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Alkaline Phosphatase Sensors Based on Amplified Quenching of Conjugated Polyelectrolytes

Permanent Link: http://ufdc.ufl.edu/UFE0024109/00001

Material Information

Title: Alkaline Phosphatase Sensors Based on Amplified Quenching of Conjugated Polyelectrolytes
Physical Description: 1 online resource (79 p.)
Language: english
Creator: Huang, Lijuan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: amplified, conjugated
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: We investigated chemiluminescent and fluorescent sensors for alkaline phosphatase (ALP) based on the amplified quenching of functionalized poly(para-phenylene ethynylene)s, including poly(phenylene ethynylene) carboxylate and a cationic poly(phenylene ethynylenes) which has dendritic charged amino groups. Although they relied on two different emission methods, these two sensors shared the same quenching-unquenching mechanisms and both featured high sensitivity and good selectivity. First, a chemiluminescent sensor was developed to detect ALP based on the amplified quenching of PPE-CO2- by Cu2+ and the interaction between Cu2+ and pyrophosphate, a substrate of ALP. A peroxyoxalate chemiluminescence (POCL) system which consists of bis(2,4,6-trichlorophenyl) oxalate (TCPO) as aryl oxalate, imidazole as catalyst, H2O2 as oxidant, and PPE-CO2- as fluorophore has been developed and optimized. The quenching of chemiluminescence of PPE-CO2- by Cu2+ was more sensitive than that of fluorescence under the same conditions. This chemiluminescent sensor has been successfully applied to qualitatively and quantitatively detect ALP activity. Second, a convenient fluorescent assay was developed to detect the pyrophosphatase activity of ALP based on the direct interaction between PPE-dNH3Cl and pyrophosphate. The fluorescence of cationic dendritic PPE-dNH3Cl was sensitive to anions with more negative charges including pyrophosphate, ATP and ADP, while not affected by anions with less negative charges including phosphate and AMP. The PPE-dNH3Cl/PPi system has been employed to detect the enzymatic activity of ALP by monitoring the hydrolysis of pyrophosphate which induces rapid and sensitive fluorescence responses. The calibration plot was directly derived from the linear range of Stern-Volmer plot, which easily converted real-time fluorescence plots into enzymatic reaction processing curves. This ALP turn-on assay allowed the derivation of kinetic parameters and inhibition constant of phosphate for the ALP activities.
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 Lijuan Huang.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Schanze, Kirk S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024109:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024109/00001

Material Information

Title: Alkaline Phosphatase Sensors Based on Amplified Quenching of Conjugated Polyelectrolytes
Physical Description: 1 online resource (79 p.)
Language: english
Creator: Huang, Lijuan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: amplified, conjugated
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: We investigated chemiluminescent and fluorescent sensors for alkaline phosphatase (ALP) based on the amplified quenching of functionalized poly(para-phenylene ethynylene)s, including poly(phenylene ethynylene) carboxylate and a cationic poly(phenylene ethynylenes) which has dendritic charged amino groups. Although they relied on two different emission methods, these two sensors shared the same quenching-unquenching mechanisms and both featured high sensitivity and good selectivity. First, a chemiluminescent sensor was developed to detect ALP based on the amplified quenching of PPE-CO2- by Cu2+ and the interaction between Cu2+ and pyrophosphate, a substrate of ALP. A peroxyoxalate chemiluminescence (POCL) system which consists of bis(2,4,6-trichlorophenyl) oxalate (TCPO) as aryl oxalate, imidazole as catalyst, H2O2 as oxidant, and PPE-CO2- as fluorophore has been developed and optimized. The quenching of chemiluminescence of PPE-CO2- by Cu2+ was more sensitive than that of fluorescence under the same conditions. This chemiluminescent sensor has been successfully applied to qualitatively and quantitatively detect ALP activity. Second, a convenient fluorescent assay was developed to detect the pyrophosphatase activity of ALP based on the direct interaction between PPE-dNH3Cl and pyrophosphate. The fluorescence of cationic dendritic PPE-dNH3Cl was sensitive to anions with more negative charges including pyrophosphate, ATP and ADP, while not affected by anions with less negative charges including phosphate and AMP. The PPE-dNH3Cl/PPi system has been employed to detect the enzymatic activity of ALP by monitoring the hydrolysis of pyrophosphate which induces rapid and sensitive fluorescence responses. The calibration plot was directly derived from the linear range of Stern-Volmer plot, which easily converted real-time fluorescence plots into enzymatic reaction processing curves. This ALP turn-on assay allowed the derivation of kinetic parameters and inhibition constant of phosphate for the ALP activities.
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 Lijuan Huang.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Schanze, Kirk S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024109:00001


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1 ALKALINE PHOSPHATASE SENSORS BASED ON AMPLIFIED QUENCHING OF CONJUGATED POLYELECTROLYTES By LIJUAN HUANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Lijuan Huang

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3 To my parents

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4 ACKNOWLEDGMENTS I would like to express my grat itude to all those who helped, encouraged and instructed me during my past years of MS studies in UF. Withou t them, I could not have completed this thesis. First of all, I would like to expr ess my great appreciation to my s upervisor, Dr. Kirk S. Schanze, for his encouragement and support throughout this wo rk. He guided me to this fantastic area of conjugated polymers and biosensors, which I barely knew before I joined his group. He encouraged me to learn new techniques and be an independent thinker. His enthusiasm for science and his kindness to people have a remarkab le impact on my current studies and future careers. I also would like to thank current and form er members of the Schanze group for creating an excellent working environment. Special thanks are given to Dr. Yan Liu, who trained me to use the instruments, and also shared her experience and knowledge in the biosensor research. I have learnt a lot from her. I would like to thank Dr. Xiaoyong Zhao for synthesis of the wonderful conjugated polymers and discovered their sensing potentials. His work is the basis of the research described in this th esis. I would also like to tha nk Dr. Hui Jiang for his willingness to teach and help others. Whenever I have questio ns in the photochemistry, he is always there with answers. I give my thanks to Julia keller and Abigail Shelton for managing the orders for the group. I also want to thanks many other pe ople, Dr. Kye-Young Kim, Johnathan Sommer, Dr. John Peak, Yongjun Li, Jarret Vella, Emine De mir, Eunkyung Ji, Seuong-Ho Lee, Zhuo Chen, Dongping Xie, Chen Liao, Dr. Richard Farley, Dr. Katsu Ogawa. My research and life in the lab become easier with their help and kindness. I am also thankful to Dr. Ben Smith and Dr. Linda Bloom for being my committee members. Finally, I am grateful to my parents. They have provided me the best conditions for my education and always teach me to be an optimis tic person. I would not be able to be anywhere

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5 without their unselfish love and support. I want to thank my boyf riend for always being there for me in the past years, especially in hard times.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF FIGURES................................................................................................................ .........8ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION..................................................................................................................12 Conjugated Polyelectrolytes...................................................................................................12Fluorescence Quenching.........................................................................................................15Amplified Quenching of C onjugated Polyelectrolytes...........................................................18Aggregation of CPEs............................................................................................................ ..22Applications of Conjugated Po lyelectrolytes as Sensors........................................................24Quencher Induced Quenching-Unquenching Mechanism...............................................25Chain Conformation Perturbation Mechanism................................................................27Fluorescence Resonance Energy Tr ansfer (FRET) Mechanism.....................................29CPE-based Real-time Enzymatic Activity Assay...................................................................32Alkaline Phosphatase Assay...................................................................................................342 ALKALINE PHOSPHATASE SENSORS USING CHEMILUMINESCENT CONJUGATED POLYMER..................................................................................................37Introduction................................................................................................................... ..........37Results and Discussion......................................................................................................... ..40Photophysical Property of PPECO2.................................................................................40Chemiluminescence of PPECO2......................................................................................40Quenching Studies...........................................................................................................42Alkaline Phosphatase Chemiluminescence Turn-off Assay............................................44Discussion..................................................................................................................... ...48Experimental................................................................................................................... ........49Chemicals...................................................................................................................... ..49Chemiluminescence Measurements................................................................................50Quenching Behavior........................................................................................................50Alkaline Phosphatase Chemiluminescence Assay with PPECO2...................................513 ALKALINE PHOSPHATASE SENSORS USING FLUORESCENT DENDRITIC POLYMER........................................................................................................................ .....52Introduction................................................................................................................... ..........52Results and Discussion......................................................................................................... ..55Overview of Alkaline Phosphatase/PPi Assay................................................................55Quenching Studies of PPE-dNH3Cl by PPi.....................................................................58

