions containing PPE-CO2 and Cu2+. Specifically, Figure 3-6A illustrates the fluo rescence spectra of solutions that contain 3 M PPE-CO2 and 200 M Cu2+ in Tris-HCl buffer (10 mM, pH 7. 5) with [PPi] ranging from 0 to 300 M. The red spectrum in Figure 3-6A shows th e fluorescence of the initial (quenched) PPECO2 -/Cu2+ solution, where the intensity at 525 nm is quenched 95% relative to fluorescence of the (unquenched) pure PPE-CO2 -. As can be seen from the figure, addition of PPi induces a significant increase in the polymers fluorescence intensity. When [PPi] = 300 M, the fluorescence is recovered to 86% of the initial intensity of the unquenched polymer, and relative to the initial solution containing PPE-CO2 and Cu2+ the fluorescence intensity at 525 nm is increased 26-fold.

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110 Figure 3-6. Fluorescence changes upon tit ration of PPi into solution of PPE-CO2 and Cu2+. A) Fluorescence spectroscopic changes of 3 M PPE-CO2 -/200 M Cu2+ solution observed upon titration of PPi B) Fluorescence recovery, Ir/ Iq, at 525 nm of 3 M PPE-CO2 -/Cu2+ solution titrated with PPi at different concentratison of Cu2+ in 10 mM Tris-HCl buffer (pH 7.5) at 37 C, ex = 390 nm. Reprinted with permission from Liu et al .211 A more detailed series of titrations was car ried out wherein the fluorescence of PPE-CO2 -/Cu2+ solutions was studied as a function of added [PPi], for five different [Cu2+] ranging from

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111 25 200 M. As shown in Figure 3-6B, plots of the fluorescence recovery ratio ( Ir/Iq, Iq and Ir are fluorescence intensities of PPE-CO2 -/Cu2+ before and after addition of PPi) as a function of the PPi to Cu2+ concentration ratio ([PPi]/[Cu2+]) at different c oncentration of Cu2+ display a sigmoidal shape. Interestingly, regardless of the [Cu2+], in each case 50% of the fluorescence recovery occurs at same ratio of [PPi] and [Cu2+]. Figure 3-7. Logarithm of fluores cence recovery efficiency, Log ( Ir/ Iq), at 525 nm of 3 M PPECO2 -/Cu2+ solution titrated with PPi at different [Cu2+] in 10 mM Tris-HCl buffer (pH 7.5) at 37 C, ex = 390 nm. Reprinted with permission from Liu et al .211 In order to obtain an approximate linear corre lation that can be used as a calibration curve to relate the change in fluorescence intensity to [PPi], fluorescence intensity data was plotted as shown in Figure 3-7, which shows correlations of Log( Ir/Iq) vs. [PPi]/[Cu2+] for five different [Cu2+]. Each of the plots exhibits an approximately linear increase in Log( Ir/Iq) with [PPi] until the end point is reached ([PPi]/[Cu2+] = 1) at which point the plots level off.

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112 Figure 3-8. Calibration curve for PPE-CO2 fluorescence recovery indu ced by addition of PPi. Conditions: solution contains 3 M PPE-CO2 -, 200 M Cu2+ and 10 mM Tris-HCl buffer (pH 7.5) at 37 C, ex= 390 nm, em= 525 nm. Reprinted with permission from Liu et al .211 Plots of Log( Ir/Iq) vs. [PPi] can be used as calibra tion curves, providing the basis for calculation of [PPi] from the chan ge in fluorescence intensity as shown in Equation 3-1 in the experimental section. For example, Figur e 3-8 illustrates a ca libration plot (Log (Ir/ Iq) vs. [PPi]) for a solution that contains 3 M PPE-CO2 -/200 M Cu2+ over a [PPi] range of 0 225 M. A linear least squares fit of the data passes through the origin and affords a correlation coefficient R2 = 0.99, indicating a reasonable fit. Note that the applicability of Equation 3-1 for determining the [PPi] during the enzyme assays is independent of [Cu2+]. However, the range of [PPi] over which the calibration curve is linear varies with [Cu2+], and the range over which the calibration is linear is 0 < [PPi]/[Cu2+] < 1. In view of this fact, the ra nge of PPi (the enzyme substrate)

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113 concentration that can be studied is adjustable by varying the [Cu2+] in the sensor solution. The necessary condition is that the initial [ PPi] must be less than or equal to [Cu2+]. Another important point is that the fluorescence sensor response increases with [Cu2+]. Keeping all of these variables in mind, in the course of the de tailed investigation of AL P activity sensing, we selected [Cu2+] = 200 M as the quencher concentration as a reasonable compromise between sensor response and the [PPi] needed to produce a response. From this point onward in the text, we will refer to a solution containing 3 M PPE-CO2 -, 200 M Cu2+ and 10 mM Tris-HCl buffer (pH 7.5) as the standard assay solution used in the ALP assays. ALP Turn-off Assay Several control experiments were carried out be fore the ALP assays were examined. First, the effects that addition of substrate, PPi, and enzyme, ALP, have on the fluorescence of solutions of PPE-CO2 were explored and these tests reveal ed that these species elicited little effect when added individually to the polymer so lution. Next, the effect that addition of the hydrolysis products, Pi (PO4 3-, HPO4 2and H2PO4 -), have on the fluorescence of the quenched (PPE-CO2 -/Cu2+) or recovered (PPE-CO2 -/Cu2+/PPi) assay solutions was studied. In each case, addition of Pi up to a concentra tion of 2 mM had little effect on the fluorescence intensity. These control experiments rule out th e possibility that the fluorescence changes observed during the ALP assay arise due to interference by the various components added to the solution. Introduction of ALP into solutions that contain PPE-CO2 -, Cu2+ and PPi initiates hydrolysis of PPi and induces a concomitant decrease of the polymers fluorescence intensity. Figure 3-9A shows typical fluorescence spectroscopic chan ges observed during an end-point ALP assay carried out in the standard assay solution at 37 C. First, the initial fluorescence from 3 M PPECO2 is quenched significan tly by the addition of 200 M Cu2+ (indicated by arrow 1). Second, the quenched fluorescence is recove red to ~70% of the initial in tensity by the addition of 150 M

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114 PPi to the solution (indicated by arrow 2). Th ird, following addition of 100 nM ALP to the solution, hydrolysis of PPi is signaled by a decr ease of the fluorescence intensity with increasing incubation time (indicated by arrow 3). The decrea se in the fluorescence arises because free Cu2+ is produced as a result of the ALP-catalyzed PPi hydrolysis, and the free Cu2+ complexes with PPE-CO2 resulting in quenching of the fluorescen ce. The fluorescence intensity decreased to ~17% of the initial value after 120 minutes of incubation. The fluorescence bandshape does not vary much during the assay, suggesting that the changes arise primarily due to the quenchunquench-quench mechanism caused by the ch ange in the concentration of free Cu2+ in the solution. In order to demonstrate the f easibility of using the PPE-CO2 -/Cu2+/PPi -based fluorescence sensor as a real-time assay for ALP activity, we studied the ALP-catalyzed hydrolysis of PPi as a function of time at several different enzyme concentrations. These real-time assays were conducted with 150 M PPi in the standard assay solution at 37 C. Figure 3-9B illustrates the decrease of fluorescence intensity at 525 nm for solutions with ni ne different ALP concentrations ranging from 0 300 nM, where the fluorescence intensity was measured every 10 sec as a function of time. (Note that the time-dependent fl uorescence intensity values shown in this figure have been corrected for photobleachi ng by measuring the blank intensity Ibt, as described in experimental section and appendi x.) As expected, the reaction ra te increases with increasing [ALP], and the fluorescence intensity decreases approximately linearly with time for [ALP] < 50 nM, indicating that under these conditions the reaction rate is constant over the time range studied. However, for [ALP] 50 nM the reaction rate decreases with increasing time, and this effect becomes more pronounced at higher enzyme concentration. We believe that the decrease in reaction rate with time is caused by Pi product inhibition.193

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115 Figure 3-9. Fluorescence changes observed in the ALP turn-off assay. A) Fluorescence spectroscopic changes observed in an endpoint ALP turn-off assay. () Initial fluorescence of 3 M PPE-CO2 in 10 mM Tris-HCl buffer (pH 7.5) at 37 C, ex = 390 nm; ( ) fluorescence after step (1): addition of 200 M Cu2+; ( ) fluorescence after step (2): addition of 150 M PPi; fluorescence intensity as a function of time after step (3): addition of 100 nM ALP: 1 ( ), 5 (), 15 ( ), 40 ( ), 60( ), 120 () minutes. B) Decrease of fluorescence intensity at 525 nm recorded every 10 sec during the real-time ALP turn-off assays with varying c oncentration of ALP. PPi (150 M) in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. Reprinted with permission from Liu et al .211

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116 In order to determine the initial ra te of the ALP-catalyzed reaction (v0), the calibration defined by Equation 3-1 (shown in experimental section) was applied to the time-varying fluorescence data, affording plots of the substrate concentration [PPi]t as a function of time as shown in Figure 3-10. These plots are linear at early time, and the slopes of the plots were computed to afford values of v0 at the nine different ALP concen trations investigated. As shown in Figure 3-11, v0 is directly proportional to [ALP] in the range of 0 300 nM, which indicates that the reaction is kinetically controlled by ALP. This experiment also demonstrates that it is possible to use the PPE-CO2 -/Cu2+/PPi assay to detect ALP activity in less than one minute, even at very low [ALP]. Figure 3-10. Decrease of [PPi] during the re al-time ALP turn-off assays with varying concentration of ALP. 150 M PPi in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. Reprinted with permission from Liu et al .211 An analytical detection limit of ~5.0 nM ALP is obtained from the calibration curve plotted at low [ALP] (see inset, Figure 3-11). Even though the sens itivity may not be as high as

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117 in assays with more reactive substrates such as pNPP195 or 4-MUP203, or other assay formats such as chemiluminescence204 or electrochemical method205, the sensitivity is considerably better than that of other reported assays that use PPi as the substrate.206,207 Figure 3-11. Dependence of initial rate of enzymatic reaction ( v0) on ALP concentration. Inset: Plot of v0 vs. [ALP] at lower concen tration of ALP. PPi (150 M) in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. Reprinted with permission from Liu et al .211 Determination of ALP Catalyzed PPi Hydrolysis Kinetic Parameters To investigate the suitability of the ALP turn-off assay for kinetic study, Figure 3-10 was converted to Figure 3-12, which illustrates a plot of Ln[PPi]t vs. time for seven [ALP]. As described above, in these expe riments [ALP] was varied from 10 to 300 nM and the initial substrate concentration ([PPi]0) was constant at 150 M. The kinetic parameter, Vmax/ Km, was derived from the slope of each plot in Figure 3-12, and the resulting parameters are plotted as a func tion of [ALP] in the inset to Figure 3-12. It is

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118 evident that Vmax/ Km varies linearly with enzyme concentration (Equation 3-3). The slope of this linear plot affords a value for the specificity constant, kcat/ Km = 3.13 103 M-1s-1, which is in very good agreement with the range of kcat/ Km (1.6 102 ~ 8.9 104 M-1s-1) obtained by a discontinuous assay using PPi as substrat e but using ALP from a different source.215 This analysis demonstrates that it is possible to use the PPE-CO2 -/Cu2+/PPi assay to rapidly detect ALP activity in real-time and that the assay affords the ability to quantitatively recover the enzyme kinetic parameters. Figure 3-12. Natural logarithm of concentrati on of PPi as a function of incubation time for various ALP concentrations. Inset: Dependence of kinetic parameters ( Vmax/ Km) on ALP concentration. PPi (150 M) in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. Reprinted with permission from Liu et al .211 In a second set of experiments, a series of assays was carried out in which the [ALP] was maintained at 100 nM, but the PPi substrate conc entration was varied over the range of 80 200

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119 M. (Note that this [PPi] is less than the millimolar range of Km values reported in the literature.193,216-218) The standard assay solution was used in these experiments. The Equation 3-1 (shown in experimental section) calibration was used to conver t raw fluorescence intensity data to [PPi]t, and these values were further convert ed to the product concentration, [Pi]t, by using the reaction stoichiometry, PPi 2Pi. Figure 3-13 illustrates the resulting plot of [Pi]t vs. time at six initial substrate concentrations [PPi]0. This presentation clearly shows that the reaction rate increases with [PPi]0, as expected when [PPi]0 is less than Km. The difference between the signals observed at the different [PPi]0 values is easily resolved in less than 1 minute of reaction time. Nevertheless, it is seen that the error in the signal becomes noticeable when [PPi]0 < 80 M. Figure 3-13. Concentrations of hydrolyzed produc t, Pi, as a function of time at various initial PPi concentrations with 100 nM ALP in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. Reprinted with permission from Liu et al .211

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120 Inhibition of the ALP Catalysis The active site in ALP is re latively unselective: it binds to the phosphate unit in the substrate, regardless of the remainder of the subs trate structure. Conseque ntly, any substrate that contains the phosphate group can bind to the ALP active site acting as a competitive inhibitor for the reaction of interest. As not ed above, in the ALP-catalyzed PPi hydrolysis reactions the reaction rate slows with increas ing reaction time suggesting that phosphate (Pi), which is the product of hydrolysis of PPi, is acting as a competitive inhibitor of th e enzyme. In order to explore this effect more fully, a series of assays were conducte d with increasing [Pi], using the standard assay solution with 100 nM ALP and initial [PPi] concentrations of 150 M and 200 M. Figure 3-14. Inhibition of ALP turn-off assay by Pi. Reciprocal of initial reaction rate (1/v0) as a function of Pi concentration for two PPi s ubstrate concentrations and derivation of Ki from intersection point. Assay conditions: PPi (150 or 200 M) and 100 nM ALP in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. Reprinted with permission from Liu et al .211

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121 Figure 3-14 shows the Dixon plot219 of Equation 3-4 (shown in experimental section) which exhibits two linear curves of the reciprocal of the initial reaction rate, 1/ v0 vs. [Pi] for each [PPi]. The intersection points of thes e two liner plots affords a value of Ki = 21.2 M, which is comparable to the value obtained by using 4-MU P as substrate and monitoring the enzymatic hydrolysis by fluorometric method (16.5 M).212 This inhibition study dem onstrates that ALP is very sensitive to Pi inhibition, and it underscores the need for us e of an amine buffer such as Tris-HCl rather than phosphate buffer in the re action medium. Furthermore, it demonstrates that the decrease in reaction velocity observed durin g the ALP catalyzed PPi hydrolysis reaction arises due to product inhibition of the enzyme. Specificity of the ALP Turn-off Assay In order to test the response of the PPE-CO2 -/Cu2+/PPi assay to other pr oteins, a series of experiments were carried out using ALP and six control pr oteins including phospholipase A2 (PLA2), glucose oxidase (GOx), peroxidase from horseradish (HRP), peptidase (PEP), bovine serum albumin (BSA) and avidin ( AVI). None of the control prot eins has a specific interaction with PPi. In these experiments, 200 M Cu2+ and 150 M PPi in the standard assay solution was assayed with 100 nM of ALP or one of the above control proteins (for PE P the concentration was 16 g-mL-1). As shown in Figure 3-15, in contrast to th e behavior seen for ALP where there is a continuous decrease in PPE-CO2 fluorescence following addition of the enzyme, addition of the control proteins induces a rapi d decrease in the polymers fluorescence intensity (< 5 minutes). Following this initial change, the fluorescence intensity remains constant. The decrease in the intensity upon addition of the prot ein to the assay solution likely arises from non-specific binding between the protein and PPE-CO2 or the Cu2+/PPi complex, disturbi ng the equilibria which control the polymers fluorescence intensity.58