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7 Real Time ALP Turn-on Assay.......................................................................................60Kinetic Studies of ALP Turn-on As say for Pyrophosphatase Activities........................62Inhibition Studies of the ALP Activity............................................................................64Selectivity of PPE-dNH3Cl/PPi Turn-on Assay for ALP................................................65Discussion..................................................................................................................... ...66Experimental................................................................................................................... ........67Materials...................................................................................................................... ....67Instrumentation................................................................................................................68Fluorescence Assays........................................................................................................68Calculation of Kinetic Parameters and the Inhibition Constants.....................................694 CONCLUSION..................................................................................................................... ..71Chemiluminescent Conjugated Polyelectrolyte......................................................................71Fluorescent Real-Time ALP Assay........................................................................................72LIST OF REFERENCES............................................................................................................. ..74BIOGRAPHICAL SKETCH.........................................................................................................79

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8 LIST OF FIGURES Figure page 1-1 Structures of commonly used CPs.....................................................................................131-2 Examples of PPEs with diffe rent structural modifications................................................141-3 Mechanism of fluorescence quenching..............................................................................151-4 The S-V plot of the combined dynamic and static quenching...........................................171-5 Structures of neutral PE m onomer (1) and neutral PPEs (2).............................................181-6 Mechanism of amplified quenching...................................................................................191-7 Amplification of fluores cence quenching sensitivity........................................................211-8 Absorption (left) and fluorescence (right) of PPE-SO3 -....................................................231-9 Proposed aggregation modes of cationic polymers...........................................................241-10 Biosensor application based on QTL system.....................................................................261-11 Formation of polythiophene/singl e-stranded nucleic acid duplex and polythiophene/hybridized nucleic acid triplex forms........................................................281-12 The PNA/CPE assay for ss-DNA detection.......................................................................291-13 Mechanism of POCL reaction...........................................................................................311-14 Enzyme kinetics measured by using PPE SO3/Bz-FVR-pNA/thrombin assay system......331-15 Mechanism of fluorescent ALP assay................................................................................362-1 Optimization of CL signals................................................................................................382-2 Structures of PPECO2 (a) and TCPO (b)...........................................................................402-3 CL intensity of PPE-CO2 -..................................................................................................412-4 Fluorescence intensity A) and chemiluminescence intensity B) of PPE-CO2 upon titration of Cu2+ and S-V plots C)......................................................................................432-5 Mechanism of CL turn-off assay for ALP.........................................................................442-6 Chemiluminscence intensity quenched by Cu2+ and then recovered by PPi.....................452-7 Changes of chemiluminescence intensity observed in the turn-off assay for ALP...........46

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9 2-8 Changes of CL intensity as a function of incubation time (0 30 min) after addition of 400 nM ALP during turn-off assay for ALP.................................................................473-1 Four major subclasses of dendritic polymers.....................................................................523-2 Structure of PPE-dNH3Cl...................................................................................................533-3 Absorption (a) and fluorescence (b) spectra of PPE-dNH3Cl in water as a function of pH............................................................................................................................. ..........543-4 Absorption and fluorescence spectra of PPE-dNH3Cl upon titration of PPi and Pi...........553-5 Mechanism of ALP turn-on sensor using PPE-dNH3Cl.................................................573-6 Fluorescence changes upon titration of PPi into PPE-dNH3Cl and Stern-Volmer plot.....593-7 Fluorescence changes observed in the ALP turn-on assay...............................................603-8 Decrease of [PPi] during the enzymatic reaction in the ALP turn-on assay with varying ALP concentrations..............................................................................................613-9 Natural logarithm of the concentration of substrate for ALP activ ity as a function of reaction time plotted for diffe rent enzyme concentrations................................................633-10 Concentration of the hydrolysis product Pi as a function of tim e at various initial substrate concentrations in ALP catalyzed reaction system..............................................633-11 Inhibition of ALP activity by inorganic phosphate...........................................................643-12 The fluorescence responses of PPE-dNH3Cl/PPi (10 M/20 M) to various proteins.....65

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10 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ALKALINE PHOSPHATASE SENSORS BASED ON AMPLIFIED QUENCHING OF CONJUGATED POLYELECTROLYTES By Lijuan Huang May 2009 Chair: Kirk S. Schanze Major: Chemistry We investigated chemiluminescent and fluores cent sensors for alkaline phosphatase (ALP) based on the amplified quenching of functionali zed poly(para-phenylene ethynylene)s, including poly(phenylene ethynylene) car boxylate and a cationic poly(phenylene ethynylenes) which has dendritic charged amino groups. Although they re lied on two different emission methods, these two sensors shared the same quenching-unquenc hing mechanisms and both featured high sensitivity and good selectivity. First, a chemiluminescent sensor was devel oped to detect ALP based on the amplified quenching of PPE-CO2 by Cu2+ and the interaction between Cu2+ and pyrophosphate, a substrate of ALP. A peroxyoxalate chemiluminescence (P OCL) system which consists of bis(2,4,6trichlorophenyl) oxalate (TCPO) as ar yl oxalate, imidazole as catalyst, H2O2 as oxidant, and PPE-CO2 as fluorophore has been developed and optimized. The quenching of chemiluminescence of PPE-CO2 by Cu2+ was more sensitive than that of fluorescence under the same conditions. This chemiluminescent sensor has been successfully applied to qualitatively and quantitatively detect ALP activity. Second, a convenient fluorescent assay was developed to detect the pyrophosphatase activity of ALP based on the di rect interaction between PPE-dNH3Cl and pyrophosphate. The

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11 fluorescence of cationic dendritic PPE-dNH3Cl was sensitive to anions with more negative charges including pyrophosphate, ATP and ADP, while not affected by anions with less negative charges including phosphate and AMP. The PPE-dNH3Cl/PPi system has been employed to detect the enzymatic activity of ALP by mon itoring the hydrolysis of pyrophosphate which induces rapid and sensitive fluorescence respons es. The calibration plot was directly derived from the linear range of Stern-Volmer plot, wh ich easily converted real-time fluorescence plots into enzymatic reaction processing curves. This ALP turn-on assay allowed the derivation of kinetic parameters and inhibition consta nt of phosphate for the ALP activities.

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12 CHAPTER 1 INTRODUCTION Conjugated Polyelectrolytes Conjugated polymers (CPs) have been develope d and widely used since Shirakawa et al.1 discovered in 1977 that polyacetylenes obtaine d unusual conductivity, as high as 10 million times, upon halogen doping. The stable charge-transfer complexes were believed to be formed during the halogen doping to achieve the systematica lly controllable electri cal properties. As the first step of making plastics elec trically conductive, this discovery led to the 2000 Nobel Prize in Chemistry, which was awarded to Hideki Shirakwa of the University of Tsukuba in Japan, Alan MacDiarmid of the University of Pennsylvani a at Philadelphia and Alan Heeger of the University of California at Santa Barbara.2,3 The past decades w itnessed revolutionary applications of conducting polymers, such as flat panel displays using OLEDs,4 light-emitting electrochemical cells (LECs),5 polymer solar cells,6 field-effect transistors(FETs),7 plastic lasers,8 and chemical and bio-sensors.9,10 Conjugated polymers (CPs) are chain-like compounds with alternating double and single bonds as their backbones. CPs feature the fant astic optoelectronic/re dox properties because whenever excess charges are on the polymer chains, the charges can hop along the conjugated backbones easily. Neutral CPs are normally wide band gap organic semiconductors that exhibit efficient absorption or emission at the band edge.1 The strong luminescence is related to the delocalization and polariza tion of the electronic structure. Du e to the extraordinary photophysical and electrochemical properties induced by the ch emical doping of CPs, many CPs (Figure 1-1) have been synthesized and investigated: polyacetylene,3 poly(para-phenylene) (PPP),11 poly(para-phenylene vinylene) (PPV),4,12 poly(para-phenylene ethynylene) (PPE),12 polythiophene (PT),7 and polypyrrole (PPy).13