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122 Figure 3-15. Changes in fluorescence emission in tensity at 525 nm as a function of incubation time after addition of 100 nM ALP, PLA2, GOx, HRP, AVI, BSA, or 16 g-mL-1 PEP. 150 M PPi in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. Reprinted with permission from Liu et al .211 Figure 3-16 compares the overall response of th e assay solution after a 60 min incubation period in the presence of the di fferent proteins. The assay soluti on that contains ALP exhibits greater than a 60% decrease in fluorescence intensity at 525 nm following 60 min incubation. By contrast, solutions containing the different control proteins exhi bit in the range of 4% ~ 25% decrease in fluorescence intensity; importantly this change occurs within the first 5 minutes of incubation due to non-specific interactions descri bed above. Note that th e non-specific effects are most pronounced when the control protein is charged. For example BSA, which is negatively charged at neutral pH and is capable of binding to Cu2+220 and PPi,221 exhibits nearly the greatest non-specific effect on the PPE-CO2 fluorescence intensity. Note that despite the fact that significant non-specific effects are observed some of these control expe riments, a negligible

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123 decrease (< 0.5%) in fluorescence intensity is obse rved in the first 5 min when ALP is added to a solution that contains PPE-CO2 and Cu2+, but no PPi substrate. Therefore, it is safe to conclude that the decrease of fluorescence intensity noted in the ALP turn-off assay arises from the specific enzymatic activity of ALP. Figure 3-16. Specificity of AL P turn-off assay. Different changes in fluorescence emission intensity at 525 nm after 60 minutes of incubation of 100 nM ALP, PLA2, GOx, HRP, BSA AVI, or 16 g-mL-1 PEP. PPi (150 M) in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. Reprinted with permission from Liu et al .211 Discussion By taking the advantage of the selective response of the PPE-CO2 --Cu2+ complex to PPi, we have developed a novel fluorescence turn-off assay for ALP catalyzed PPi hydrolysis. To the best of our knowledge, this is the first report of a real-tim e assay for ALP activity using its physiological substrate, PPi. Compared with assays that use other synthetic substrates, the ALP assay with PPi as a substrate offers the possi bility to develop a di agnostic ALP assay for pathological conditions. The prev iously reported ALP assay usi ng PPi as a substrate was based on periodic measurement of [P i] by converting the liberated Pi to a chromogenic phosphate

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124 derivative.207 Compared with this assay, the PPE-CO2 --based ALP turn-off assay exhibits high sensitivity, high selectivity and most importa ntly, flexibility of end-point and real-time measurements to easily study enzyme activity and inhibition. The assay can be carried out with initial substrate conc entration in the 100 M range, which is much lower than the reported Km. 193,216-218 Moreover, the assay is easy to implemen t, and it provides the ability to quantify enzyme kinetic and inhibition parameters. Im portantly, the method is based on fluorescence intensity and therefore it is directly amenable to being us ed in a high-throughput screening (HTS) format using a mu lti-well plate reader.68 While the PPE-CO2 --based ALP turn-off assay has many advantages, nonetheless it has some disadvantages. The influence of non-specific proteins on the fluorescence intensity of the PPE-CO2 -/Cu2+/PPi system represents a possible source of interference if th e assay is to be carried out in complex biological milieu. In general, proteins that have high charge density are likely to interact with PPE-CO2 -, Cu2+ and/or PPi, and therefore are likely to exhibit non-specific effects on the fluorescence intens ity of the sensor system. A second problem is that the fluorescence signal from the assay is low when the [PPi] is much lower than the [Cu2+] ([PPi]/[Cu2+] < 0.6). This feature can be overcome by carefully optimizing the relative concentrations of the assay components as approp riate for the needs of th e assay. Moreover, the experiments described in this paper provide valuable guidance concerning the effects of varying the concentrations of the various a ssay components. Finally, the PPE-CO2 --based assay can only be used with PPi as a substrate, since it relies specifically on the ability of PPi to complex with Cu2+. Other phosphate-containing substrates that are unable to recover the Cu2+ quenched fluorescence of the polymer, such as pNPP and 6-glucose phosphate, cannot be used as ALP substrates with this assay.

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125 Experimental Materials All stock solutions were prepared with water th at was distilled and th en purified by using a Millipore purification syst em. Tris-HCl buffer solution (10 mM, pH 7.5) was prepared with Tris base and hydrochloric acid. An aqueous stock solution of PPE-CO2 was diluted with Tris-HCl buffer solution to a final concentration of 3 M. All chemicals were used as received, unless otherwise noted. Coppe r (II) chloride (CuCl2), was obtained from Sigma-Aldrich, sodium pyrophosphate (Na4P2O7), was purchased from J. T. Baker Chemical Company and sodium phosphate tribasic (Na3PO4), was obtained from Fisher Scientif ic. Each reagent was dissolved in water and the concentration of the stock solutions was adjusted to 50 mM. Alkaline phosphatase from bovine intestinal mucosa (ALP) and the control proteins were purchased from SigmaAldrich. The six control pr oteins include phospholipase A2 from bovine pancreas (PLA2), glucose oxidase from Aspergillus niger (GOx), peroxidase from horseradish Type I (HRP), peptidase from porcine intestinal mucosa (PEP), bovine serum albumin (BSA) and avidin from egg white (AVI). All of the enzymes and proteins were dissolved in Tris-HCl buffer solution and adjusted to 20 M except for PEP with 3.2 mg-mL-1 as stock concentration. The enzyme and protein stock solutions were prepared immediat ely before their use in the fluorescence assays. Instrumentation Fluorescence spectra were re corded on a spectrofluorometer from Photon Technology International and corrected by using correction factors generated with a primary standard lamp. The 1-cm fluorescence cuvette was placed in a cu stom-built thermostatted cell holder which was maintained at 37 C during the assay and wa s equipped with a micro-submersible magnetic stirrer.

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126 General Methods Fluorescence turn-off assay procedure The enzyme assays were carried out in 10 mM Tris-HCl buffer (pH 7.5) at 37 C. For the real-time assays, the fluorescence intensity was recorded with excitation and emission wavelengths of 390 nm and 525 nm, respectively. All measurements were conducted with stirred solutions. A typical procedure was carried out as follows: First, a 2.0-mL aliquot of the PPECO2 solution was placed in the cuvette and allowed to thermally equilibrat e at 37 C. The initial polymer fluorescence intensity ( Ii) at 525 nm was recorded. The Cu2+ solution was then added, the solution was incubated for 15 minutes, and the quenched fluorescence intensity ( Iq) at 525 nm was measured. Subsequently, the PPi solution was introduced to another 2.0-mL aliquot of PPE-CO2 -/Cu2+ solution, this mixture was incubated fo r 15 minutes, and the solution was placed in the spectrophotometer, and the fluorescence intensity ( Ibt) as a function of time was recorded every 10 sec as a blank. The signal Ibt decreased with time due to photobleaching of polymer (see appendix for more detail). Another freshly prepared 2.0-mL aliquot of PPE-CO2 -/Cu2+/PPi solution was incubated at 37 C, and then it wa s quickly pipetted into a cuvette containing microliter range aliquot of the ALP solution and the fluorescence intensity ( It) of the solution was measured every 10 sec as a function of time. The signal It was corrected for photobleaching by using the blank intensity Ibt at each time t as described in a previous report (see correction procedures and validation in appendix). Th e assay and blank fluorescence intensity ( Ibt and It, respectively) were recorded under identical c onditions. After correction for photobleaching, the corrected fluorescence intensity Itc at each time t was derived, which is used for the calculation of initial rate of enzymatic reaction. For monitoring the assays in an end-point format, the fluorescence intensity ( Ii, or It) versus wavelength profiles were recorded with excitation wavelength at 390 nm. Because fresh

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127 assay solution was used for measurements at each time t, there was no need to correct for photobleaching. Calculation of initial rate of reaction ( v0) The corrected fluorescence intensity, Itc, was converted to substr ate concentration [PPi]t at 10 sec intervals during the assay by using follo wing Equation 3-1 which is derived from the calibration which relates the fluorescence intens ity to [PPi] (Figure 3-7B and Figure 3-8). tc q t0 0c q() [PPi][PPi] () I Log I I Log I (3-1) where [PPi]0 is the initial substrate concentration, [PPi]t is the substrate concentration at time t, Iq is the fluorescence intensity of the PPE-CO2 solution after addition of Cu2+, I0c is the initial corrected fluorescence intensity at t = 0 (i.e., the fluorescence intensity after addition of substrate PPi but before addition of the enzyme), and Itc is the corrected fluores cence intensity at time t after enzyme addition. A plot of [PPi]t vs. time was then derived and v0 was calculated from the slope of the plot by using the da ta from the region where [PPi]t is a linear function of time. Calculation of kinetic a nd inhibition parameters As mentioned in Chapter 2, Michaelis-Menten equation (Equation 2-2) is generally used to derive kinetic parameters in the single-substrate enzymatic reaction. However, the substrate (PPi) concentration used in our ALP turn-off assay is much lower than the reported Km of ALP with PPi as substrate,193,216-218 so the Michaelis-Menten equation reduces to cat 000 m()[E][S] k v K (3-2) where [E]0 is the initial enzyme concentration and kcat is catalytic constant or turnover number, and defined as dividing Vmax with [E]0.219 kcat/ Km is called as specificity constant and determines

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128 the relative rate of reaction at low substrate c oncentration. In order to derived the specificity constant of ALP turn-off assay, E quation 3-3 which relates the ratio Vmax/ Km with [E]0 was derived based on definition of kcat ( kcat = Vmax / [E]0) maxcat 0 mm()[E] Vk KK (3-3) By using Equation 3-3, a linear curve of Vmax/ Km vs. [E]0 was plotted and kcat/ Km was calculated from the slope of the curve. The inhibition of the ALP catalysis by the comp etitive inhibitor, Pi, was studied. As shown in Equation 3-4, a modified Michaelis-Menten equa tion in the presence of a competitive inhibitor was used to calculate the inhibition parameters where [I] is the inhibitor concentration and Ki is the inhibition constant. The value of Ki was obtained using Dixon plot219 which is the graphical presentation of Equation 3-4 in a linear form. max0 m0 i[S] [I] (1)[S]0V v K K (3-4) Synthetic Procedures of PPE-CO2 PPE-CO2C12H25 A solution of 2,5-bis(dodecyloxycarbonyl methoxy)-1,4-diiodobenzene (204 mg, 0.25 mmol) and 1,4-diethynylbenzene (32 mg, 0.25 mmol) in 10 mL of dry THF/Et3N (v:v = 2:1) was outgassed with argon for 15 minutes. Then 8.7 mg of Pd(PPh3)4 (7.5 mol) and 1.4 mg of CuI (7.5 mol) were added under argon. Th e reaction was stirred at 60 C for 18 hours. The obtained viscous suspension was poured into 150 mL of me thanol, resulting in the precipitation of the ester precursor polymer as light yellow fibers. The polymer was fu rther purified by two repeated cycles of dissolution in THF and precipi tation in methanol. Yield: 135 mg (79%). 1H NMR (CDCl3): = 0.88 (br, t, 3H), 1.25 (br, m, 36H), 1.68 (b r, m, 4H), 4.24 (br, m, 4H), 4.74 (br, s,

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129 4H), 7.02 (br, s, 2H), 7.56 (br, s, 4H). GPC (THF, polysty rene standards): Mn 127 kg-mol-1, Mw 286 kg-mol-1 (PDI = 2.25). PPE-CO2 To a solution of 135 mg of PPE-CO2C12H25 (0.20 mmol) in 30 mL of dioxane/THF (v:v = 5:1) was added 1.5 mL of 1 M (n-Bu)4N+OHin methanol, and the resulting mixture was stirred at room temperature for 24 hours. During the course of the hydrolysis reaction, 2 mL of water were systematically added in order to keep the solution clear. Then a solution of 0.20 g of NaClO4 (1.6 mmol) in 3 mL of water was added to the hydrolyzed polymer solution, and the resulting mixture was poured into 400 mL of co ld acetone, resulting in the precipitation of PPECO2 as a fine yellow powder. The polymer was then dissolved in 50 mL of deionized water and the material was purified by dialysis into deionized water using a regenerated cellulose membrane (12 kD MWCO). After dialysis, the polymer solution was filtered through 1.0 m glass fiber filter (Fisher Scientific ) and stored as a stock solution. 1H NMR (D2O): = 4.99-5.11 (br, s, 4H), 7.35 (br, s, 2H), 7.88 (br, s, 4H).

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130 CHAPTER 4 ADENYLATE KINASE ASSAY USING POLY(PHENYLENE ETHYNYLENE) CARBOXYLATE Introduction Adenylate kinase (ADK) is a ubiquitous ATP:AMP phosphotransferase which distributes in variety of organisms and catalyzes the intercon version of the adenine nu cleotides as shown in chemical Equation 4-1222 (4-1) where ATP is adenosine 5 -triphosphate disodium, AMP is adenosine 5 -monophosphate sodium and ADP is adenosine 5 -diphosphate sodium. ADK composes the adenylate system with ATP, ADP, and AMP and plays a unique buffering role in maintenance of equilibrium am ong the adenine nucleotides, thereby maintain constant energy charge and facilitating the storage and use of the high energy of the adenine nucleotides.223 The enzyme also serves as a regulator y factor of many enzy matic reactions in which the adenine nucleotides may participate as a substrate, activator, or inhibitor.224,225 In addition, recent research reveal s that ADK plays an important ro le in cell metabolism, including the synthesis of DNA and RNA molecules.226 Adenylate kinases can also phosphorylate nucleoside analogs used in the trea tment of cancer and viral infection.227,228 Because of the importance of ADK in biological systems, th e enzyme has been extensively studied in enzymology, human genetics, protein chemistry, crystallography, and molecular spectroscopy. Several ADK assays have been developed based on chromatographic separation,229 pH-stat titration,230 bioluminescence,231-233 and coupled enzyme methods.234,235 The chromatographic separation-based assay offers direct measurement of changes in the amount of the adenine nucleotides; however, it is la borious and time-consuming.222 The pH-stat assays suffer from low

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131 sensitivity and the inherent disadvantage that large quantities of enzymes and substrate are required.236 Figure 4-1 illustrates the mechanism of bioluminescent assay for bacterials ADK reported by Sanders and Squirrell in 1998.231 The conversion of ADP to ATP catalyzed by ADK was signaled by ATP bioluminescence with additi on of luciferin and luciferase. Although the bioluminescence assay is sensitive and rapid, it is not widely used because it requires coupling with firefly luciferase. Figure 4-1. Bioluminescent assay for ADK. Reprinted with permission from Blasco, et al .231 The most popular and commercial available as says are colorimetric with coupling the ADK catalyzed reaction with other enzymes.237 As shown in Figure 4-2, the assay to detect the forward reaction activity of ADK (Equation 4-1) was coupled with pyruvate kinase and lactate dehydrogenase and conducted by monitoring the absorbance change of NADH at 340 nm which is concomitant with its oxidation. In a similar wa y, the assay to detect the reverse activity of ADK was coupled with hexokinase and glucose-6-phosphate and carried out by monitoring the absorbance change of NADPH at the same wa velength accompanying th e reduction of NADP.234 The colorimetric enzyme-coupled assays offe r simple operation and potentially real-time detection, however, they are less sensitive compared with fluorometric assay and the accuracy of the assay depends on other system factors such as activity of coupled enzymes.