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13 n n R R R R n R R n R n R R n PA PPP PPV PPE PT PF Figure 1-1. Structures of commonly used CPs Conjugated polyelectrolytes (CPEs) retain the high absorption co efficient (excellent light harvesting properties) and hi gh fluorescent quantum yields, which originate from their conjugated backbones.14 However, with attached ionic solub ilizing side-chains, such as sulfonate (-SO3 -), carboxylate (CO2 -), phosphate (PO3 2-) and alkyl ammonium (NR3 +), CPEs are soluble in water and other polar solvents. CPEs feature am plified quenching effect credited both to strong association to small molecular que ncher with opposite charges and to efficient exciton transport to quencher sites.9 These unique properties make CPEs a ttractive materials for highly sensitive fluorescence-based sensors for biol ogical and chemical targets.15-17 Poly(arylene ethynylene)s (PAEs) comprise an important segmental CPEs family. PAEs share the same backbone of c onjugated ethynyl linked aromatic or heteroaromatic rings. Typically PAEs are insulators in the neutral state but became conductive by either oxidization or reduction of the polymers -electron system. Their semiconduc ting properties have generated some interest in device applications of electroluminescent polymers. However, PAEs photophysical properties and correspondi ng applications in TNT detection18,19 and biological sensors20,21 make them one of the most important cla sses of conjugated polymers. Considered as

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14 a molecular wire for exciton transport, PAEs ar e sensitive to small perturbations in their band structure and act as antennae for harvesting optical energy.22 As a representative class of PAEs, PPEs have been well studied and applied to many sensory systems, including methyl viologen salt sensors,21,23 TNT sensors,18,19 metal ion sensors,24-26 and PPi sensors.27 The PPEs not only feature superior photostability compared to other CPEs, such as PPVs, but they also demons trate different electroni c and optical properties from parent molecules upon structural modificat ions. The main chains of PPEs have three isomers: ortho-, metaand para-, defined by th eir different connectivity via the alkyne groups. Different aromatic building blocks can also be introduced into the c onjugated backbone to engineer the electronic properties (Figure 1-2).28 Also, variable side chains can be introduced to modify the polymer structures. All these strate gies are meant to make the PPEs amphiphilic, water-soluble, self-assembling, and able to form he lical structures or attached with receptors for targets. O CO2Na O NaO2C n O R n O n O HN O NH NH3 + NH3 + +H3N NH3 + +H3N +H3N O O O O O O O O R n O Figure 1-2. Examples of PPEs with di fferent structural modifications

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15 Fluorescence Quenching Fluorescence is widely used in chemical sensing not only because its sensitivity but also because of the availability of the diverse tran sduction schemes, which are based on changes in fluorescence intensity, fluorescence lifetime, and excitation or emission wavelength. The main reason for employing CPEs in the chemical sensory scheme is their amplified fluorescence quenching response to small perturbations. There are usually two fluorescence quenching mechanisms: static quench ing and dynamic quenching.29 Static and dynamic quenching both require molecular contact between the fluorophore and quencher. Ho wever, static quenching is due to the formation of a non-fluorescent comple x between the fluorophore and the quencher. On the other hand, dynamic quenching, which is also called collisional quenching, is due to random collisions between fluorophore and quencher mol ecules. So for static quenching the quencher must diffuse to the fluorophore within the lifetime of the excited states. (a) (b) (c) Figure 1-3. Mechanism of fluorescence quenchi ng. A) Dynamic quenching. B) Static quenching. C) Combination of dynamic and static quenching.29

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16 Figures 1.3a and 1.3b describe dynamic quenchi ng and static quenchi ng, respectively. In these figures, F* represents the excited fluorophore, Q represents quencher, and kq is the bimolecular quenching rate constant. There are several ways to dis tinguish these two mechanisms. First, the lifetime in static quenching does not change, because the fluoresce nce occurs from the uncomplexed fluorophore, which remains the same during the quenching process. However, the lifetime in dynamic quenching decreases in proportion to the intensit y. Second, static quenching is decreased at higher temperature due to disso ciation of weakly bound comple xes formed in the quenching process, while dynamic quenching is increased at higher temperature due to faster diffusion and collision. The Stern-Volmer (SV) equation is used to describe the emission intensity quenching: = 1 + Ksv [Q] (1-1) where I0 and I are fluorescence intensities in the abse nce and presence of quencher, respectively; [Q] is the quencher concentration; and Ksv is the Stern-Volmer quenchi ng constant. Both static and dynamic quenching can be expressed in term s of the SV equation. In dynamic quenching, the SV equation can be also written as: = = 1 + kq 0 [Q] = 1+ KD [Q] (1-2) where 0 and are the fluorescence lifetimes in the absence and presence of the quencher respectively. Here, Ksv is replaced by KD, which is equal to kq0. For diffusion-controlled quenching, the bimolecular quenching constant kq cannot exceed the diffusi on rate constant (ca. 1010 M-1 s-1). If kq is greater than 1010 M-1 s-1, usually static quenching is occurring. In static

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17 quenching, Ksv is represented by Ka as shown in the following e quation, which is the association constant for formation of the ground state complex [FQ]. = 1+ Ka [Q] (1-3) where Ka is the binding constant of the ground stat e association between the fluorophore and the quencher. Figure 1-4. The S-V plot of the co mbined dynamic and static quenching.29 When either static quenching or dynamic que nching dominates the quenching process, the SV plot of I0/I vs. [Q] is linear according to equati ons 1-1 and 1-2. When static quenching dominates, the slope of the plot equals Ka; while when dynamic quenching dominates, the slope gives KD. But in many cases fluorescence quenching involves both static and dynamic quenching mechanisms, as shown in Figure 1-3 the SV plots are nonlinear, upward-curved(Figure 1-4). A modified SV equation has been formulated to fit the quenching data in the upward-curving SV plots. It shows the combination of static and dynamic quenching effect on the relationship of I0/I and quencher concentration [Q]: = (1 + KD [Q]) (1+ KS [Q])

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18 The upward-curving quenching is sometimes e xplained by sphere of action. This means that the fluorophore and quenche r do not actually form a ground-state complex. Instead, when the quencher is adjacent to the fluorophore at th e moment of excitation, these closely spaced fluorophore-quencher pairs are im mediately quenched. So within the sphere of action, the probability of quenching is unity. In the fluorescence quenching of conjugated polymers, this effect is called superlinear quenching, which can arise from a variety of processes, including mixed static and dynamic quenching, variation in the association c onstant with quencher concentration, and chromophor e (or polymer) aggregation. Amplified Quenching of Conjugated Polyelectrolytes The dominant attribute that has driven interest in CPE-based sensors is their ability to produce superior amplified signal gains in response to minor perturbations, compared to small molecule indicators. The amplified fluoresce nce quenching of CPs has been called the molecular wire effect, which was fi rst described by Swagers group in 1995.22 The authors believed that the amplification is a result of the ability of the CPs delocalized electronic structure (i.e., energy bands) to facilitate efficient energy migr ation over large distances. To prove this proposal, the authors conduc ted parallel studies on neutral PPEs ( 2 ), featuring the bis( p -phenylene)-34-crown-10 (BPP) group on each re peating unit, as we ll as the corresponding monomer ( 1 ). Since BPP is an exceptional receptor of paraquat, a well-known electron transfer quenching agent, both the polymers and monome r displayed quenching resulting from the binding of paraquat, while polymer demonstrated greatly enhanced sensitivity compared to the monomer. Figure 1-5. Structures of neutral PE monomer (1) and neutral PPEs (2).