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132 Figure 4-2. Mechanism of coupled enzyme assays for ADK. In this chapter, we describe a CPE-based c ontinuous and sensitive fluorescent assay for ADK which affords the ability to detect AD K activity both in the forward and reverse transphosphorylation reactions. As we described in Chapter 3, the Cu2+-quenched fluorescence of PPE-CO2 (structure shown in Figure 1-10) is efficiently recove red by addition of pyrophosphate,208 which provides the basis for the CPEbased ALP turn-off assay. However, titration of three adenosine phosphates (ATP, ADP and AMP) individually into the polymer/Cu2+ solution exhibits fluorescence recovery to differe nt extent, which affords the platform for the fluorescent ADK assay. In particul ar, quenched fluorescence was rec overed to the greatest extent by ATP, contrasting by a much smaller rec overy by ADP and no recovery by AMP. The different recovery efficiency arises from distin ct association abilities of adenosine phosphates with Cu2+: ATP with more negative charges possesse s the highest associ ation constant for binding with Cu2+. As a result, the polymer-metal complex is disrupted by ATP, leading to the recovery of the fluorescence intensity, which is demonstrated to be a quantitative index of ATP concentration in the solution. The real-time fluorescence assa y for ADK was designed based on

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133 monitoring the amount of ATP which serves either as the substrate in the forward reaction or as the product in the reverse reaction. The assay to sense the forward reaction is a turn-off assay. By introducing ADK into a solution of PPE-CO2 -/Cu2+/Mg2+/ATP/AMP and applying the ex-situ calibration, the decrease of the fluorescence intens ity is quantitatively related to the decrease of ATP concentration as a function of incubation time. In this assay, Mg2+ is the activator and AMP is the other substrate. The assay to monitor the reverse reaction is a turn-on assay, where the increase of fluorescence intensity monitors an increase in the ATP concentration. The assay is carried out at physiological pH, prov ides flexibility of either endpoint or real-time measurement, allows determination of enzyme kinetic parameters, and offers comparable sensitivity. Results and Discussion Overview of ADK Turn-off Assay The polymer, PPE-CO2 (structure shown in Figure 1-10) is used in the ADK assay. The synthesis and characterization of th is polymer is described in the experimental section in Chapter 3. In 10 mM HEPES buffer solution (pH 7.5), PPE-CO2 exhibits a broad fluorescence band with an emission maximum at 525 nm, suggesting the polymer is aggregated in buffer solution. A divalent metal ion is required for ADK activity.222 Magnesium ion, Mg2+, was chosen in our experiments, because it is reported to be the most effective activator compared with other metal ions, e.g. calcium (Ca2+) and manganese (Mn2+).237 The cartoon in Figure 4-3 illustrates the m echanism of the ADK turn-off and turn-on assays, which depends on the different fluorescence recovery efficiency of Cu2+ quenched polymer by ATP, ADP and AMP. The fluorescence of PPE-CO2 is quenched efficiently by Cu2+. The quenched fluorescence is recovered to the greatest extent upon addition of ATP, however, it is recovered to a distinctive smalle r extent by introducing ADP and to a neglectable extent by introducing AMP. In a turn-off approach the ATP and AMP substrates are added into

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134 the PPE-CO2 -/Cu2+ solution containing the activator, Mg2+. AMP has no effect on the polymermetal complex; however, the complex is disrupted by ATP due to stronger association of ATP with Cu2+, thus leading to the recovery of the fluor escence intensity. Intr oduction of ADK to the mixture of PPE-CO2 -/Cu2+/Mg2+/ATP/AMP initiates the forwar d transphosphorylation producing ADP. With less negative charge density, the product, ADP, binds to Cu2+ more weakly than ATP. As the reaction proceeds, the amount of ATP available to associate with Cu2+ decreases, and the fluorescence of PPE-CO2 is quenched by the Cu2+ that is released. Thus, ADK catalytic activity is signaled by the fluorescence switching from the on state to the off state. The changed fluorescence intensity is related to the s ubstrate concentration, [ATP], as a function of time in the enzymatic reaction (see Equation 42 experimental section), thereby allowing quantitiative investigation of ADK activity. Figure 4-3. Mechanism of ADK turn-off and turn-on assay.

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135 In a turn-on approach as shown in Figure 4-3, the procedure is reversed compared with the turn-off approach in terms of steps and on-off sw itch. The ADP substrate is added into the PPECO2 -/Cu2+ solution containing Mg2+. In contrast to ATP, ADP has a lesser ability to effectively restore the quenched fluorescence of PPE-CO2 -. Consequently, the mixture maintains its fluorescent off state. Addition of ADK to the mixture of PPE-CO2 -/Cu2+/Mg2+/ADP initiates the reverse transphosphorylation producing ATP, which induces the recovery of the quenched fluorescence of PPE-CO2 -. Therefore, the activity is monitored by the increase of the fluorescence intensity of PPE-CO2 -. Fluorescence Recovery by ATP, ADP and AMP In the previous communication,208 we reported that PPE-CO2 shows an amplified quenching effect by Cu2+ in HEPES buffer with a Stern-Volmer constant ( KSV) of 106 M-1. The quenching mechanism is suggested to be singlet exciton quenching via charge and/or energytransfer facilitated by coordination of Cu2+ with the carboxylate groups of polymer.209,210 The Cu2+ quenched fluorescence of PPE-CO2 is efficiently recovere d by PPi due to the strong binding between phosphate groups on PPi and the me tal ion, thus disrupting the polymer-metal complex. Adenine nucleotides (ATP, ADP and AMP), bearing the similar phosphate groups as PPi, should give rise to a similar recovery effect. A series of titrations of ATP, ADP or AMP over concentration ranging from 0 to 40 M into a HEPES buffer (10 mM, pH 7.5) solution containing 5 M PPE-CO2 and 10 M Cu2+ were carried out to investigate the the recovery e ffect. Figure 4-4 shows the recovery efficiency, Ir/Iq as a function of concentration of ATP/ADP/AMP. ( Iq and Ir are respectively the fluorescence intensities of PPE-CO2 -/Cu2+ at 525 nm before and after addition of adenosine phosphates.) At the same concentration level, the recovery effi ciency increases in the order of ATP > ADP > AMP, which is correlates with the decrease of negative charge density of the anions. With the

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136 largest negative charge density, ATP features the strongest electrosta tic association with Cu2+, thus showing the greatest recovery efficiency among three adenosine phos phates. Similar to PPi recovery as shown in Fi gure 3-6, the plot of Ir/Iq of ATP displays as the sigmoidal curve: increasing slowly at very low [ATP] and rising sharply with increase of [ATP] until reaching a plateau. In the same concentration range, ADP w ith one less negative charge than ATP exhibits a much lower recovery efficiency, and AMP with th e lowest charge density exhibits negligible recovery efficiency. When the concentra tions of ATP, ADP and AMP are all at 40 M, the fluorescence is recovered to 77%, 25%, and 0% of initial intensity of pure polymer, or as a 17, 5, and 0-fold of increase relative to the quenched fluorescence, respectiv ely. It is important to point out that ADP is able to substantially recover the quenched fluores cence when its concentration is very high (e.g. 600 M ADP induces a 15-fold of fluorescence intensity increase), but AMP does not give rise to notable recovery even at concentrations as high as 3 mM. Figure 4-4. Comparison of fluor escence intensity increase at 525 nm upon titration of ATP, ADP and AMP, respectively. 5 M PPE-CO2 and 10 M Cu2+ in 10 mM HEPES buffer (pH 7.5) at 37 C, ex= 390 nm.

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137 The different response among ATP, ADP and AMP to Cu2+ quenched fluorescence of PPE-CO2 provides the basis for development of an ADK assay. In principle, both turn-off and turn-on approaches shown in Figure 4-3 are applicable; however, the turn-off approach is much easier to implement in practice due to followi ng two factors: (1) easie r control of reaction direction; (2) more sensitive signal detection. Concerning the first factor, the apparent equilibrium constant, Kapp (= [AMP] [ATP]/[ADP]2) for the interconversion reaction shown in Equation 4-1 was reported to be ~0.82,222 so it is desired to control the reaction and insure that the reaction only or mainly occurs in one direction. Generally this is realized by manipulating the amount of AMP, either by adding excess AMP in the forward direction of Equation 4-1 (turn-off approach) or by eliminating gene rated AMP in the reverse direc tion (turn-on approach). It is obvious that adding excess AMP is much simpler than eliminating generated AMP. The second factor arises from the sigmoidal shape of curv e for ATP as shown in Figure 4-4 and the relative stronger fluorescence recovery by ADP at higher c oncentration. At the beginning of the turn-on assay as shown in Figure 4-3, the florescence r ecovery by small amount of newly-formed ATP is obscured by the relatively stronger recovery effect induced by larg e amount of substrate, ADP. Consequently, it is difficult to monitor the [A TP] at the beginning of the turn-on assay by detecting the changed fluorescence. On the contra ry, in the initial time period of the turn-off assay, the assay solution has high level of ATP and very low amount of ADP. Thus, the change of fluorescence intensity accurately represents th e decrease of ATP concentration. Because of these two factors, we emphasize on the turn-o ff assay and describe the assay process, Mg2+ effect, kinetic study, inhibition and specificity in vestigation in detail below. We will briefly discuss the turn-on assay at the end of the chapter.

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138 Calibration Curves of ADK Turn-off Assay Magnesium ion, Mg2+ is a required activitor for ADK activity.222 However, addition of Mg2+ into the polymer solution al so induces quenching of PPE-CO2 but with a much smaller KSV (4.6 102 M-1) compared with that of Cu2+. In addition, the presence of Mg2+ changes the ionic environment of the solution, affecting the association among PPE-CO2 Cu2+ and ATP. Therefore the effect of Mg2+ needs to be considered when the amount of ATP is quantified by fluorescence intensity. A series of titrations of ATP into a HE PES buffer (10 mM, pH 7.5) solution of 3 M PPECO2 and 10 M Cu2+ in the presence of Mg2+ at various concentrations were carried out and the plots of recovery efficiency (Ir/Iq) as a function of [ATP] displays the similar sigmoidal curves as shown in Figure 4-4. In order to obtain an appr oximate linear correlation that can be used as a calibration curve to related the change in fluorescence intensity to [ATP], fluorescence intensity data was plotted as shown in Figure 4-5, which shows correlations of Log( Ir/Iq) vs. [ATP] at different concentrations of Mg2+. Note that as the Mg2+ concentration increases, more PPi is needed to recover the fluorescence. This is consistent with the quenching effect of Mg2+, as well as the weaken complexation between ATP and Cu2+ in the presence of Mg2+. Importantly, each of the plots which corresponds to different [Mg2+] exhibits a linear increase in Log( Ir/Iq) with [ATP] over the concentration range from 0 to about 1.2 equivalent of [Mg2+]. For example, in the presence of 300 M Mg2+, the linear range of [ATP] is from 0 to 360 (1.2 300) M. This suggests that each plot can be used as a cal ibration curve which linearly relates fluorescence intensity to ATP concentration. These calibra tion curves are used to derive Equation 4-2 which is applied to determine [ATP]t during the ADK turn-off assay (see e xperimental section). Note that the applicability of Equation 4-2 is independent of [Mg2+], but the range of [ATP] over which the calibration curve is linear varies with [Mg2+]. In addition, this linear range of [ATP] is also

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139 affected by Cu2+ concentration. Therefore, the range of a pplicable substrate concentration can be adjusted by the amount of Mg2+ or Cu2+ in the system. Figure 4-5. Calibration curves, logarithm of fluorescence recovery efficiencies (Log ( Ir/ Iq)) as a function of [ATP], at diff erent concentration of Mg2+. 3 M PPE-CO2 and10 M Cu2+ in 10 mM HEPES buffer (pH 7.5) at 37 C, ex= 390 nm, em= 525 nm. In order to control the reacti on to be favorable in the forw ard direction, excess AMP was added in the ADK turn-off assay. The control expe riments revealed that the AMP elicited little effect on the calibrations curves even when added at the millimolar range. In the following discussions, we will refer to a solution containing 3 M PPE-CO2 -, 10 M Cu2+, 2 mM AMP and 10 mM HEPES buffer (pH 7.5) as the standard assay solution used in the ADK turn-off assays. ADK Turn-off Assay In order to eliminate the possi bility that the fluorescence of the polymer is influenced by unexpected factors, several cont rol experiments were conducted. The effects that addition of three adenosine phosphates, ATP, ADP, AMP a nd the enzyme, ADK, have on the fluorescence of solution of Cu2+-free PPE-CO2 were explored and little effects were observed when these

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140 species were added individually to the polym er solution. Introduction of ADK into solution containing PPE-CO2 -, Cu2+, Mg2+, ATP and AMP initiates the transphosphorylation from ATP to AMP and forms ADP. The enzyme reaction is monitored by the decrease of the polymers fluorescence intensity. Figure 4-6A shows typica l fluorescence spectroscopic changes observed during an end-point ADK turn-off assay carried out in the standard soluti on at 37 C, and inset photograph illustrates these fluorescence changes accompanying the assay. In particular, the quenched fluorescence from solution 1 which contains 200 M Mg2+ in the standard solution is significantly enhanced in intensity by adding 200 M ATP, forming solution 2 (change indicated by arrow 1 2). Following introduction of 500 nM ADK into the solution 2 and initiating the enzymatic reaction, consumption of ATP is signa led by a decrease of the fluorescence intensity with increasing incubation time (change indicated by arrow 2 3). In solution 3, the fluorescence of the polymer is quenched by the free Cu2+, which is produced as a result of ADKcatalyzed enzymatic reaction. The fluorescence intensity declines very fast initially (~50% in the first 3 minutes), then reduces gradually as the reaction is approaching equilibrium, and decreasing by ~83% of recovered fluorescence after 60 minutes of incubation time. The decrease of the fluorescence intensity is faster at the beginning of the assay, because the enzymatic reaction shown in Equation 4-1 is dominated by th e forward reaction in the presence of ATP and AMP substrates. However, as the reaction pro ceeds, the generated ADP induces the reverse reaction which competes with the forward reaction and offsets its rate; therefore, a slower decrease of fluorescence intensity is observed al ong with the assay. In addition, the non-specific effect due to the addition of ADK into the assa y solution also contributes to the decrease of fluorescence intensity during the initial period of assay. We will discuss the non-specific effects in detail below.

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141 Figure 4-6. Fluorescence changes observed in the ADK turn-off assay. A) Fluorescence spectroscopic changes observed in an end-point ADK turn-off assay. ( ) fluorescence of solution 1: 200 M Mg2+in the standard assay solution at 37 C, ex = 390 nm; ( ) fluorescence of solution 2: addition of 200 M ATP into solution 1; fluorescence intensity as a function of tim e of solution 3: addition of 500 nM ADK into solution 2 and incubation for 3 ( ), 5 ( ), 8 ( ), 15 ( ), 60 ( ) minutes. Inset: Photographs of solution 1, 2 and 3 (60min) illuminated with near UV light illustrate the polymer fluorescence under the different condition of the assay. B) The decrease of fluorescence intensity at 525 nm recorded every 1 sec during the realtime ADK turn-off assays with varying concentration of ADK. 200 M Mg2+ and 200 M ATP in the standard assay solution at 37 C, ex = 390 nm.

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142 In order to test the feasibility of using PPE-CO2 --based fluorescence assay as a real-time turn-off assay for ADK, we monitored the contin uous fluorescence change as a function of assay time at varying ADK concentrations. The real time assays were carried out with 200 M Mg2+ and 200 M ATP in the standard assay solution at 37 C. Figure 4-6B illu strates the continuous decrease of fluorescence intensity at 525 nm for solutions with different ADK concentration ranging from 0 500 nM, where the fluorescence in tensity was measured every second. Note that as the concentration of ADK increases, the reaction rate increa ses. The fluorescence intensity decreases linearly with time during the initial period of incubation, indicating that the reaction rate is a constant over this time range However, the reaction rate decreases as the reaction continues, which is in a good agreement with what we observed in the end-point ADK turn-off assay (Figure 4-6A). This effect becomes more pronounced at higher enzyme concentration. Figure 4-7. Decrease of [ATP] during first 60 sec of the real-time ADK turn-off assays with varying concentration of ADK. 200 M Mg2+ and 200 M ATP in the standard assay solution at 37 C, ex = 390 nm

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143 Figure 4-8. Dependence of initial rate of reaction ( v0) on ADK concentration. 200 M Mg2+ and 200 M ATP in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. The concentration of ATP during the assay, [ATP]t was calculated from the time-varying fluorescence intensity in Figure 4-6B by using the calibration e xpressed by Equation 4-2 (see experimental section). Figur e 4-7 illustrates the [ATP]t at 5 second increments during the first 60 seconds of the ADK turn-off assay at different con centrations of ADK. It is evident that these plots are linear and the slope s of the plots were comput ed to afford values of v0 at 7 different ADK concentration investigated. Figure 4-8 displays a plot of v0 as a function of ADK concentration in the rang e of 0 500 nM. The initial rate of reaction rises approximately linearly with [ADK] when the enzyme concentration is below 300 nM, which indicates that the reaction is kinetically controlled by ADK. The correlation flattens at hi gher concentration of ADK due to inhibition of the forward re action by ADP which is genearated quickly when [ADK] is high. This experiment also demonstrates th at it is possible to use the PPE-CO2 --based assay to detect ADK activity in less than one minute, even at lo w [ADK]. An analytical detection limit of ~42

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144 nM ADK is obtained from the linear range of plot in Figure 4-8. This sensitivity is far below the concentration range of ADK which is generally assayed by commonly used coupled enzyme assays in previous applications.226,234,238-240 Note that the fluorescence intensity values us ed here were not corrected for photobleaching as was done in Chapters 2 and 3. Because photobleaching of PPE-CO2 in HEPES is neglectable, less than 2% of the initial fl uorescence intensity is lost upon 5-min of continuous excitation. Effect of Mg2+ on ADK Activity in Turn-off Assay The ADK-catalyzed reaction requires a nucleotide complexed with Mg2+ as one substrate and a free nucleotide as the second substrate.237 In particular, in the forward reaction as shown in Equation 4-1, Mg2+ combines with ATP to form a complex (MgATP) which acts as a substrate and occupies one of two active substrate sites in the enzyme. The other substrate site is bound with the metal-free substrate, AMP. In the reverse reaction, the two substr ate sites are occupied by complex MgADP and metal-free ADP.237 Therefore, MgATP/AMP and MgADP/ADP are generally used to represent the substrates in the ADK catalyzed in terconversion reaction. In order to optimize the concentratio n of the activator, the effect of Mg2+ on the ADK activity was investigated. In particular, the dependence of the initial rate of reaction, v0, on [Mg2+] was examined with the standard assay solution containing 200 nM ADK and concentrations of ATP ranging from 50 to 500 M. At a given [Mg2+], the linear [ATP] range on the calibration curve is from 0 to 1.2 equivalents of [Mg2+], therefore, the range of [Mg2+] in this experiment was chosen to begin at 0.83 equivale nt of the ATP concentration (= [ATP]/1.2). For example, if 300 M ATP was used in the assay, the concentration of Mg2+ started at 250 M (= 0.83 300). Figure 4-9 illust rates the effect of Mg2+ on v0 at different concentrations of ATP. It is evident that at each [ATP], v0 reaches a maximum rate when [Mg2+] equals [ATP] and decreases quickly when [Mg2+] is in excess of [ATP] even to a small extent. The observed

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145 dependence of v0 on [Mg2+] is consistent with the previous kinetic studies of ADK activity,225,237 which showed the maximum enzyme activity when [Mg2+]/[ATP] is one and the strong inhibition when [Mg2+] is in excess. This result further confirms that Mg2+ combines with ATP to form the stoichiometric complex, MgATP, wh ich acts as a substrate occupying the substrate site. Figure 4-9. Effect of concentration of Mg2+ on initial rate of reaction ( v0) at different concentrations of ATP. 200 nM ADK in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. Determination of ADK Catalyzed ATP Tran sphosphorylation Kinetic Parameters The mechanism of ADK catalysis is a random Bi -Bi reaction and the reaction is interpreted as the binding of MgATP and AMP at independent sites.222,237 The PPE-CO2 --based fluorescence turn-off assay was used to determine the kinetic parameters ( Km and Vmax) for the ADK catalyzed transphorphorylation using the Michaelis-M enten equation (Equation 2-2). Because two substrates (ATP and AMP) are involved in th e enzymatic reaction, th e concentration of one substrate is required to be in excess so as to obtain the kinetic pa rapmeters for the second

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146 substrate. Therefore, the kinetic experiments we re carried out by fixing the concentration of one substrate, AMP, and varying the concentration of the other, ATP. In order to maintain the maximum activity of ADK, the stoichiometry of Mg2+ and ATP was chosen to be 1:1 and the Mg2+-ATP complex concentration, [M gATP] was varied from 0 to 500 M. The experiment was conducted with 300 nM ADK in the standard assay solu tion at 37 C. The values of initial rate of reaction, v0, were obtained and plotted as a function of initial [MgATP ] as shown in Figure 4-10. Figure 4-10. Dependence of initial rates of reaction ( v0) on substrate conc entration [MgATP] ranging from 0 and 500 M. Km app. (MgATP) and Vmax app (MgATP) values were derived by nonlinear regression of these data. 300 nM ADK in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. After the plot of v0 vs. [MgATP] was fitted with the Michaelis-Menten equation (Equation 2-2), the apparent kineti c parameters for MgATP, Km app.and Vmax app.were obtained from the fitted curve with the values of 104 15 M and 0.48 0.02 M-s-1 (4.53 0.22 mol-min-1-mg-1), respectively. The value of the apparent Km is in good agreement with the value obtained using a coupled enzyme assay ( Km = 160 30 M),238 however the apparent Vmax is about 14-fold smaller compared with the value reported from the same coupled enzyme assay ( Vmax = 63 6

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147 mol-min-1-mg-1).238 It is likely that the lower Vmax arises due to the effect of Cu2+ on sulfhydryl groups of the enzyme. As a potent sulfhydryl reagent,241 Cu2+ is capable of oxidizing sulfhydryl groups on ADK molecule which are essential fo r maximum enzyme activity and involved in maintaining the necessary c onfiguration of the enzyme.237 However, the [Cu2+] (10 M) used in this assay is low enough to mainta in most activity of the ADK. Inhibition of the ADK Catalysis in Turn-off Assay In order to further demonstrate that the obs erved fluorescence intensity decrease that is induced by addition of ADK arises due to ADK catalyzed transphosphorylation, the effect of the known inhibitor, Ag+, on the ADK activity was examined. Ag+ is a sulfhydryl reagent and its inhibition towards ADK arises from its effect on two sulfhydryl groups of the enzyme.235 The inhibition experiments were conduc ted at 37 C with the standa rd assay solution containing 200 M Mg2+, 200 M ATP, 300 nM ADK and Ag+, the concentration of which was varied from 0 mM to 1.0 mM. Figure 4-11. Inhibition of ADK turn-off assay by Ag+. Plot illustrates the initial rate of reaction ( v0) versus inhibitor concentration: 200 M Mg2+, 200 M ATP and 300 nM ADK in the standard assay solution at 37 C,ex = 390 nm, em= 525 nm.

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148 As shown in Figure 4-11, Ag+ inhibits the ADK activity from 20% to 60% when its concentration varies from 10 M to 500 M. While the overall inhibition increases with increasing inhibitor concentration, the inhib ition efficiency is largest at low inhibitor concentration. The inhibition expe riments provide very strong eviden ce that the assay is based on the specific ADK catalyzed tr ansphosphorylation reaction. Specificity of the ADK Turn-off Assay In order to test the specific ity of ADK turn-off assay, the response of the assay to other proteins was examined including peroxidase (HRP), peptidase (PEP), phospholipase D (PLD), glucose oxidase (GOx), bovine seru m albumin (BSA). None of the control proteins has a specific interaction with ATP. In this experiment, 400 M Mg2+ and 400 M ATP in the standard assay solution was assayed with 300 nM ADK or one of th e control proteins (for PEP the concentration was 48 g-mL-1). Figure 4-12 compares the fluorescen ce intensity changes at 525 nm observed after a 60 min incubation period in the presen ce of the different proteins and the inset photographs illustrates the differe nt fluorescence response accompa nying the assay. The assay with ADK exhibits a more than 70% decrease in fluorescence intensity, while the assays containing the control proteins (with the exception of BSA) exhibit in the range of 8% ~ 35% decrease in fluorescence intens ity. The decrease in the intensity upon addition of the control protein to the assay solution arises from a non-specific interaction be tween species in the solution including protein, PPE-CO2 -, Cu2+ and adenosine phosphates (ATP, ADP and AMP). This effect results in a disturba nce of the electrostatic balance as well as the equilibria which control the polymers fluorescence intensity.58 Note that the non-specific effect is most pronounced when the control protein is charged, which is demonstr ated by the ~70% decrease in fluorescence intensity by negatively charged BS A. The presence of a non-specific effect is further demonstrated by the real time assay with ADK and BSA added individually as shown in

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149 Figure 4-13. In contrast to th e behavior seen for ADK where th ere is a continuous decrease in PPE-CO2 fluorescence following addition of the enzy me, addition of ADK assay, addition of BSA induces a rapid decrease in the fluorescence intensity in one minute, following this initial change, the fluorescence intensity remains constant, as a new electrostatic balance in the solution is attained. Figure 4-12. Specificity of AD K turn-off assay. Changes in fluorescence emission intensity at 525 nm after 60 minutes of incubation of 300 nM ADK, HRP, PLD, GOx, BSA or 48 g-mL-1 PEP. 400 M Mg2+ and 400 M ATP in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm. Photographs of solutions illuminated with near UV light illustrate the polymer fluorescence under the ADK turn-off assay and control assays. Due to the existence of the non-specific effects, it is of interest to measure the contribution of non-specific effect to the overall decrease in the fluorescence intens ity during the ADK turnoff assay. The control assay was carried out under the same conditions used for Figure 4-6B except that no AMP was added. The shape of intens ity decrease curve in th is control experiment

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150 is very similar to that of BSA curve shown in Fi gure 4-13 and this decrease arises due to the nonspecific effect caused by addition of ADK. The hypothetical initia l rate of reaction, v0c, was calculated from the decrease of fluorescence inte nsity. The absence of AMP eliminates the catalytic effect of ADK; nevertheless, the value of v0c is ~30% of the value of v0 obtained in Figure 4-8 at each concentration of ADK. It s eems that the non-specific effects accounts for 30% of fluorescence intensity decrease, however, its contribution in the real ADK turn-off assay with AMP added is believed to be smaller than 30%. That is because the pr esence of AMP initiates the catalytic function of ADK and changes the affinity of ADK towards ATP. As a result, the non-specific effect is decreased. In summary, it is apparent that the decrease in the fluorescence intensity noted in the ADK turn-off assay arises mainly from the specific enzymatic activity of ADK and the non-specific interactio n only accounts for small part of this fluorescence intensity decrease. Figure 4-13. Changes in fluorescence emission in tensity at 525 nm as a function of incubation time after addition of 300 nM ADK and BSA. 400 M Mg2+ and 400 M ATP in the standard assay solution at 37 C, ex = 390 nm, em= 525 nm.

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151 ADK Turn-on Assay In order to examine the f easibility of using polymer/m etal ions/adenosine phosphates complex as the platform for an ADK turn-on assa y to detect the revers e reaction as shown in Equation 4-1, the effect of added ADK on the pol ymers fluorescence intensity was examined in the presence of Cu2+, Mg2+ and ADP which in this case is regarded as the substrate for ADK. The results of turn-on assay are illustrated in Figure 4-14. Initially the fluorescence of 3 M PPE-CO2 is quenched by 10 M Cu2+ in the presence of 100 M ADP and 50 M Mg2+ in 10 mM HEPES buffer (pH 7.5) at 37 C. (Note that [Mg2+] is 0.5 equivalent of [ADP], because the two substrates occupied the active site of ADK are complex MgADP and metal-free ADP.222) After introduction of 300 nM ADK, the fluorescence increases as a function of assay time and is doubled in fluorescence intensity after 180 min of incubation. Compared with the ADK turn-off assay, the turn-on assay is less sensitive in term s of rate of reaction and scale of the intensity change. This low sensitivity is consistent with our expectation descri bed in the section of fluorescence recovery by ATP, ADP and AMP. Figure 4-14. Change of fluorescence intensity observed during ADK turn-on assay. After addition of 300 nM ADK into the solution containing 3 M PPE-CO2 -, 10 M Cu2+, 100 M ADP and 50 M Mg2+ in 10 mM HEPES buffer (pH 7.5) at 37 C, fluorescence intensity increas es as a function of incubation time (0 ~ 180 min), ex = 390 nm, em= 525 nm.