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19 Optical excitation creates an electron-hole pair, which migrates throughout the polymer (Figure 1-6). The electron transfer quenching occu rs when the electron encounters a receptor site occupied with a PQ2+ group. So the polymer needs only a small fraction of receptor sites occupied to effect complete quenching. Howe ver, for the monomer, every receptor must be occupied for complete quenching. The degree of sensitivity enhancement is determined by the radiative lifetime and the mobility of the excitatio ns in the polymer. Longer lifetimes and higher mobilities will produce longer average diffusion le ngths. If energy migration is rapid with respect to the fluorescence decay, then a single bi nding site to quencher is able to change the entire emission dramatically. If the diffusion length exceeds the polymers length, then an increase in molecular weight wi ll produce greater enhancements. Figure 1-6. Mechanism of amplified quenching by whic h the molecular wire receptor assembly can produce an enhancement in a fluores cence chemosensory response. Reprinted with permission from Swager et al.9 Swager et al.9 also demonstrated that the energy mi gration-based amplification is much greater in thin films than in solution. The phys ical state of the CPE has profound effects on its ability to amplify. In solution, we can presume that the polymers are in random coil form, and the excitons therefore under go a one-dimensional random walk, which is inefficient for amplification, because the exciton visits the sa me receptor many times. If the exciton can be

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20 made to undergo vectorial transport in a given di rection, then much highe r amplification factors can be achieved. Hence, either polymer aggregat es or very thin films are preferential for increasing rates of energy transfer in a thre e-dimensional manner. Close proximity of the neighboring polymer chains facil itates interchain ener gy migration, and CPEs often exhibit more planar conformations in thin films and aggregat es, and these conformations appear to promote exciton diffusion. This concept later led to th e development of sensors for nitroaromatics by Swagers group and commercialization of an explosive detectors, Fido R by ICx Technologies, Inc.15 Another striking discovery of the amplified quenching was reported by Whitten et al.16 in 1999 on their study of the fluorescen ce quenching of MPS-PPV by MV2+ (Figure 1-7). This is the first report of the amplified quenching of CP Es, in which the use of an anionic conjugated polymer leads to greater than million-fold amplification of the sensitivity relative to that of corresponding small conjugated molecu les with similar structures. The absorption and fluorescence spectra of M PS-PPV are similar to those studies of trans stilbene and its derivatives, but are shifted to longer wavelength due to the conjugation in polymers. The fluorescence of stilbene anal ogues can be quenched by electron-deficient MV2+ by the formation of don or-acceptor complexes.17 The quenching efficiency is enhanced when trans -stilbene or its amphiphilic derivatives are in corporated into anionic assemblies, such as micelles or bilayer vesicles. This can be attributed to a concen tration enhancement effect, in which the stilbene and viologen are assemble d by a combination of Coulombic and entropic interactions in a microphase, such that their local concentrations are greatly enhanced. Remarkably, addition of very low concentrations of MV2+ leads to noticeable changes in the absorption spectrum and a dramatic quenching of its fluorescence. The quenching constant (Ksv)

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21 is nearly four orders of magnitude greater than that for stilbene in micelles and six orders of magnitude greater than that for dilute stilbe ne solutions. Approximately, one molecule of MV2+ quenches an entire polymer chain. Figure 1-7. Amplification of fluorescence quenching sensitivity. Reprinted with permission from Chen et al.17 By using a combination of steady-state and ultrafast spectr oscopy, Whitten et al.18 established that the dramatic quenching re sults from weak complex formation [polymer (-) quencher (+)], followed by ultrafast electron transfer fr om excitations on the entire polymer chain to the quencher, with a time constant of 650 fs. The ultrafast exc iton decay involves two competing quenching mechanisms: aggregation que nching caused by formation of interchain states and electron-transfer quenching caused by the MPS-PPVMV2+ complex. The divalent cation MV2+ plays a dual role here: formation of th e donor-acceptor co mplex and inducement of polymer aggregation. Th is is the reason why MV2+ is a more effective quencher than Mg2+ or Ca2+, which can only induce the aggregation of poly mers by electrostatic fo rces, which acts only as an electron acceptor. The amplification of the fluorescence quenching can be further optimized by extending the intrinsic lif etimes of polymer excited states, 17 and controlling the directional transport of excitons. Many other groups have also st udied the mechanism of fluores cence quenching in CPEs in the following years. It has been shown that th e quenching constant (Ksv ) can be affected by a

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22 variety of factors, such as polymer concentration,19 quencher properties,20-22 solution properties,2325 and presence of additives.26-29 Aggregation of CPEs CPEs are inherently amphiphilic materials and, they tend to aggregate in aqueous solution, even at very low concentrations. In a se ries of investigatio ns, Schanze et al.30, 31 have examined the aggregation of PPE and the influence of a ggregation on electronic charge carrier transport and energy migration. The photophysics data show th at the solvent polarity plays an important role in aggregation of CPEs.As the increasing amount of water, the absorption a nd fluorescence are red-shifted; the fluorescence spectra and que nched intensity appear as very broad bands. (Figure 1-8) However, in methanol, the absorpti on and fluorescence spectra are very similar to those of structurally similar, organic-sol uble neutral PPEs in good solvents, where the aggregation is expected to be minimal. This sugg ests that in methanol the polymer exists in a non-aggregated state, while in water the polymer is strongly aggregated. The fluorescence decay time in methanol is dominated by a short-lif etime component with =420 ps (amplitude = 97%), while in water the fluorescence decay is biexponential and wavelength dependent, where the fluorescence ma ximum corresponds to a longer lifetime. The red-shifted absorption band and broad, less efficien t, long-lived fluorescence observed from PPE in water suggests an excimer-like state, which is presumably formed via inter-chain interactions. Schanze et al.30 believe that the increased structural order and conjugation length arise due to face-to-face -stacking between phenylene rings in adj acent chains. The chains align with their long axes parallel to optimize -stacking, so that the phenylene ri ngs in each chain should be nearly co-planar. This aggregate conformati on reduces the hydrophobic interactions between adjacent polymer chains and, allows the polar sulfonate groups to extend into the aqueous solvent. A larger SV for the fluorescence que nching of PPE is observed in water than in

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23 methanol, suggesting that intrach ain exiton migration occurring in the aggregates may lead to further amplification of quenching response. Figure 1-8. Absorption (left) a nd fluorescence (right) of PPE-SO3 in methanol, methanol:water (50:50) and water. Reprinted w ith permission from Tan et al.16 Bazan et al. studied the solvent-dependent aggregation of a CPE and the influence on energy transfer to chromophore.31 They reported different aggr egation tendencies of water soluble cationic poly [9, 9-bis (6 -N, N, N-trimethylammonium-he xyl) fluorine diiodide] (poly1) in aqueous solutions with varying amounts of THF as well as changes in the fluorescence resonance energy transfer (FRET) of poly1 to dye-labeled DNA (Figure 1-9). Since the backbones and alkyl side chains are hydrophobic moieties, while the cationic charged quaternary amines control electrostatic interactions, th e resulting amphiphilic ch aracteristics lead to different aggregation conformati ons in different solvents. Two aggregation states have been proposed based on the photophysical measurements conducted in the water/THF mixtures. When the THF content is in the range from 30% to 80% the polymer shows single chain behavior, or weak aggregation. In pure water, the polymers form tight aggr egates, which are dominated by the interchain hydrophobic interactio ns, resulting in lower fluorescence emission intensities due

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24 to interactions. When the THF content is higher than 80%, the ionic in teractions of charged groups with the non-polar medium lead to buryi ng of these groups within a new aggregate structure. This aggregation is dominated by the el ectrostatic interactions of charged quaternary amine groups and iodide counter ions. The aggregation states of polymers influence the contact extent between the polymer and dye-labeled DNA and the distance between the polymers and dyes. So the FRET efficiencies are de pendent on the aggregation states. Figure 1-9. Proposed aggregation modes of catio nic polymers in water with different THF content. Reprinted with permission from Wang et al.31 Applications of Conjugated Polyelectrolytes as Sensors In recent years, the conjugated polymers unique optical properties have triggered tremendous exploration of their uses for sensing chemical and biological materials. For example, the conjugated polymers have been effectivel y employed as sensors to detect metal ions,24,25 anions,32,33 explosives,34,35 small biomolecules,36,37 proteins and DNA,38,39 etc. Due to the amplification occurred from the conjugated back bones of these polymers, the chemical and biosensors are able to achieve extraordinary sens itivity. Typically, the detection limits for CPEbased biosensors are in the nanomolar range. In a few cases, sensors can even detect the target analytes at the zeptomole level.