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152 Discussion The assay described herein takes advantag e of the selective re sponse of the PPE-CO2 --Cu2+ complex to adenosine phosphates and affords the ability to detect ADK activity both in forward and reverse transphosphorylation reaction. Compared with routine ADK enzyme coupled assays,234,235 this method offers comparable sensitivity in terms of amount of enzyme required. However, in contrast to the mo st popular couple-enzyme methods, it eliminates the use of other enzymes and prevents the influence from their activ ity. In addition, it outperforms with respect to the amount of substrate used. It allows the assa y to be carried out at hundreds of micromolar range of initial substrate concen tration which is much lower compared with literature reported range in the other assays.226,234,238-240 Moreover, the PPE-CO2 based fluorescence assay affords the ability to carry out real time detection of the enzyme activity and to measure kinetic parameters and inhibition behavior. Finally, the assay is easy to implementation and gives rapid response to different enzyme ac tivity. The fluorescence-based met hod offers the applicability of adapting it to high-throughput screening (HTS) format.68 While the PPE-CO2 based ADK assay has offered many advantages, nonetheless this method still has some disadvantages. The existe nce of non-specific inte raction with various proteins and other charged solutes could interf ere with the sensor re sponse, especially if quantitative (kinetic) data is need. As a result, when applying this assay with real biological samples, such as serum samples, it would be nece ssary to purify the ADK to eliminate the source of non-specific interactions induced by different salts, proteins or other bi ological components. It also prevents its application to investigate th e inhibition of the enzyme by inhibitors (e.g., EDTA) which could interrupt the electrostatic balance of the system. In addition, the two sulfhydryl groups on the enzyme could be affected by Cu2+ in the solution, which may lead to a

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153 decrease in the activity of the en zyme. Therefore, the amount of Cu2+ should be added at a low concentration in orde r to maintain activity of the enzyme. Experimental Materials All stock solutions were prepared with water th at was distilled and th en purified by using a Millipore purification system. HE PES buffer solution (10 mM, pH 7.5) was prepared with 4-(2hydroxyethyl)-1-piperazineethanesu lfonic acid and hydrochloric acid. The aqueous stock solution of PPE-CO2 was diluted with HEPES buffer solution to a final concentration of 3 M. All chemicals were used as received, unless otherwise noted. Coppe r (II) chloride (CuCl2), adenosine 5 -triphosphate disodium salt (ATP), adenosine 5 -diphosphate sodium salt (ADP) and adenosine 5 -monophosphate sodium salt (AMP) were obtained from Sigma-Aldrich. Magnesium chloride was purchased from Fisher Chemical. Silver nitrate was obtained from Mallinckrodt. Each reagent was dissolved in HE PES buffer and the concentration of the stock solutions was adjusted to 100 mM. Adenylate ki nase (ADK, myokinase from rabbit muscle) and the control proteins were purchased from Sigm a-Aldrich. The five c ontrol proteins include peroxidase from horseradish Type I (HRP), pept idase from porcine intestinal mucosa (PEP), phospholipase D from Arachis hypogaea (peanut) (PLD), glucose oxida se from Aspergillus niger (GOx), bovine serum albumin (BSA). All of the enzymes and proteins were dissolved in HEPES buffer solution and adjusted to 20 M except for PEP with 3.2 mg-mL-1 as stock concentration. The enzyme and protein stock solutions were prepared immediately before their use in the fluorescence assays. Instrumentation Fluorescence spectra were re corded on a spectrofluorometer from Photon Technology International and are corrected by using correctio n factors generated with a primary standard

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154 lamp. The 1-cm fluorescence cuvette was placed in a custom-built thermostatted cell holder which was maintained at 37 C during the assay and was equipped with a micro-submersible magnetic stirrer. General Methods Fluorescence assay procedure The enzyme assays were carried out in 10 mM HEPES buffer (pH 7.5) at 37 C. For the real-time assays, the fluorescence intensity was recorded with excitation and emission wavelengths of 390 nm and 525 nm, respectively. All measurements were conducted with stirred solutions. In the turn-off approach, ATP was used as a substrate. A typical proc edure was carried out as follows: First, a 2.0-mL aliquot of the solution containing PPE-CO2 -, Cu2+, Mg2+ and AMP was placed in the cuvette and allowed to therma lly equilibrate at 37 C The quenched polymer fluorescence intensity (Iq) at 525 nm was recorded. Subsequently, the ATP solution was introduced into another 2. 0-mL aliquot of PPE-CO2 -/Cu2+/Mg2+/AMP solution, this mixture was incubated for 15 minutes and quickly pipetted into a cuvette containi ng microliter range of aliquot of the ADK solution and the fluorescence intensity ( It) of the solution was measured every second as a function of time. In the turn-on approach, ADP was used as the substrate. Therefore, the procedure was the same as the turn-off approach, except that it was initiated with measurement of Iq of PPE-CO2 -/Cu2+/Mg2+ solution and followed by the addition of ADP solution before introducing ADK and recording It. For monitoring the assays in an end-point format, the fluorescence intensity ( It) of the assay solution versus wavelength spectra were recorded with excitation wavelength at 390 nm.

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155 Calculation of initial rate of reaction ( v0) In the turn-off approach, fluorescen ce intensity of the assay solution, It, was converted to substrate concentration [ATP]t at one-second intervals during th e assay by using Equation 4-2. Equation 4-2 is derived from the calibration wh ich relates the fluorescence intensity to [ATP] (Figure 4-5). t q t0 0 q() [ATP][ATP] () I Log I I Log I (4-2) where [ATP]0 is the initial substrate concentration, [ATP]t is the substrate concentration at time t, Iq is quenched fluorescence intensity of the PPE-CO2 solution in the presence of Cu2+, Mg2+ and AMP, I0 in the initial fluorescence intensity at t = 0, that is, the fluorescence intensity after addition of substrate ATP but befo re the addition of enzyme, and It is the fluorescence intensity at time t after enzyme addition. A plot of [ATP]t vs. time was then derived and the v0 was calculated from the slope of the plot by us ing the data from the region where [ATP]t is a linear function of the time. Calculation of kinetic parameters Michaelis-Menten Equation (Equation 2-2) was used to derive the kinetic parameters in the CPE-based ADK turn-off assay (forward reaction as shown in Equation 4-1). By fixing the concentration of AMP in excess and maintaining the stoichiometry of [Mg2+] and [ATP] as 1:1, a plot of v0 vs. [MgATP]0 was fitted with the Michaelis-Menten equation using a non-linear regression routine, the values of apparent Km and Vmax for ATP were obtained.

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156 CHAPTER 5 CHEMILUMINESCENT CONJ UGATE POLYELECTROLYTES Introduction Much of the current analytical interest in chemiluminescence arises from its requirement for simpler instrumentation without the need fo r an excitation source and higher sensitivity compared with photoluminescence, which is due to the elimination of the noise caused by light scattering, background emission and source instability.140,242 Although the photoluminescence of conjugated polyelectrolytes (CPEs) have been investigated in depth, the chemiluminescence of CPEs has never been studied to our best knowledge. Therefore, it is of in terest to investigate chemiluminescence of CPEs as we ll as its sensor application. Figure 5-1. Three steps in peroxyoxalate chemiluminescence. Peroxyoxalate chemiluminescence is one of the most efficient and versatile chemiluminescence processes available at present.133 As shown in Figure 5-1, peroxyoxalate chemiluminescence consists of three steps, (1) re action between an aryl oxalate and an oxidant, typically hydrogen peroxide (H2O2), to form a high-energy intermediate; (2) excitation of the fluorophore via energy transfer from the intermed iate; (3) chemiluminescence emission from the fluorophore (which is spectroscopically identical to its fluo rescence). By using various fluorophores, peroxyoxalate chemiluminescence reacti ons have been demonstrated as useful and sensitive detection methods for sensi ng chemical and biological species.144 However, the application of peroxyoxalate chemiluminescence in detection of biological analytes is limited to those which involve H2O2 in the reactions, either as the s ubstrate or product. By applying a CPE

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157 as the fluorophore in peroxyoxalate chemilumine scence and taking advantage of its biosensor potential, it is possible to expa nd chemiluminescence applications to include biological targets that are unrelated to H2O2. Meanwhile, the amplified quenching effect of CPEs and the intrinsic high sensitivity of the chemiluminescence technique are expected to improve the property of possible biosensors. In this chapter, we describe the first chemiluminescence system for CPEs which utilizes bis(2,4,6-trichlorophenyl) oxala te (TCPO) as peroxyoxalate chemiluminescence reagent, H2O2 as oxidant, imidazole (ImH) as catalyst a nd two anionic CPEs as fluorophores in aqueous/acetonitrile (CH3CN) solvents. The process was carried out simply by mixing aqueous solutions of CPE/H2O2/ImH and CH3CN solution of TCPO, and si multaneously recording the chemiluminescence signal by a spectrometer. The effects of concentration of each reagent and solvent composition on the chemiluminescence signals were evaluated. Subsequently, in order to test the property of chemiluminescence of th e CPEs, the chemiluminescence quenching of the CPEs was studied. In comparison to phot oluminescence quenching, chemiluminescence quenching shows similar amplified quenching propert y with comparable quenching efficiency in the same solution environment. Finally, as an example application of chemiluminescence of CPE, we demonstrated the suitability of chemiluminescence-based CPE as biosensor by detecting the activity of two different enzymes. Specifically, chemiluminescence was utilized to qualitatively and quantitatively detect the hydro lysis of quencher-labeled peptide catalyzed by peptidase,87 and hydrolysis of inor ganic pyrophosphate cataly zed by alkaline phosphatase (ALP).208 The sensor response is expected to be further improved by using water stable chemiluminescence reagents to avoid the need for organic solvents and by applying more efficient sample mixing met hods, such as flow injection243 and stopped-flow.244

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158 Results and Discussion Photophysical Property of CPEs Two different CPEs, BpPPE-SO3 (structure shown in Figure 2-2) and PPE-CO2 (structure shown in Figure 1-10) were used in this work. The synthesis and characterization of these two polymers are included in the experimental sections of Chapter 2 and 3, respectively. Both of the polymers exhibit similar solvent dependent abso rption and emission properties as other CPEs.30 For example, BpPPE-SO3 exhibits absorption and emission maximum at 397 nm and 416 nm (Figure 5-2), respectively in methanol soluti on, similar spectral prope rties are seen in CH3CN solution. With increasing volume fraction of H2O in the organic solvents, the absorption and emission bands progressively red shift and the em ission band becomes broader. In pure water, BpPPE-SO3 absorbs at = 419 nm and exhibits a broad fluorescence with a maximum at = 503 nm. The solvent-dependent photophysical properties arise due to the aggregation of the polymer in aqueous solution.30 Figure 5-2. Normalized absorpti on and fluorescence spectra of 1 M BpPPE-SO3 in methanol () and water ( ).

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159 Chemiluminescence of CPEs Two common oxalic acid derivatives, 1,1oxalyldiimidazole (ODI) and TCPO were initially selected as the chem iluminescence reagents. Even though ODI is more reactive with H2O2, its low stability and solubility in aqueous-rich media (or even in pure CH3CN) hampered its use in our experiments. By c ontrast, TCPO is more stable than ODI but it is less reactive and therefore a basic catalyst is required to increase the rate of its reaction with H2O2. In comparison with other amine bases, ImH has the greatest effect on improvement of chemiluminescence yield.142,143 Therefore, a combination of TCPO and ImH was chosen as the chemiluminescence reagents for the experi ments described herein. Before carrying out the chemiluminescence m easurements, initial studies were done to demonstrate that the luminescence of BpPPE-SO3 or PPE-CO2 is not influenced by the presence of H2O2, ImH and TCPO in the solution. These expe riments indicated that the reagents do not lead to appreciable quenching of the CPE luminescence at the concentrations used for the chemiluminescence work. Because TCPO under goes hydrolysis in aqueous solution to trichlorophenol (TCP), the reagent was dissolved in CH3CN as stock solution. The reaction between TCPO and H2O2 in the presence of ImH catalyst is rapid, so the sequence of adding a CH3CN solution of TCPO into the aqueous polymer solution was chosen so that H2O2 and ImH can be incubated with polymer in aqueous solu tion before the chemiluminescence reaction is initiated by addition of the TCPO. Similar re sults were obtained from chemiluminescence measurements of BpPPE-SO3 or PPE-CO2 -; therefore, only BpPPE-SO3 data are presented herein. Figure 5-3A illustrates the chemiluminescen ce intensity vs. wavelength profile of a solution containing 250 M BpPPE-SO3 with 2.5 mM H2O2, 2.5 mM ImH and 0.25 mM TCPO in H2O/CH3CN (50/50). The chemiluminescence profile is identical to the photoluminescence

PAGE 160

160 spectrum (data not shown) of BpPPE-SO3 in the same solvent condition with a maximum emission at = 450 nm. Figure 5-3. The chemiluminescence signal profiles. A) Chemiluminescence signal vs. wavelength spectrum. 250 M BpPPE-SO3 -, 2.5 mM H2O2, 2.5 mM ImH and 0.25 mM TCPO in H2O/CH3CN (50/50) obtained 2 sec after mixing. B) Chemiluminescence signal vs. time profile. 20 M BpPPE-SO3 -, 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO in H2O/CH3CN (50/50). Inset: Photographs of chemiluminescence of BpPPE-SO3 (same condition as B) and corresponding control without TCPO.

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161 Figure 5-3B shows the chemiluminescen ce intensity vs. time profile of 20 M BpPPE-SO3 at the emission wavelength of = 450 nm. The reaction is carrie d out in a solution containing 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO in H2O/CH3CN (50/50). The point at about 5 sec indicted by an arrow is the mome nt of addition of the TCPO CH3CN solution. After this point, the chemiluminescence profile is characteri zed by two components, rise and fall components; the intensity rises sharply to the maximum within 5 sec and decays gradually until falling to almost zero. The entire chemilumine scence signal lasts for only ~30 sec. The rise component corresponds to the produ ction of high-energy intermedia te coupled with excitation of the CPE fluorophore, whereas the relatively slower fall component is due to disappearance of TCPO in the chemiluminescence reaction solu tion. It should be emphasized that the chemiluminescence data exhibit goo d reproducibility when the tests are repeated multiple times. As shown by the inset photograph in Figure 5-3B, the chemiluminescence of BpPPE-SO3 is strong enough to be observed by eye in a dark room. Following the addition of the TCPO CH3CN solution to the aque ous solution of BpPPE-SO3 with H2O2 and ImH, the chemiluminescence is readily observed (right vi al) compared with the control in which CH3CN solution without TCPO is added (left vial). Optimization of Chemiluminescence Signal The chemiluminescence signal depends on a number of experimental factors, such as the purity of the solvents and reagents, reagent co ncentration, and solven t composition. Thus, in order to optimize the chemiluminescence signal, BpPPE-SO3 was used to investigate the effects of each factor on the intensity and duration of the chemiluminescence signal. The objective was to find the conditions compatible with the sensor work (see below) under which the chemiluminescence yield (intensi ty and duration time) is optimized. The influence of the concentrations of each reagent (H2O2, ImH, TCPO and BpPPE-SO3 -) was first studied and

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162 carried out by fixing concentrations of three reagents and recording chemiluminescence time profiles as a function of concentration of the other one. The chemiluminescence intensity vs. time profiles were recorded in a mixture of H2O and CH3CN (50/50) at = 450 nm. Figure 5-4 illustrate s the effects of [H2O2] on the chemiluminescence signal. The [H2O2] was varied in the range 0 125 mM in reaction media containing 375 M BpPPE-SO3 -, 1.5 mM ImH and 0.5 mM TCPO. The chemiluminescence intensity increases linearly with [H2O2] and reaches a plateau at about 30 mM H2O2, which is reasonable and explains why peroxyoxalate chemiluminescence is often used to detect H2O2 quantitatively. The chemiluminescence duration time increases with [H2O2] at low concentration and it reaches a maximum at 4 mM H2O2, which is a moderate excess relative to the TCPO. At higher concentration, the chemiluminescence duration time decreases significantly because the chemiluminescence reaction rate increases with excess of H2O2.245 Therefore, 8 mM H2O2 was chosen for the following experiments as a compromise between chemiluminescence intensity and duration time. Figure 5-4. Effect of H2O2 concentration on chemiluminescence intensity and duration time. 375 M BpPPE-SO3 -, 1.5 mM and ImH 0.5 mM TCPO in H2O/CH3CN (50/50).