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25 The strategy of CPE-based se nsors relies not only on the el ectron transport and energy migration along the conjugated backbone, but also on the conformational changes of polymeric chains. Thus, CPEs can be used to study c onformational changes of proteins and DNA at a fundamental level. Based on this platform, CPEs have been employed for a variety of commercial and scientific applications, includin g the identification of genetic mutations or single-nucleotide polymorphisms (SNPs),40 the sensing of amyloid fibril formation,40 mercurysensing based on conformational change of mercury-specific oligonucleotide (MSO),41 etc. The CPEs can be fabricated by several methods to meet the needs of different sensing targets: introduction of functional groups to side chains for selectively bi nding to metal ions or forming helical structures;42 attachment of receptors,39 peptide linkers or aptamers.43 The formats of CPE sensors can be homogenous soluti on, layer-by-layer assemblies, glass-supported materials and nanoparticle-supported materials. Despite the variet y of sensing targets and sensor formats, the underlying sensing strategies are usually divide d into three categorie s: quencher induced quenching-unquenching, chain conformation pe rturbation, and fluorescence resonance energy transfer. Quencher Induced Quenching-Unquenching Mechanism Some CPE-based sensors take advantage of the superquenching behavior of CPEs by electron or energy-accepting quenchers. For example, MV2+ is an important electronic acceptor quencher for MPS-PPV first repor ted by Chen and Whitten in 1999.17 They constructed a quencher-tether-ligand (QTL) sy stem by covalently linking MV2+ via a flexible tether chain to biotin. When this quencher-tet her-ligand(QTL) system is mixed with a solution of PPV-SO3 -, the fluorescence is quenched at very low concentrations of QTL due to the superquenching effect. Addition of small amount of avidin results in recovery of the fluores cence, because the binding of avidin to biotin disrupts the association between polymer and QTL system. As a result, a

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26 fluorescence response is produced when the ligands bind to their specific targets. Using this strategy, Whitten and co-workers have devel oped a sensor platform based on CPE-coated polystyrene microsphere for detecting enzymatic activity and DNA hybridization.17 (Figure 1-10) Figure 1-10. Biosensor application based on QTL system. Reprinted with permission from Chen et al .17 Zhao and Schanze discovered that Cu2+ is an efficient quencher of PPE-CO2 -.27 The SternVolmer constant (Ksv) for Cu2+ is much higher than that of ot her metal ions, and is comparable to that of methyl viologen. The st rong and selective quenching by Cu2+ likely arises because the metal coordinates with the carboxylate groups of PPE-CO2 -, and it efficiently quenches the singlet exciton via charge and/ or energy-transfer mechanisms By taking advantage of the efficient quenching of PPE-CO2 by Cu2+, a turn-on sensor for anions that coordinate with Cu2+ was developed. Some ions with the diphosphate moiety, such as PPi, ATP and ADP, would effectively sequesters the metal ion, disrupts its ability to bind to the carboxylate groups, and recovers the fluorescence intensity of PPE-CO2 -. This fluorescence response is highly selective to pyrophosphate, compared to other an ions including monovalent anions (H2PO4 F-, Cl-, Br-, I-, etc.) and divalent anions (HPO4 2-, SO4 2-, etc.). This platform ca n also be extended to a bioanalytical application by monito ring the activity of alkaline phos phatase (ALP) in real-time. Since ALP catalyzes the hydrolysis of pyrophospha te to monophosphate at physiological pH, the author used PPE-CO2 -/Cu2+ fluorescent sensor to detect the enzymes activity.

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27 Chain Conformation Perturbation Mechanism CPE-based sensors that transduce the r ecognition event via the chain conformation perturbation mechanism are widely a pplied into DNA hybridization detection,44 protein conformation studies,45 etc. This strategy usually does not require any chemical reaction of the probes or the analytes. Instea d, it is based on different elect rostatic interactions and conformational structures. Poly(t hiophene) derivatives are commonly used in CPE-sensors of this type because they display chromatic and fluorescent output upon the formation of different conformational structures. Leclerc pioneered the ap plication of water soluble cationic regioregular poly (thiophene) in the detection of DNA.43 Poly (3-alkoxy-4-methylthiophene) s were synthesized and formed complexed with single-stranded oligonucleotides or double-strande d (hybridized) nucleic acids. Originally, these polythiophenes are yellow so lution with maximum absorption at a short wavelength (Figure 1-11). This corresponds to a random-coil conformation, since any twisting of the conjugated backbone leads to a decrease in th e effective conjugation length. Upon adding 1.0 equivalent amount of oligonucleotides, the soluti on becomes red, because of the formation of a so-called duplex (highly conj ugated, planar conformation) be tween the polythiophene and the oligonucleotide probe. After adding 1.0 equi valent of the perfectly complementary oligonucleotide, the solution retu rns to yellow, presumably caused by the formation of a new complex termed a triplex (less conjugated, nonplan ar conformation), which includes the polymer and the hybridized nucleic acids. For comparis on, the singleand two-mismatch oligonucleotide do not form the triplex with polythiophene and th e oligonucleic probe, so the solutions stay red when those two are added into duplex solutions. Thus, the sensor is very selective for the oligonucleotide complementary to the probe DNA.

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28 Figure 1-11. Formation of polythiophene /single-stranded nucl eic acid duplex and polythiophene/hybridized nucleic acid triplex forms. Reprinted with permission from Ho et al.43 The fluorometric detection of oligonucleotide hybridization is also possible, since the fluorescence of polymer is quenched in the pl anar, aggregated form as the duplex forms.46 When hybridization with the perfect complementary st rand takes place, the formation of a polymeric triplex leads to a fivefold increase in fluorescen ce intensity. By monitoring either the absorption spectrum or fluorescence spectrum, oligonucleotid e hybridization can be detected with a high sensitivity (10-14 M) and oligonucleotides with one mismatch can be discriminated from the perfect complementary oligonucleot ide. Leclerc and coworkers appl ied a similar strategy to other sensors, including using a cationic polythioph ene/DNA based aptamer complex to detect K+ and human thrombin. In addition, Nilsson and co-workers have a series of publications showing that this mechanism is also effective for the detection of certain protein conformational changes. For example, in 2003, Nilsson and co-workers desc ribed how the conformational changes of a synthetic peptide could alter the conformati on of an electrostatic ally bound amino acidsubstituted conjugated polyelectrolyte;46 and in 2005, Nilsson repor ted a method to detect

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29 amyloid fibril formation both with a zwitterionic conjugated oligomer and with anionic poly (thiophene).47 Fluorescence Resonance Energy Transfer (FRET) Mechanism Fluorescence resonance energy transfer is a commonly used signal tr ansduction pathway in biochemical research. Conjugated polymers have the potential to be excellent energy donors in FRET-based sensing schemes. First of all, the high extinction coefficients stemming from their delocalized backbones enable efficient light em ission. Second, the excitons generated throughout the entire polymer can migrate to a position on the chain from which FRET is efficient. Figure 1-12. The PNA/CPE assay for ss-DNA detec tion. Reprinted with permission from Liu et al.48 Bazan and Heeger have reported many examples of using CPE to detect specific DNA (or RNA) sequences via FRET to dye-labeled probe molecules.44,48 In their systems, the electrostatic attraction between the charged CPE and DNA results in short distances be tween the donor (CPE) and the acceptor (dye-labeled probe strand). The probe can be PNA, ssDNA, dsDNA or protein (Figure 1-12). The sensing system consists of th ree parts: the cationic conjugated polyelectrolyte, the probe peptide nucleic acid (PNA) labeled wi th an energy-accepting chromophore which has strong spectral overlap with the CPE, and the target DNA strand. The PNA is neutral because the phosphate linkages have been replaced with ne utral amide linkages, so the average distance

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30 between chromophore and conjugated polymer is t oo great for efficient FRET. However, when PNA forms stable Watson-Crick base pairs with the complementary single-stranded DNA (ssDNA) target, the resulting complex is strongl y negative so it can bind to the conjugated polymer, which allows the efficient FRET to take place. In comparison, noncomplementary ssDNA gives little observable FRET. Bazan and co-workers49 also developed a complementary method based on traditional double-stranded DNA (dsDNA) helix formation for the sensing recognition chemistry, instead of the more expensive PNA. In order to alleviate th e effects of nonspecific el ectrostatic interactions between the dye-labeled ssDNA probe strand and th e conjugated polymer, Bazan and co-workers used a well-known DNA intercalating dye, ethidi um bromide (EB), as an energy acceptor to improve the performance of ssDNA-based assay.44 Furthermore, they introduced fluorescein as an intermediate FRET gate into the ssDNA to a llow much more efficient energy transfer to EB, resulting in an 8-fold amplification relative to direct excitation of the intercalator. Chemiluminescence System Chemiluminescence (CL) is the light emission as a result of chemical reaction with limited emission of heat. The first CL reaction was prepared by B.Radziszewski in 1877.50 After that, many more CL reactions were discov ered during the early 20th century.51 Peroxyoxalate chemiluminescence (POCL) is one of the most ef ficient and versatile CL processes available today. Many studies have been undertaken to de termine the mechanisms of the CL reactions. It relies on the reaction between aryl oxalates and an oxidant, typically hydrogen peroxide, to form a high-energy intermediate, wher e characterization remains contr oversial. Although the structure of the intermediate is yet to be elucidated, it is believed that it is capable of exciting a large

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31 number of fluorophores.52-54 This process, known as sensitizat ion, is independent of irradiation. The chemical reactions in POCL ar e generally written as follows: oxalate + H2O2 base high-energy intermediate (1-1) high-energy intermediate + fluorophore fluorophore* (1-2) fluorophore* fluorophore + h (1-3) Commercially available bis(2,4,6trichlorophenyl)oxalate (TCPO) is widely used in the POCL. Compared to other oxa lates, TCPO features relativ ely higher stability but lower reactivity, so a weak base, generally imidazole (I mH), is employed as th e catalyst in the TCPO CL reaction.55 POCL is better than other CL systems su ch as luminal and lucigenin, because the reaction can be carried out at pH 7, the optimal pH for most enzymatic reactions. The sensitivity of POCL is reported to be a 10 to 100-fold improvement over the PL detection method.56 The mechanism of POCL reaction is demonstrated in Figure 1-13. Figure 1-13. Mechanism of POCL reaction The main advantage of CL over other techniques is there is no requireme nt of irradiation of samples with electromagnetic radiation. The absen ce of a source leads to the elimination of the noise caused by light scattering, background emi ssion and source instability. Because the CL emission can be detected against a much dark er background than fluorescence emission, the detection limit can be lower. Meanwhile, the CL r eactions are so rapid that usually the maximum

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32 intensity is reached in less than one or two seconds. So CL dete ction is useful for the rapid onsite analysis, and is suitable for assays which ne ed a large sample throug hput in a short period of time. When coupled with flow injection analysis (FIA),57 liquid chromatography (LC),58 or capillary electrophoresis (CE),59 TCPO based POCL systems can be used not only for direct detection of H2O2,60 fluorophores (e.g. polycyc lic aromatic hydrocarbons61) or fluorescent derivatized compounds (e.g., amino acids,62 carboxylic acids63 and amines64), but also for indirect determination of substrates a nd enzymes (e.g. glucose and glucose oxidase65) by detection of H2O2 which is produced through enzymatic re actions. Although the insolubility and hydrolysis of oxalate esters limit the applicati on of POCL systems, aqueous POCL was achieved by combining it with FIA and delivering oxa late solution via a separate flow line.66 CPE-based Real-time Enzymatic Activity Assay CPE-based fluorescence assays share the comm on features of being relatively easy to implement, being highly sensitiv e and giving a rapid response. In addition, the CPE-based assays are usually carried out in soluti on under physiological c onditions, so that they can provide a realtime signal and also allow determin ation of enzyme kinetic parameters at very low substrate and enzyme concentrations. In 2004, Pinto and Schanze15 used two anionic conjugated polymers, PPESO3 and PPECO2 as the signal-transduction element to develop a turn-on and turn-off sensor, respectively (Figure 1-14). The sensing mechanism relies on an electr ostatic interaction between the conjugated polyeletrolyte and a peptide substr ate that is labeled with a fluo rescence quencher. In the turn-on sensor, the assay is base d on the quenching of PPESO3 by two quencher-labeled substrates such as L-Lysp -nitroanilide dihydrobromide (K-pNA) and N -benzoyl-Phe-Val-Argp -nitroanilide hydrochloride hydrate (Bz-FVR-pNA), which can be hydrolyzed by thrombin and peptidase, respectively. The enzyme-catalyzed peptide hyd rolysis is signaled by an increase in the

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33 fluorescence from the conjugated polyelectrolyt e. The turn-on system was used to sense peptidase and thrombin activity for concentrations of enzyme and substrate in the nano-molar regime. Kinetic parameters were re covered from real-time assays. Figure 1-14. Enzyme kinetics measured by usi ng PPESO3/Bz-FVR-pNA/thrombin assay system. Reprinted from permission with Pinto et al.15 In the turn-off sensor, the PPECO2 was employed with a caged peptide substratequencher, N,Nbis(carboxybenzyloxy-L-arginine amide)rhodamine-110 dihydrochloride (RhoArg-2). This particular deriva tive is nonfluorescent and does not quench the fluorescence of PPECO2. However, when it is hydrolyzed into RhoArg, catalyzed by papain, the fluorescence intensity is quenched due to singlet -singlet energy transfer from PPECO2 to Rho-Arg. The

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34 papain activity can be monito red by a series of assays ca rried out by using the PPECO2/RhoArg-2/papain system at various concen trations of the Rho-Arg-2 substrate. Later, Zhao and Schanze developed a real-time turn-off assay to monitor the activity of alkaline phosphatase (ALP).27 Since ALP catalyzes the hydrolysi s of PPi to Pi, the authors can detect the activity of ALP by using PPECO2/Cu2+ system to sense the PPi concentration upon adding different amounts of ALP. Liu and Scha nze introduced a sensi tive fluorescent turn-off assay for phospholipase C (PLC). The assay is ba sed on the reversible change in fluorescence properties of an anionic CPE (BpPPESO3) induced by the formation of a polymer-phospholipid complex. The catalytic kinetic parameters, Km and Vmax have been determined from the assay. Alkaline Phosphatase Assay Alkaline Phosphatase (ALP) hydrolyses a wi de range of phosphate monoesters in many types of molecules, including nucleotides, protei ns, and alkaloids. The assay for ALP enzymatic activity has been the subject of considerable inte rest owing to the important role that ALP plays in the diagnostic field.67 Although ALP is present in all tissu es throughout the entire body, it is especially concentrated in the liver, kidney, bone and the placenta. Abnormal level of ALP is an important index of several diseases.68 Elevated ALP level is possi bly due to liver dysfunction (hepatitis or liver tumor), bone di sease (Pagets disease, osteosarcoma, osteomalacia, and rickets, etc.), diabetes, bile abduct cons truction, renal disease and pregnancy.69 Lower ALP levels may relate to hypophosphatasia, heart surgery, ma lnutrition, magnesium deficiency, etc. Among the broad substrates of ALP, pyrophosphate (PPi) has a lower optimum pH compared to other substrates, but the reactivit y is relatively low. ALP and PPi are both key regulatory factors in vascular cal cification, and they play important roles in clinical calcific vasculopathy and valvulophathy.70 Hydrolysis of PPi by ALP lead s to two free phosphates (Pi). The balance between levels of Pi and PPi c ontrols the formation of hydroxyapatite mineral

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35 crystals and their growth in cartilage and bone. The presence of PPi prevents soft tissues from mineralizing, whereas its degradation to Pi catalyzed by ALP facilitates crystal growth. Therefore, development of assays to monito ring the PPi and Pi levels under physiological conditions are essential to the study of cellmediated phosphate and pyrophosphate metabolism and their effects on regulating calcification. Many commercialized ALP assay methods have been developed so far. For example, an amperometric analysis method has been designed for indirect measurement of ALP activity using 3-indoxyl phosphate substrate.71 The hydrolysis catalyzed by ALP gives rise to an indigo product, which is insoluble in aqueous solutions but is eas ily converted into soluble indigo carmine. This compound is easily detected at a bare screen-pri nted electrode placed in an Flow Injection Analysis system. In colorimetrical ALP assays, p -nitrophenyl phosphate is usually used as the substrates in alkaline conditions in the pr esence of phosphate acceptors such as 2-amino-2methyl-1-propanol. A chemilumnescent immuno assay uses an adamantyl-1, 2-dioxetane phosphate derivative as a substrate for quantif ication of ALP. The dephosphorylation of the substrate catalyzed by ALP induces light emission with a max of 470 nm. Although these methods are highly sensitive, none of them have used PPi as the substrate. Therefore Liu and Schanze72,73 developed a fluorescent assay using PPi as the substrate, and this assay enables the continuous detection of PPi c oncentration (Figure 1-15). This sensor is based on the amplified quenching of PPE-CO2 by Cu2+ and the binding between PPi and Cu2+. The fluorescence of PPE-CO2 is initially quenched by Cu2+. Then the addition of PPi to the PPE-CO2 -/Cu2+ solution causes the fluores cence to recove r by disrupting the polymer-metal complex. Upon hydrolysis of PPi into Pi catalyzed by ALP, the fluorescence is quenched again, since the Pi is unable to complex with Cu2+. By monitoring the concentration