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163 The effect of [ImH] on the chemiluminescence signal is more complex and related to the kinetics of peroxyoxalate chemiluminescence reaction.244,245 Figure 5-5 illustrates the dependence of chemiluminescence intensity and duration time on [ImH] over the concentration range of 0 25 mM at constant BpPPE-SO3 -, H2O2 and TCPO concentrations of 375 M, 1.5 mM and 0.5 mM, respectively. The chemiluminescence intensity and duration time increase to a maximum when [ImH] is 2 mM and 1 mM, respectively, which indicates the need for the ImH catalyst to initiate and optimi ze the TCPO hydrolysis. However, both the chemiluminescence intensity and duration decrease as [ImH] increases from the optimum concentrations to 25 mM. The decrease of the chemiluminescence duratio n time with increasing [ImH] is due to an increase in the rate of chemiluminescence reactio n. The decrease of chemiluminescence intensity is because ImH catalyzes not only the chemilumi nescence reaction but also the hydrolyisis of TCPO. Therefore, with the ra pid lost of TCPO at high concentration of ImH, the chemiluminescence intensity decrea ses. Finally, 1.5 mM was sele cted as the concentration of ImH for the future experiment after compro mising between chemiluminescence intensity and duration time. Figure 5-5. Effect of ImH concentration on chemiluminescence intensity and duration time. 375 M BpPPE-SO3 -, 1.5 mM H2O2 and 0.5 mM TCPO in H2O/CH3CN (50/50).

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164 Regarding to the effects of [TCPO], as shown in Figure 5-6, the chemiluminescence intensity increases approximately linearly with increasing [TCPO] from 0 to 1.5 mM in the presence of 375 M BpPPE-SO3 -, 8 mM H2O2 and 1.5 mM ImH, which is consistent with the chemiluminescence yield increa sing in proportion to [TCPO].244 However, the chemiluminescence duration time is independent of [TCPO] (data no t shown), which is consistent with the peroxyoxalate chemilumines cence reaction being pseudo-first order in TCPO when H2O2 is in moderate excess compared to TCPO.135,244 In this case, [TCPO] has no effect on the rate of the peroxyoxa late chemiluminescence reaction. Howe ver, TCPO has limited solubility in H2O, so the optimum amount of TC PO is adjusted in different solution media with different amount of H2O and CH3CN so that TCPO does not precipitate after mixing H2O and CH3CN solutions. Figure 5-6. Effect of co ncentration of TCPO on chemiluminescence intensity. 375 M BpPPESO3 -, 8 mM H2O2 and 1.5 mM ImH in H2O/CH3CN (50/50), em = 450 nm. The effect of varying [BpPPE-SO3 -] on the chemiluminescence signal in solutions with 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO was also i nvestigated. As shown in Figure 5-7, The chemiluminescence intensity increases in direct proportion to [BpPPE-SO3 -] over the range 0

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165 100 M, which is in good agreement with effect of fluorophore in peroxyoxalate chemiluminescence reaction.135,246 The chemiluminescence duration time does not vary with [BpPPE-SO3 -], which confirms that BpPPE-SO3 is not involved in the chemical reactions of leading to the chemiluminescence. Figure 5-7. Effect of concentration of BpPPE-SO3 on chemiluminescence intensity. 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO in H2O/CH3CN (50/50), em = 450 nm. As mentioned before, TCPO undergoes hydrolysis to TCP in solvents containing water, so we carried out an investigation to discern how the chemiluminescence signal is affected by the amount of water in the chemiluminescence medium. The experiment was done under the optimized concentrations of H2O2 (8 mM) and ImH (1.5 mM). Th e concentration of TCPO was set at 0.3 mM, because it is the solu bility limit of the reagent in 80% H2O/20% CH3CN. Figure 5-8 shows the chemiluminescence vs. time profiles with 10 M BpPPE-SO3 in different volume fractions of H2O in H2O/CH3CN mixture, decreasing from 80% to 20%. There is no chemiluminescence signal in solutions that cont ain > 75% water, because TCPO hydrolyses very rapidly under these conditions. The chemilumines cence duration time increas es with decreasing water content, which can be explained in two ways. First, water increases the rate of base-

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166 catalyzed TCPO reaction.247 Second, the rate of hydrolysis of TCPO increases with the amount of water present in the solvent, allowing the chemiluminescence reaction to become less effectively. In addition, as shown in Figure 58, chemiluminescence intensity increases sharply when water contente in the solvent decreases fr om 80% to 60%. Then further decrease of water content causes little additional change in the chemiluminescence intensity. Two reasons accounts for the initial increase of chemiluminescence inte nsity. First, TCPO beco mes more stable with decrease the amount of water, which induces more effective chemiluminescence reaction. Second, the luminescence quantum yield of BpPPE-SO3 is higher in the solvent medium with more organic solvent. However, the maximum em ission of the polymer is blue shifted with increase of organic solvent fraction, and this effect partially offsets the increase of chemiluminescence intensity at 450 nm. Theref ore the chemiluminescence intensity hardly changes when water content in the solvent mixture decreases from 60% to 20%. Figure 5-8. Effect of solvent composition on chemiluminescence intensity and duration time. 10 M BpPPE-SO3 -, 1.5 mM ImH, 8 mM H2O2 and 0.3 mM TCPO in 20% ( ), 25% ( ), 33% ( ), 50% ( ), 66% ( ), 75% ( ), 80% ( ) of H2O content in H2O/CH3CN mixture.

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167 Quenching Study Conjugated polyelectrolytes are well known for the amplified fluorescence quenching effect by small molecular ion quenchers with opp osite charge, either in aqueous or organic solution. In order to assess th e feasiblitlity of using chemilu minescence quenching for a sensor system, a quenching study was car ried out. Methyl viologen, MV2+ (structure shown in 1-4) and Cu2+ were chosen as quenchers for BpPPE-SO3 and PPE-CO2 -, respectively, because MV2+ is a previously reported effective quencher of CPEs28 and Cu2+ is a required quencher for ALP assay implementation as described in Chapter 3. Th e quenching experiments were carried out by a series of titrations of MV2+ or Cu2+ into a solution of polymer in the presence of 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO. The solvent medium was selected as H2O/CH3CN (50/50) and Tris-HCl/CH3CN (50/50) for quenching experiments of BpPPE-SO3 and PPE-CO2 -, respectively. As a comparison, fluorescence quenching was conducted with the same concentration of CPE in the same solv ent medium except TCPO was not added. Figure 5-9A and 5-9B illustrate th e Stern-Volmer chemiluminescence and photoluminescence quenching plots for MV2+ quenching of BpPPE-SO3 and Cu2+ quenching of PPE-CO2 -. In these plots, I0 and I are the initial and quenched chemiluminescence and photoluminescence intensities, respectively, and the integrated area of the chemiluminescence and photoluminescence profiles was used as inten sity. In quenching experiments with both polymers, the chemiluminescence quenching effi ciency is comparable to that of the photoluminescence quenching in the sa me solvent mixture, with the KSV values ranging from 2 105 to 3 105 M-1. In particular, Figure 5-9A shows that the MV2+ chemiluminescence quenching efficiency of BpPPE-SO3 is only slightly less than that of the photoluminescence quenching. As seen in Figure 5-9B, the Cu2+ quenching of PPE-CO2 is superlinear for both

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168 chemiluminescence and photoluminescence, and in this case chemiluminescence quenching is slightly more efficient. Figure 5-9. Stern-Volmer plots of chemilu minescence and photoluminescence quenching of CPE. A) Chemiluminescence and phot oluminescence quenching of BpPPE-SO3 by MV2+. Chemiluminescence: 10 M BpPPE-SO3 -, 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO in H2O/CH3CN (50/50), em = 450 nm. Photoluminescence: 10 M BpPPE-SO3 in H2O/CH3CN (50/50) with 8 mM H2O2, 1.5 mM ImH, ex = 400 nm. B) Chemiluminescence and photoluminescence quenching of PPE-CO2 by Cu2+. Chemiluminescence: 25 M BpPPE-SO3 -, 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO in Tris-HCl (10 mM, pH 7.5)/CH3CN (50/50), em = 446 nm. Photoluminescence: 25 M PPE-CO2 in H2O/CH3CN (50/50) with 8 mM H2O2, 1.5 mM ImH, ex = 380 nm.

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169 It is important to note that the quenching efficiency for both chemiluminescence and photoluminescence in the H2O/CH3CN solvent mixture used here is approximately 10-fold less compared with a typical supe rlinear photoluminescence quench ing observed in pure aqueous solution ( KSV ~106 M-1). Two factors accounts for the less efficient quenching in the mixed solvent system. First, CH3CN is a good solvent for the CPEs, thus it disfavors CPE aggregation, which is known to decrease the quenching efficiency.30,44,51 In addition, the quencher ions are better solvated in the mixed solvent, which reduc es their propensity to associate with the CPE chains by electrostatic and hydrophobic intera ctions. However, the chemiluminescence quenching displays the amplified quenching proper ty with about 10 to 100 larger of quenching efficiency compared to the small model compound, PE-SO3 (structure shown in Figure 1-7).30 Peptidase Chemiluminescence Turn-on Assay with BpPPE-SO3 A sensitive CPE-based fluorescence turn-on assay for peptidase has been previously developed by our group (mechanism shown in Figure 1-25).87 In order to evaluate the feasiblitlity of biosensor applic ation of chemiluminescent CPE, we applied the same peptidase assay format but using the chemiluminescence of BpPPE-SO3 as the signal transducer. Figure 510 illustrates the mechanism of peptidase chemiluminescence assay. A cationic peptide, KpNA, which is labeled with a p-nitroanilide unit, is used as a quencher for BpPPE-SO3 as well as a substrate for peptidase. The cationic KpNA ion-pairs with anionic BpPPE-SO3 and pnitroanilide unit is able to quench the chemilu minescence of the polymer, which results in a weak chemiluminescence upon addition of the chemiluminescence reagen t. Introduction of a peptidase to the mixture of KpNA and BpPPE-SO3 induces hydrolysis of KpNA and formation of a single lysine peptide and a neutral p-nitroanilide moiety, which is unable to associate with polymer and quench the chemiluminescence effec tively. As a result, the chemiluminescence intensity increases concomitant with peptide hydrolysis.

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170 Figure 5-10. Mechanism of chemilumine scence turn-on assay for peptidase. The chemiluminescence quenching efficiency of BpPPE-SO3 by KpNA was investigated first because the sensitivity of the BpPPE-SO3 -/KpNA/peptidase chemiluminescence assay is determined partially by the quenching efficien cy. Phosphate buffer solution (PBS, 10 mM, pH 7.1) was used as the solvent for the polymer. The mixed solvent system containing 20% of PBS and 80% of CH3CN was selected to increase the durat ion of chemiluminescence. The emission wavelength to record the chemiluminescence time profiles was set at 450 nm, which is the maximum emission wavelength achieved from photoluminescence spectrum of BpPPE-SO3 in PBS/CH3CN (20/80). The chemiluminescence quenching by K-pNA was carried out by a series titration of KpNA into a PBS/CH3CN solution containing 8 mM H2O2, 1.5 mM ImH, 0.3 mM TCPO and BpPPE-SO3 at concentrations ranging from 2 10 M. The photoluminescence quenching experiment by KpNA was conducted with 1 M BpPPE-SO3 in water in the absence of CH3CN, H2O2, ImH and TCPO. The KSV values of both chemiluminescence and

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171 photoluminescence quenching by KpNA are summarized in Table 5-1. Generally, the chemiluminescence quenching is approximately 10 to 50 times less efficient than that of photoluminescence, which is likel y due to the effect of CH3CN as explained in the section of quenching studies. In addition, the chemiluminescence quenching efficiency increases with increasing polymer concentration from 2 to 6 M and then decrease slightly with further increasing polymer concentration. This effect is different from photoluminescence, which displays reversely dependence of quenching efficiency on the polymer concentration.30 This phenomenon is not fully understood yet. Based on the results shown in Table 5-1, 6 M BpPPESO3 was used in the peptidase assay. Table 5-1 Comparison of Stern-Volmer quenching of chemiluminescence and photoluminescence of BpPPE-SO3 by KpNA. Chemiluminescencea Photoluminescenceb [BpPPE-SO3 -] ( M) 2 4 6 10 1 KSV (M-1) 6.9 104 7.8 104 1.4 105 1.1 105 3.6 106 Note: a 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO in PBS (10 mM, pH 7.1)/CH3CN (20/80), em = 450 nm. b 10 mM PBS buffer, pH 7.1, ex = 400 nm. Figure 5-11 shows the change of the chemilumi nescence intensity in a typical peptidase chemiluminescence turn-on assay which is carried out in a mixed solution (PBS/CH3CN = 20/80) of polymer in the presence of 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO at 25 C. The chemiluminescence spectra were recorded at 450 nm and the integrated area of the chemiluminescence profile was used as intensity. The initial chemiluminescence intensity ( I ) from BpPPE-SO3 is normalized to 1. Then the chemiluminescence is quenched ~80% by addition of 20 M KpNA (Q). After introduction of 40 g-mL-1 peptidase which catalyses the hydrolysis of KpNA, the quenched chemiluminescence inte nsity increases as a function of incubation time until it recovers to approxamitely 50% of original chemiluminescence ( I ) at 100 min, after this point further incubation causes li ttle additional increase in the chemiluminescence

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172 intensity. The incomplete luminescence recovery which is not found in the previous work,87 is due to the non-specific quenchi ng effect induced by peptidase.58 This effect is further demonstrated by the control assay which was conduc ted with the same concentration of polymer, chemiluminescence reagents and peptidae in the same solvent medium except K-pNA was not added. As a response to the control assay, the chemiluminescence intensity decreases with incubation time upon addition of the peptidase to the control assay solution. Finally the chemiluminescence intensity is quenched by more than 30% at 100 min. In order to assess the sensitivity of chemiluminescence peptidase assay, the photoluminescence turn-on assay for peptidase us ing the same approach was conducted with 1 M BpPPE-SO3 -, 1 M KpNA and 1.7 g/mL peptidase. Note that less amount of polymer, substrate (quencher) and enzyme is used in th e photoluminescence peptidase assay, but it shows similar result as the chemiluminescence assay, i.e. the quenched fluorescence intensity is recovered to 50% of or iginal intensity after 100 min of incubation. Figure 5-11. Change of chemilu minescence intensity observed in the turn-on assay for peptidase at 25 C, em = 450 nm. Initial chemiluminescen ce (I) is normalized to 1. 6 M BpPPE-SO3 -, 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO in PBS (10 mM, pH 7.1)/CH3CN (20/80). After addition of 20 M Kp NA (Q) followed by addition of 40 g-mL-1 porcine intestinal peptidase, chemiluminescence intensity increase as a function of time from 1 to 100 minutes.

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173 Alkaline Phosphatase Chemilumine scence Turn-off Assay with PPE-CO2 A real-time fluorescence turn-off assay for ALP utilizing PPE-CO2 has been developed in Chapter 3 (Figure 3-4). The same approach was implemented using the chemiluminescence of PPE-CO2 as the signal transducer. The cartoon in Figure 5-12 illustrates the mechanism of the chemiluminescence turn-off assay for ALP. First, the chemiluminescence of PPE-CO2 is quenched efficiently by Cu2+ via a charge and/or energy transfer mechanism.209,210 Next, the quenched chemiluminescence is recovered upon addition of inorganic PPi to the PPE-CO2 -/Cu2+ solution. As mentioned in Chapter 3, emission re covery occurs because PPi strongly chelates Cu2+, thereby disrupting the PPE-CO2 -/Cu2+ complex. Finally, addition of ALP to the solution containing PPE-CO2 -/Cu2+/PPi initiates hydrolysis of PPi to Pi. The product, Pi, is unable to complex with Cu2+. As a result, the chemiluminescence of PPE-CO2 is quenched by the released Cu2+. Therefore, the activity of ALP is mon itored by decrease of the chemiluminescence intensity. Figure 5-12. Mechanism of chemiluminescence turn-off assay for ALP.

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174 The chemiluminescence turn-off assay for ALP was carried out in three steps. First, in a solution of Tris-HCl (10mM, pH 7.5)/CH3CN (50/50) containing 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO, the initial chem iluminescence intensity from 25 M PPE-CO2 is quenched significantly by the addition of 50 M Cu2+. Second, the quenched chemiluminescence is recovered to ~80% of the initia l intensity by the addition of 75 M PPi. Third, following addition of ALP to the chemiluminescence solution containing Cu2+ and PPi, hydrolysis of PPi is signaled by the decrease of the chemiluminescence intensity. The emission wavelength to record the chemiluminescence profile was set at 446 nm, which is the maximum emission wavelength achieved from the photoluminescence spectrum of PPE-CO2 in Tris-HCl/CH3CN (50/50). Figure 5-13A illustrates chemiluminescence spectroscopic changes observed after initiating the chemiluminescence ALP assay for 5 min. The assay was carried out with ALP concentrations ranging from 100 to 2000 nM at 37 C. It is evident that the change of chemiluminescence intensity after 5 min of inc ubation increases with increasing [ALP]. The inset graph displays a linear correlation between intensity change ( I0/ I ) and amount of ALP added, where I0 and I are initial and decreased chemilumi nescence intensities upon addition of ALP; the integrated area of chemiluminescen ce profile was used as the chemiluminescence intensity. The linear correlation suggests that it is possible to quantit atively determine the concentration of ALP by using the chemiluminescence assay. Figure 5-13B shows the decrease of chemiluminescence intensity in the presence of 400 nM ALP as a function of incubation time. The initial chemiluminescence intensity of PPE-CO2 at time 0 is normalized to 1. The relative chemiluminescence intensity during the assay de creases with increasing incubation time until reaching only 15% of initial intensity at 30 min. It is evident that most substrate (PPi) is hydrolyzed within firs t half of an hour.

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175 Figure 5-13. Changes of chemiluminescence intensity observed in the turn-off assay for ALP. A) Changes of chemiluminescence intensit y after 5 min of addition of different concentrations of ALP. Inset: Linear calib ration plot of chemiluminescence intensity decrease as a function of ALP concentr ation. B) Change of chemiluminescence intensity as a function of incubation ti me (0 30 min) after addition of 400 M ALP during turn-off assay for ALP. Conditions: 25 M PPE-CO2 -, 8 mM H2O2, 1.5 mM ImH, 0.3 mM TCPO, 50 M Cu2+ and 75 M PPi in Tris-HCl (10 mM, pH 7.5)/CH3CN (50/50), at 37 C, em = 446 nm. It is of interest to compare the chemilumi nescence ALP assay with the photoluminescence ALP assay which is described in Chapter 3. Both of them afford rapid detection of ALP activity

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176 with substrate in micromolar range, however, the photoluminescence ALP assay outperforms in terms of better detection limit (~5 nM ALP), which is lower than the smalle st [ALP] used in the chemiluminescence assay. Discussion We have developed the first chemilumines cence system for CPE based on the imidazolecatalyzed peroxyoxalate chemiluminescence reaction, in which TCPO is used as the aryl oxalate. It is simple in implementation, and it exhibi ts amplified quenching effect with comparable quenching efficiency to that of photoluminescence of CPE in the same solvent medium. Using superquenching mechanism, the CPE-based chemiluminescence assays were used to detect activity of peptidase and alkaline phosphatase. While the CPE-based chemiluminescence biosen sor affords accurate detection of analytes, its sensitivity is expected to be further im proved by modifying the chemiluminescence system. First, the organic solvent is required to dissolve TCPO. However it is advantageous to eliminate the organic solvent, because it induces the decr ease of the quenching efficiency and alters the buffer condition which is important to enzyme activity. Therefore, a new chemiluminescence reagent with better water solubility and stability is needed to substitute for TCPO. As shown in Figure 5-14, oxamide reagents with two su lfonate groups on the benzene rings are good candidates. They are specially designed for aqueous chemiluminescence reagent by Russell et al .248,249 Second, the chemiluminescence reaction woul d become more efficient if coupling it with advanced sample injection techniques such as flow inje ction or stopped-flow. These techniques provide rapid and reproducible mixing, th ereby affording effective chemiluminescence measurement and high sample throughput.140 By improving the sensitivity of chemiluminescence detection methods with these tw o modifications, plus its inherent advantages

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177 of simple and direct measurement, the ch emiluminescent CPE provides a new and powerful platform in the biosensor applicati on of conjugated polyelectrolytes. Figure 5-14. Structures of oxamide reagents. Experimental Materials The concentrated aqueous solution of BpPPE-SO3 and PPE-CO2 was diluted with water or buffer solution to a final con centration ranging from 10 to 750 M and 10 to 25 M, respectively. All chemicals were used as received, unless otherwise noted. Bis(2,4,6trichlorophenyl) oxalate (TCPO) and imidazole (ImH) were purchased from Acros. 1,1 Oxalyldiimidazole (ODI) was obtained from Sigma-Aldrich. Hydrogen peroxide (H2O2), 30% solution in water was purchased from Fi sher Chemical. The concentration of H2O2 was determined by titration with the solution of potassium permanganate prior to dilution. HPLCgrade acetonitrile (CH3CN) was obtained from Fisher and us ed without further purification. Water was distilled and then purif ied by using a Millipore purifica tion system. Stock solutions of 2.0 mM TCPO in CH3CN and 10.0 mM ImH in H2O were prepared immediately before their use. TCPO solution was stored in da rk prior to dilution and analysis.

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178 Methyl viologen dichloride hydrate (MV2+) and copper (II) chloride (Cu2+) were obtained from Sigma-Aldrich. Stock solutions of MV2+ and Cu2+ in water were prepared before the quenching test and adjusted to 1.0 mM. The substrate for alkaline phosphatase, sodium pyrophosphate (PPi), was purchased from J. T. Baker Chemical Company. The substrate for peptidase, L-lysine p-nitroanilide dihydrobromide (KpNA) and two enzymes, peptidase from porcine intestinal mucosa and alkaline phosphatase bovine intestinal mucosa (ALP) were purchased from Sigma-Aldrich. Bu ffer solution was prepared with reagent-grade material from Fisher. Stock solutions of enzymes in the approp riate buffers were prepared immediately before their use in the assays. Prior to peptidase chemiluminescence assay, Kp NA and peptidase were dissolved in the phosphate buffer solution (PBS, 10 mM, pH 7.1) and adjusted to 3.0 mM and 0.5 mg-mL-1, respectively as stock solutions. The peptid ase assay was conducted in the same buffer solution. Prior to ALP chemiluminescence assay, PPi and ALP were dissolved in Tris buffer solution (Tris-HCl, 10 mM, pH 7.5) and adjusted to 2.0 mM and 20 M as stock solution. The ALP assay was carried out in the same buffer solution. General Methods Chemiluminescence measurements In a typical chemiluminescence measurement, a 0.6 2.4 mL aliquot of aqueous solution containing 10 750 M BpPPE-SO3 or 10-25 M PPE-CO2 -, 10 40 mM H2O2 and 1.875 7.5 mM ImH was pipetted into the cuvette which was placed in spectrometer. A 0.6 2.4 mL aliquot of 0.375 mM 1.5 mM TCPO CH3CN solution was pipetted into above solution in dark to initiate the chemiluminescence reaction. The emitted light was detected simultaneously. The profile of chemiluminescence intensity vs. wavelength was recorded on a home-built spectrometer without switching on the excitation source. The spect rometer was equipped with a Triax 180 spectrograph (ISA -Spex) with liquid N2 cooled silicon CCD detector (EEV CCD chip,

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179 1024 128 pixels). The profile of chemiluminescence intensity vs. time was measured at a specific wavelength on a JOBI N YVON-SPEX Industries Fluor olog-3 spectrofluorometer (Model FL3-21) with the lamp turned off. The emission wavelength to record time profiles was chosen from maximum wavelengt h in the photoluminescence spectrum measured under the same condition but with the lamp turned on. The ch emiluminescence signal (intensity and duration) were acquired as a function of the co ncentration of each reagent (BpPPE-SO3 -, H2O2, ImH and TCPO) and ratio of solvents (H2O/CH3CN). The chemiluminescence intensity is the peak intensity in the chemiluminescence vs. time profile, and the chemiluminescence duration refers to the range of time from additi on of TCPO to the moment when the intensity stops decreasing. The concentration of each reagent shown in the results and discussion se ction is the calculated concentration after mixing different ratio of aqueous and CH3CN solutions. Quenching behavior The chemiluminescence quenching test was done by a series of titr ations of aqueous polymer (BpPPE-SO3 or PPE-CO2 -) solution containing H2O2 and ImH by the quenchers including MV2+, Cu2+ and KpNA. It was followed by addition of CH3CN solution of TCPO and measurement of chemiluminescence vs. time profile. The integrated area of profile was used as chemiluminesnce intensity. The photolum inescence quenching was conducted by the same method except TCPO was not added. Peptidase chemiluminescence assay with BpPPE-SO3 The peptidase assay was conducted at 25 C. The initial chemilumine scence intensity of BpPPE-SO3 was measured first. The substrate, KpNA was then added to another aqueous solution containing BpPPE-SO3 -, H2O2 and ImH, and this solution was incubated for 10 minutes. The quenched chemiluminescence intensity was then recorded. Subsequently, an aliquot of the peptidase solution was added to a nother fresh solution of BpPPE-SO3 -/H2O2/ImH/Kp NA, and

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180 the chemiluminescence profiles were measured as a function of incubation time. The incubation time refers to the time after the addition of pep tidase into the aqueous solution but before mixing with CH3CN solution of TCPO to initiate chemilumi nescence reaction. The integrated area of profile was used as the chemiluminescence intensity. Alkaline phosphatase chemiluminescence assay with PPE-CO2 The ALP assay was carried out at 37 C. The initial chemiluminescence intensity of PPECO2 was recorded first. The Cu2+ solution was then added to another PPE-CO2 -/H2O2/ImH mixture, the solution was incubated for 10 min, and the quenched chemiluminescence intensity was measured. Subsequently the PPi solution was introduced to another fresh solution containing PPE-CO2 -/H2O2/ImH/Cu2+, this mixture was incubated for 10 min, and the recovered chemiluminescence intensity was recorded. Finally, an aliquot of ALP solution was added to another aqueous soluti on containing PPE-CO2 -/H2O2/ImH/Cu2+/PPi, and the chemiluminescence profiles were measured as a function of incubation time or as a function of the concentration of ALP. The incubation time refers to the period afte r the addition of ALP into the aqueous solution but before mixing with CH3CN solution of TCPO to initiate chemiluminescence reaction. The integrated area of profile was used as the chemiluminescence intensity.