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36 of PPi as a function of enzymatic reaction time, which is calibrated with fluorescence intensity, the kinetics of ALP activity can be investigat ed and Pi inhibition can also be studied. Figure 1-15. Mechanism of fluorescent ALP assa y. Reprinted with permission from Liu et al.73

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37 CHAPTER 2 ALKALINE PHOSPHATASE SENSORS USIN G CHEMILUMINESCENT CONJUGATED POLYMER Introduction Although the photoluminescence (PL) of CPE has been investigated in depth, the chemiluminescence (CL) of CPE has never been studied to our best knowledge. The POCL system achieves superior sensitivity and detec tion limits compared to PL detection methods. However, POCL-based assays for biological analyt es can be applied only to those which involve H2O2 in enzymatic reactions. While CPEs are em ployed as fluorophores in POCL systems, the biosensor potential of CPEs ma ke it possible to expand CL app lication to biological targets which are not related to H2O2. The combination of the amplifie d quenching effects of CPE with the intrinsic high sensitivity of CL is expected to improve the properties of possible biosensors. In previous work in the Schanze research group, Liu developed the first CL system for CPE utilizing TCPO as CL reagent, H2O2 as oxidant, ImH as catalyst and an anionic CPE (BpPPESO3) as fluorophore in aqueous/acetonitrile (CH3CN) solvent mixtures. The results showed that the luminescence of BpPPESO3 is not influenced by the presence of H2O2, ImH or TCPO in the solution, an essentia l prerequisite for CPE-based CL system. The CL intensity vs. wavelength profile is identical to the PL spectrum of BpPPESO3 in the same solvents with maximum emission at 450 nm. The CL intensity vs. time profile showed that the intensity rises sharply to the maximum value within 5 s and deca ys gradually until falling to almost zero after about 30 s. The optimum reagent concentrations and solvent composition were determined by evaluating their effects on CL signals, taking in to consideration both maximum intensity and signal duration. Figure 2-1a dem onstrates the effects of [H2O2] on the signal, with 375 M BpPPESO3, 1.5 mM ImH and 0.5mM TCPO in 50/50 THF/H2O solvent system. The CL

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38 intensity increases linearly with [H2O2] and reaches a plateau at about 30 mM H2O2. The CL duration time increases with [H2O2] at low concentrations until it reaches its maximum at 4 mM H2O2, a moderate excess relative to the oxalate. Therefore, 8 mM H2O2 was chosen for subsequent experiments as a compromise of the CL intensity and duration time. Figure 2-1. Optimization of CL signals ( em = 450 nm). (a) Effect of H2O2 concentration on CL intensity and duration time. (b) Effect of ImH concentration on CL intensity and duration time. (c) Effect of solvent com position on CL intensity and duration time. These experiments were carried out by Liu in the Schanze research group. The effect of [ImH] on CL signal is illustrate d in Figure 2-1b. The CL reaction conditions were 375 M BpPPESO3, 1.5mM H2O2 and 0.5 mM TCPO in 50/50 THF/H2O. The CL intensity and duration time increase to their maximum when [ImH] is 2 mM and 1mM, respectively. However, they both decrease as [ImH] increases from 2mM to 25 mM. This is probably due to the decrease of CL quantum yield with excess Im H, from the perspective of kinetics of CL reaction. Another possibility is that excess ImH results in the breakdown of TCPO to generate

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39 1,1-Oxalyldiimidazole (ODI) which is confirmed to be the main precursor formed in the TCPOCL reaction. Finally, 1.5 mM was selected as the concentration of ImH after compromising between CL intensity and duration time. Regarding effects of [TCPO], CL intensity in creases almost linearly with increasing of initial [TCPO] from 0 to 1.5 mM in the presence of 375 M BpPPESO3, 8mM H2O2 and 1.5 mM ImH. However, CL duration time is independe nt of [TCPO], because the POCL reaction is pseudo-first order when H2O2 is in moderate excess compared to TCPO. In this case, [TCPO] has no effect on the rate constant of the POCL reaction. Since TCPO ha s limited solubility in H2O, TCPO is dissolved in CH3CN and then mixed with other reactants in water solution to initiate the CL reaction. Effects of water conten ts on the CL signals have also b een investigated. Figure 2-1c shows the CL vs. time profiles with 10 M BpPPESO3 using different volume fractions of H2O in H2O/CH3CN mixtures, decreasing from 80% to 20%. There is no reasonable CL signal when more than 75% of H2O is present, because TCPO hydrolyses rapidly before i nducing CL reaction at such high water content. The CL intensity incr eases with decreasing of water content and then remains almost unchanged from 50% to 20% H2O. The reasons may be the increased stability of TCPO in the less aqueous environment, as well as increased qua ntum yield of BpPPESO3 in the organic solvent. The CL durati on time also increases with decr easing water content, which may be due to the increase in rate of the based-catalyzed TCPO reacti on at high water concentrations or decreased consumption of TCPO from hydr olysis. However, in order to develop a chemiluminescence-based assay for enzyme, a hi gher water content is preferred to mimic the physiological conditions. Therefore, 50% water content was chosen for the CL-based assays that will be described later.

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40 Results and Discussion Photophysical Property of PPECO2 PPECO2 is an anionic CPE having carboxylate si de groups (Figure 2-2a). The synthesis and characterization of this polymer is included in the early literatures.27 Similar to other CPEs, PPECO2 shows solvent-dependent absorption and em ission properties. In an organic solvent (methanol), PPECO2 exhibits an absorption maximum at 417 nm and an emission maximum at 437 nm. With increasing water amount, the polyme r shows a red-shift an d narrowing absorption spectrum, as well as a significant red-shift and broadening of the fluorescence spectrum. In pure water, it absorbs at maximum of 435 nm a nd exhibits an emission maximum at 520 nm. The solvent-dependent photophysical properties of CPEs are due to the aggregation of the polymer in aqueous solution. In methanol, the ab sorption and emission spect ra of this polymer correspond well with the spectra of structurally analogous polymers that contain alkyl or alkoxysolubilizing groups in good solvents. In water, the spectra display spec tral changes that are characteristic of aggregate formation as seen in the solid films of the structurally similar organic polymers. O CO2Na O NaO2C n Cl Cl Cl O C O C O O Cl Cl Cl a b Figure 2-2. Struct ures of PPECO2 (a) and TCPO (b). Chemiluminescence of PPECO2 The chemiluminescent reaction based on PPECO2 is not affected by the presence of ImH, H2O2 and TCPO, which is the prerequisite for this CL system. Since TCPO hydrolyzed easily in

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41 water, it is dissolved in CH3CN first, and then mix with th e buffer solution containing PPECO2, ImH, and H2O2 to initiate the CL reaction. The CL intens ity is recorded instantaneously when the TCPO/CH3CN solution is added into th e aqueous mixture. The CL in tensity vs. time profile of 25 uM PPECO2 is measured at the emission wavelength of 435 nm. The optimum conditions for the CL system has been investigated by Liu and, the final concentrations are set to be 25M PPECO2, 8 mM H2O2, 1.5 mM ImH and 0.3 mM TCPO in H2O/CH3CN (50/50) solvent system. Time (s) 010203040 Chemiluminescence 0 5e+4 1e+5 H 2 O HEPEs buffer Tris buffer (a) (b) Figure 2-3. CL intensity of PPE-CO2 -. (a) The photos of CL system (right) and control system (left). Condition: 25 M PPE-CO2 -, 8 mM H2O2, 1.5 mM ImH, 0.3 mM TCPO (no TCPO in the control system) in Tris-HCl/CH3CN (50/50) solven t. (b) The CL intensity in different buffer/CH3CN system. Conditions: 25 M PPE-CO2 -, 8 mM H2O2, 1.5 mM ImH, 0.3 mM TCPO in Tris-HCl/CH3CN(50/50) ( ), H2O/CH3CN (50/50) ( ), HEPEs/CH3CN (50/50) ( ), em = 446 nm. The CL profile is characterized by two pro cesses: the intensity rises sharply to the maximum within 5 seconds, and then decays gradually until falling to nearly zero. The rise is due to the formation of high-energy intermediate as well as excitation of fluorophore. The decay is related to the loss of TCPO in the CL reaction. The CL measurement shows good reproducibility upon repeating the tests. The CL intensity of PPECO2 is strong enough to be observable in the dark, compared to the control whic h has no TCPO adde d (Figure 2-3a).