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181 CHAPTER 6 CONCLUSION In the previo us chapters, the design and development of optical biosensors using functionalized poly(para -phenylene ethynylene) (PPE) have been presented. Chapter 2 4 described the real-time fluorescent assays for phospholipase C(PLC), alkaline phosphatase (ALP) and adenylate kinase (ADK), respectiv ely using two PPE de rivatives, biphenyl poly(phenylene ethynylene ) solfonate (BpPPE-SO3 -) and poly(phenylene ethynylene) carboxylate (PPE-CO2 -). These assays were designed based on two different sensor mechanisms: conformation change and superquenching mechanis ms. By taking advantage of amplified signal response of conjugated polyelectro lyte (CPE) to aggregation and/ or the presence of charged fluorescence quenchers, all of three CPE-based a ssays exhibit good sensi tivity, good specificity and most importantly, flexibility of end-point and real-time measurements to easily study enzyme activity and inhibition. In Chapter 5, in order to expand the PPE-based optical sensor to the chemiluminescence field, the first chemiluminescence system for water soluble CPE was developed based on the imidazole-catalyzed peroxyoxalate chemilumine scence reaction. The chemiluminescence system of CPE is simple in instrumentation with no need for excitation source and allows for fast and direct measur ement activities of peptidase and alkaline phosphatase. Fluorescence PLC Assay Based on Conformation Change The BpPPE-SO3 --based fluorescence turn-off assay for PLC is developed based on the reversible interaction between BpPPE-SO3 and the natural substrate of PLC, phosphatidylcholine. The fluores cence intensity of BpPPE-SO3 in water is increased substantially by the addition of the phospholipid du e to the formation of a CPE-lipid complex. Incubation of the CPE-lipid complex with the enzyme PLC causes the fluorescence intensity to

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182 decrease (turn-off sensor); the response ar ises due to PLC-catalyzed hydrolysis of the phosphatidylcholine, which effectively disrupts the CPE-lipid complex. This novel fluorescence turn-off assay affords real-time detection of PL C activity with good specifi city and functions at substrate concentration in the micromolar range and at very low enzyme concentrations with detection limit of 0.5 nM PLC. The high sensitivity of the sensor is due to the ability of the fluorescent CPE to exhibit amplified res ponse to aggregation change induced by phosphatidylcholine. By using an ex-situ calibration curve, the real-time fluorescence intensity from BpPPE-SO3 can be converted to substrate concentration, allowing PLC catalyzed reaction rates, kinetic parameters as well as the activati on and inhibition behaviors to be determined. The kinetic parameters are found to be consistent with values found in previous literature. Although the work demonstrates the turn -off assay with a specific conjugated polyelectrolyte, substrate and enzyme, this met hod is quite general and can very likely be extended to other lipases and to different lipid substrates. For example, a similar conjugated polyelectrolyte based turn-off assay can be design ed to detect the activity of sphingomyelinase using sphingomyelin as a substrate. If an anionic phospholipid, phosphatidylinositol biphosphate is used as a substrate to monitor phosphatidy linositol phospholipase C activity, a cationic CPE can be utilized. It may also be possible to deve lop a similar assay to monitor DNA transfection, since some natural and synthetic cationic lipi ds have been utilized in gene transfer.250,251 Fluorescent ALP and ADK Assays Based on Superquenching The PPE-CO2 --based fluorescence assays for ALP and ADK are developed based on fluorescence recovery of Cu2+-quenched CPE by polyphosphates. In particular, The fluorescence of PPE-CO2 is quenched very efficien tly via the addition of Cu2+, and addition of PPi or ATP into the weakly fluorescent solution of PPE-CO2 and Cu2+ induces recovery of the polymers fluorescence; the recovery occurs b ecause PPi or ATP complexes with Cu2+, effectively

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183 sequestering the ion so that it cannot bind to the carboxylate groups of the polymer. The recovery response is very selective to PPi and ATP over the other phosphate derivatives such as Pi, ADP and AMP. An ex-situ calibration curve is developed that relates the extent of fluorescence recovery to concentration of PPi or ATP. Using the PPE-CO2 --Cu2+ system as the signal transducer, the real-time fluorescence assays for ALP and ADK are developed, respectively. PPi is used as the natural substrates in the ALP turn -off assay, and ATP is applied as either natural substrate (turn-off assay) or pr oduct (turn-on assay) in the ADK a ssay. The assays operate with PPi or ATP in the micromolar range and offer a straight-forward and rapi d detection of ALP or ADK activity with the enzyme present in the nano molar concentration rang e, operating either in an end-point or real-time format. The high sensitiv ity of the biosensors is due to the amplified response of PPE-CO2 to the presence of quencher (supe rquenching effect). The analytical detection limit for ALP and ADK is 5 nM and 42 nM, respectively. Kinetic parameters and activation/inhibition behaviors are derived by converting time-de pendent fluorescence intensity into PPi or ATP concentration, thus allowing the calculation of the initial reaction rates ( v0). Non-specific fluorescence response is observed concomitant to addition of other proteins to the assay solution; however, the signal response is de monstrated to arise from the specific enzyme catalyzed reaction. Similar to the fluorescence PL C assay method, the CPE-Cu2+-polyphosphate sensing platform is quite general and can be extended to other substrate/enzyme systems in which PPi or adenosine phosphates participate as substrate, ac tivator or inhibitor. For example, the similar turn-off assay could be developed for a hexokinase which is capable of transferring a phosphate group from ATP to hexoses (e.g., D -glucose, D -mannose, and D -fructose) and producing ADP and corresponding suga r-phosphate derivative.252 Note that the assays operate with natural

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184 substrates in physiological condition; therefore, it is also possible to utilize these assays to study the function of enzymes in vivo and to develop a diagnostic t ool for certain enzyme-related process (e.g. ALP-related skeletal mineralization196,197 and vascular calcification198,199) and diseases (e.g. ADK-related hemolyitic anemia) Chemiluminescent Conjugated Polyelectrolytes The first chemiluminescence system for CPE is developed based on the imidazolecatalyzed peroxyoxalate chemiluminescence reaction, in which bis(2,4,6-trichlorophenyl) oxalate (TCPO) is used as the aryl oxalate and BpPPE-SO3 and PPE-CO2 are served as fluorophores. The effects on chemiluminescence signals of con centrations of oxalate, catalyst, oxidant and fluorophore as well as solvent composition are demonstr ated to be consistent with the kinetics of peroxyoxalate chemiluminescence reaction. The ch emiluminescence of CPE exhibits amplified quenching by small oppositely charged quenchers w ith comparable quenching efficiency to that of photoluminescence of CPE in the same solv ent medium. The amplified chemiluminescence quenching of CPE provides a platform for its applic ation in biosensor area s. For example, using superquenching mechanism, the activities of two enzymes, peptidase and alkaline phosphatase are detected by measuring the ch ange of chemiluminescence intens ity of the polymer during the turn-off assays. The chemiluminescence-based biosensor using CPE as the signal transducer is simple in instrumentation with no need for ex citation source and allows for fast and direct measurement. It successfully extends the chemiluminescen ce biological application beyond the H2O2-related detection. The sensing sensitivity of CPE-ba sed chemiluminescence assay could be improved by using water stable chemiluminescence reagents and applying more e fficient sample mixing techniques such as flow injection and stopped-flow.

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185 Outlook of CPE-based Sensor Application In light of the optical biosensor work presen ted in this dissertation as well the sensory systems developed by other research groups, CPEs ha ve been clearly demonstrated as one of the most useful chemical platform in the sensor technology. The signal amplification displayed by these fascinating materials is the most important advantage over the other sensory methods. Apparently, the field of CPE-based optical se nsor is far from mature, and there are many potential analytes that would be detected with this sensor sy stem. For example, the CPE-based saccharide sensor developed by Schanze et al displays selectivity towards D -fructose over D glucose.83,84 Therefore, the fluorescence assay could be developed to detect enzymes which catalyze the conversion between fructose and glucose, such as glucose isomerase or phosphoglucose isomerase. In addition, we focuse d on the the fluorescence enzyme assays based on conformation change and superquenching mech anisms in this dissertation, however, the enzyme assay could also be designed based on fluorescence resonance energy transfer (FRET) mechanism. A specially synthesized substrate la beled with a dye donor or acceptor is needed to interact with CPE. For exam ple, a modified phospholipid w ith nitro-benzoxadiazole (NBD)labeled acyl chains could associate with CPE, initate FRET and induce the optical change. Hydrolysis of NBD-labeled phosph olipid catalyzed by phospholipase A2 (PLA2) disfavors the FRET. Therefore, the activity of PLA2 could be monitored by the fluorescence change of the polymer. In summary, CPE-based optical sensor is very flexible in design, quite simple in implementation and highly sensitive in dete ction. Eventhough the pr esence of non-specific interaction is the major challenge to extend its ap plication to the real-world samples; we still believe that there is a great promise for the inco rporation of CPE-based optical sensors into the routine protocols in the chem istry or biology laboratories.

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186 APPENDIX A FLUORESCENCE INTENSITY CORRECTIO N FOR PHOTOB LEACHING OF CPES Photobleaching of CPEs is observed when CPE is irradiated continuously in the fluorescence spectrophotometer for a long period of time. With the increase of the concentration of CPEs or irradiation time, photobleaching is more pronounced. In addition, the photobleaching rate is increased in concentrated Tris bu ffer solution. For example, in the BpPPE-SO3 --based PLC turn-off assay, 10 min of continuous irra diation induces ~8% of photobleaching for an aqueous solution of BpPPE-SO3 (c = 15 M); however, 20% of photobl eaching is observed for 50 mM Tris-HCl buffer solution (pH 7.4) w ith the same concentration of BpPPE-SO3 -. Added Ca2+ and/or 10CPC result in the increase of photobl eaching to a small extent (< 5%). When the initial rate of the reaction, v0, is very low (e.g. < 0.1 M/s), the contribution of photobleaching to the decrease of time-dependent fluorescence intensity curve is significant, so the correction for photobleaching is necessary. The correcti on was carried out by Equation A-1, b 0 tct b t I II I (A-1) where Ib0 and Ibt are blank fluorescence intensities at time 0 and time t of a blank assay in which no enzyme is added. It and Itc are uncorrected and corrected fluorescence intensities at time t during the assay in which a speci fic amount of enzyme is introduced. As mentioned before, the blank fluorescence intensity, Ibt, decreases with time because of photobleaching of polymer. The decrease ratio at each time t is derived by dividing the Ib0 (t = 0) by each Ibt at time t. The assay fluorescence intensity after addition of enzyme, It, is corrected for photobleaching by multiplying the corresponding decreasing ratio of Ibt at each time t. Both Ibt and It were recorded under the same conditions except for the addition of enzyme into the system to measure It. After correction for photobleaching, the correct ed fluorescence intensity Itc at each time t is derived.

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187 To validate the reliability of the correction method, the BpPPE-SO3 --based PLC turn-off assay was monitored by sampling the fluorescence in tensity by another method. Specifically, the fluorescence from the assay solution was intermitte ntly monitored every 20 sec in 10 min instead of measured continuously. Therefore, the fluor escence intensity was re corded every 20 sec instead of every second. The constant excita tion and emission wavelengths were kept 400 nm and 460 nm, respectively. The photobleaching problem is strongly alleviated under these conditions because of the considerably lower ex posure time. The data is used to calculate v0. The rates calculated from this intermittent irradiatio n pattern are compared with those derived from the continuously irradiation assay solu tions. Table A-1 shows the comparison of v0 derived by two methods. It is shown that two sets of da ta are in a good agreement, which validates the reliability of the method used for correcting photobleaching. Table A-1 Comparison of initial rates of reaction derived by two patterns. [PLC] (nM)a v0 ( M/s) Continuous Irradiation Patternb Intermittent Irradiation Patternc 10 0.0562 0.0587 15 0.0843 0.0853 20 0.1250 0.1286 Note: a 15 M of BpPPE-SO3 and 30 M of initial 10CPC in 50 mM Tris-HCl (pH 7.4) with 2 mM Ca2+ at 37 C, ex = 400 nm. b The assay solution was continuously irradiated for 10 min. c The assay solution was irradiated every 20 sec for 10 min.

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188 APPENDIX B LINEAR FITTED VS. SIGMOIDA L FITTED CALIBRATION PLOTS In the as says for PLC, ALP and ADK as disc ussed in Chapter 2 4, the linear fitted calibration curves (Figure 2-5, 3-8 and 4-5) were used to derive time-dependent substrate concentration from fluorescence intensity. However, it is more proper to regard the change of fluorescence intensity with substrate concentrat ion as sigmoid curves due to the cooperative interaction between substrate and other species, such as polymer (PLC assay) and polymer-metal complex (ALP and ADK assay). Therefore, the da ta shown in Figure 2-5 (PLC assay) and Figure 3-8 (ALP assay) were fitted using si gmoid function which is expressed as 0x-x () ba y 1 e (B-1) where x is the substrate concentration ([10CPC] or [PPi]), y is the fluorescence intensity change ( I / Ip or Log ( Ir/ Iq)), and a, b, x0 are three parameters. Figure B-1 illustrates the sigmoidal fitted calibration plots for PLC and ALP assays, respectively. The values for three parameters (a, b and x0) and correlation coefficient (R2) are summarized in Table B-1. It is evident that both plots feature a very good fit and afford R2 ~ 0.99. Table B-1 Parameters and correlation coefficient for sigmoidal fitted calibration plots in PLC and ALP Assay Assays PLC Assay ALP Assay a 9.198 1.540 b 24.254 46.548 x0 46.4487 127.656 R2 0.995 0.989

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189 Figure B-1. Sigmoidal fitted calibration plots. A) PLC assay: fluorescence intensity increase of 15 BpPPE-SO3 observed upon titration of 10CPC in 50 mM Tris-HCl (pH 7.4) with 2 mM Ca2+ at 37 C, ex = 400 nm. B) ALP assay: fluor escence recovery induced by addition of PPi. Conditions: solution contains 3 M PPE-CO2 -, 200 M Cu2+ and 10 mM Tris-HCl buffer (pH 7.5) at 37 C, ex= 390 nm, em= 525 nm.

PAGE 190

190 In order to compare the accuracy of liner f itted and sigmoidal fitted calibration plots, we calculated the relative error (% Er ror) using Equation B-2 as follows Measured Value Fitted Value % Error 100% Fitted Value (B-2) where % Error represents the deviation of measured value from fitted value and serves as a direct measurement of fitted accuracy. Figure B-2 displays % Error at different substrate concentrations in the PLC and ALP assays using both fitting met hod. Note that regardless of fitting function used, the fitting errors are relatively larger at lo wer substrate concentrations compared with those at higher concentrations. In addi tion, % Error generated from linear fitted method is greater than that from sigmoidal fitted method, especially in the ALP assay (F igure B-2B), where % Error is up to 20% when PPi is 50 M. Therefore, sigmoidal fitted calibration curve affords better accuracy. However, the values of three parameters (a, b and x0) vary with many factors including buffer, polymer and activator concentrations. C onsequently, a certain equation with constant parameters to calculate substrate concentrati on from fluorescence intensity is unrealiable. In contrast, the linear fitted calibration curve is mu ch simpler to incorporate with enzyme assays. Even though the only parameter (k, slope of the curve) in the linear f unction also depends on similar influencing factors, the equations for de termining substrate concentration (Equation 2-1 and 3-1) cancel this parameter. As a result, the applicability of these equa tions is independent of assay conditions. Therefore, we used linear f itted calibration plots in our experiments.

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191 Figure B-2. Comparsion of relative error at different substrat e concentrations from linear fitted and sigmoidal fitted calibraton plots. A) % Error vs. [10CPC] in the PLC assay. B) % Error vs. [PPi] in the ALP assay.

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205 BIOGRAPHICAL SKETCH Yan (Nancy) Liu was born in Shandong province, China. She lived in Jina n, the City of Spring, until she graduated from Jinan Foreign Language School in 1997. In September of that year, she went to Shanghai, the biggest city in China, where she started her academic career in chemistry. She obtained her bachelors degree in pharmaceutical engineering in 2001 and her masters degree in applied chemistry in 2004, from East China University of Science and Technology. On January 2, 2004, three days afte r her defense for the masters degree, Yan (Nancy) came to the University of Florida and joined the gr oup of Dr. Kirk S. Schanze to continue her education and pursue her Ph.D. in anal ytical chemistry. In the past five years, she researched in the amazing area of fluorescent/chem iluminescent biosensor applications of watersoluble conjugated polymers. In addition, Yan (Nancy) also obtained a masters degree in management in August 2008, from the same unive rsity. After finishing her Ph.D., Yan (Nancy) will start her industrial career in Syngenta, an agribusiness company, located in Greensboro, NC.