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42 In order to utilize this CL system into biosen sor, the buffer effect on CL signal needs to be studied. The CL intensities of PPECO2 were measured in different buffers/CH3CN (50/50) mixtures (Figure 2-3b). It turns ou t that the CL intensity of PPECO2 is around three times higher in Tris-HCl than that in Hepes, while two times higher than that in water. The reason for that is not fully understood, but it might results from stabilizing TCPO by Tris-HCl. Quenching Studies The amplified quenching of CPE by small molecules with opposite charges has been extensively studied, either in aq ueous or organic solvents. The mechanism is attributed to a combination of ion-pairing between CPE and quen cher with energy and ex citon rapid migration and/or delocalization within the polymer chain to the quencher binding site. Therefore, quenching study is the most convenient method to evaluate the CPE-based TCPO-CL system. Cu2+ has been reported to be a quencher for PPECO2 and is a requirement in ALP assay application. The quenching experiment was carried out by a series of Cu2+ titration of the CL reaction system in Tris-HCl buffer/CH3CN solvent (including 25 M PPECO2, 8mM H2O2, 1.5 mM ImH and 0.3 mM TCPO). As a comparison, th e PL quenching experiment was conducted in the same solvent mixture with the same concentr ation of each reactant ex cept for the absence of TCPO. Figure 2-4A and Figure 2-4B illustrates the quenching of PPECO2 by Cu2+ in the PL and in the CL system, respectively. The difference is for PL quenching, emission vs. wavelength profiles are used; for CL quenching, emission vs. time profiles are used. The results of SternVolmer plots for the quenching of PPECO2 in PL and CL system have been illustrated in Figure 2-4C. The integrated areas of CL /PL profiles are used as signal intensities. The Ksv for the PL and CL system are 1.9 105 and 4.5 105 M-1, respectively. The quenching efficiency of CL is higher than that of PL in the same solvent mixture.

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43 [Cu]Wavelength (nm) 450500550600650700 Fluorescence 0 1e+6 2e+6 3e+6 0uM 5uM 10uM 15uM 20uM 25uM 30uM 35uM [Cu]Time (s) 010203040 Chemiluminescence 0 5e+4 1e+5 0uM 1uM 2uM 4uM 6uM 8uM 10uM 15uM 20uM 25uM 30uM 50uM 3 [Cu 2+ ] (uM) 0204060 I 0 /I 0 3 6 9 Chemiluminescence Fluorescence Figure 2-4. A) Fluorescence intensity and B) chemiluminescence intensity of PPE-CO2 upon titration of Cu2+. Fluorescence: 25 M PPE-CO2 in Tris-HCl (10 mM, PH 7.5) /CH3CN (50/50) with 8 mM H2O2, 1.5 mM ImH,0.3 mM TCPO, ex = 380 nm. Chemiluminescence: 25 M PPE-CO2 in Tris-HCl (10 mM, PH 7.5)/CH3CN (50/50) with 8 mM H2O2, 1.5 mM ImH, em = 446 nm. (c) Stern-Volm er plots of fluorescence and fluorescence quenching of PPE-CO2 by Cu2+. A B C

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44 Although PPECO2 shows amplified quenching prope rty beyond the linear range, the CL/PL quenching efficiency in the mixture of aqueous and CH3CN is about 10-fold smaller compared with a typical supe rlinear PL quenching as seen in water solution with no H2O2, ImH or TCPO added (Ksv ~ 106 M-1). Two factors account for the less efficiency of quenching: first, the presence of organic solvent, CH3CN, induces less aggregation of CPE, less ion-pairing forces and less hydrophobic intera ction between CPE and small charge quencher; second, the presence of ImH, H2O2 or TCPO may change the polymer condition or solution environment. However, the quenching of CL still demonstrated the amplified quenching property of CPE with about 10 to 100 times larger than the quenchi ng efficiency of the small model compound. Alkaline Phosphatase Chemiluminescence Turn-off Assay A real-time fluorescence turn-off assay for ALP utilizing PPECO2 has been developed in the previous work of our group. The same approach was ready to be implemented by means of TCPO-CL. Figure 2-5 shows the mechanis m of the CL turn-of assay for ALP. Figure 2-5. Mechanism of CL turn-off assay for ALP. The CL of PPECO2 is quenched efficiently by Cu2+ via charge and/or energy transfer mechanism. The quenched CL is recovered upon the addition of inorganic PPi, which is due to

PAGE 45

45 the strong association of PPi with Cu2+, thus disrupting of PPECO2/Cu2+ complex. Introduction of ALP to the mixture of PPECO2/Cu2+/PPi initiates the hydrolysis of PPi to Pi, which is lack of ability to complex with Cu2+. As the reaction proceeds, the amount of PPi to associate with Cu2+ decreases. As a result, the CL of PPECO2 is quenched by the free Cu2+ again. Therefore, the presence and activity of ALP is detected by the d ecline of CL intensity. Time (s) 051015202530 Chemilumnescence 0 5e+4 1e+5 a. 25 uMPPECO 2 b. a+50 uM Cu c. b+25 uM PPi d. b+ 50uM PPi e. b+75 uM PPi Figure 2-6. Chemiluminscence intensity quenched by Cu2+ and then recovered by PPi. Conditions: 25 M PPE-CO2 -, 8 mM H2O2, 1.5 mM ImH, 0.3 mM TCPO in TrisHCl/CH3CN (50/50), em = 446 nm. The CL quenching experiment of PPECO2 by Cu2+ was discussed in the section of quenching studies. Typically, with mixt ure of Tris-HCl (10mM, pH 7.5)/CH3CN (50/50) containing 8 mM H2O2, 1.5 mM ImH, 0.3 mM TCPO, the initi al CL intensity of 25uM PPECO2 is completely quenched by 50 M Cu2+. The quenched CL was recovered to 80% of initial intensity upon addition of 75 M PP i; (Figure 2-6) and the recovery curve was level off after this amount. Introduction of ALP into the Tris-HCl solution containing PPECO2/H2O2/ImH/Cu2+/PPi and incubation of it for some time allows the hydrolysis of PPi by ALP. It would result in the decrease of CL intensity generated by mixing Tris-HCl solution with TCPO CH3CN solution. The above concentrations of each species were c hosen as the condition of CL turn-off assay for

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46 ALP. The emission wavelength to record the CL time profiles was set at 446 nm, because it is the maximum emission wavelength achie ved from PL spectrum of PPECO2 in Tris-HCl/CH3CN (50/50). [ALP] (nM) 0200400600800100 0 I 0 /I 0 1 2 3 4 Figure 2-7. Changes of chemiluminescence intensity observed in the turn-off assay for ALP. A) Changes of chemiluminescence intensity after 5 min of addition of different concentrations of ALP. B) Linear calib ration plot of chemiluminescence intensity decrease as a function of ALP c oncentration. 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), em = 446 nm, at 37 C. Figure 2-7A illustrates CL spectroscopic changes observed with 5 min of incubation of different concentration of ALP with substrate in the turn-off assay at 37 C. It shows clearly that the CL intensity decreases with increase of in itial ALP concentration in a rage of 100 2000 nM. Figure 2-7B displays a linear co rrelation between intensity cha nge and amount of ALP added, A B

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47 where I0 and I are initial and decreased CL intensiti es upon addition of ALP; the integrated area of CL profile was used as CL intensity. It s uggests that it should be possible to quantitatively determine the level of ALP by this CL assay. Time(s) 0102030 Chemiluminescence 0 5e+5 1e+6 2e+6 0 min 5 min 10 min 20 min 30 min 20 hr Relative Intensity 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 min 5 10 20 30 i n c u b a t i o n t i m e w i t h A L P Figure 2-8. Changes of CL intensity as a functio n of incubation time (0 30 min) after addition of 400 nM 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. Figure 2-8 shows the decrease of CL intens ity in the presence of 400 nM ALP as a function of incubation time. The initial CL intensity of PPECO2 which was displayed as 0 min of assay was normalized to 1. The relative CL in tensity keeps decreasing with incubation time, approaching about 15% of initial in tensity at 30 min of incubation a nd rarely changing after this. B A

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48 It is demonstrated that most PPi is hydrolyzed and enzymatic reaction almost completes in the first half of hour. Discussion Compared with PL turn-off assay for ALP w ith detection limit of ~ 5.0 nM ALP within one minute of response, the CL ALP assay is less sensitive. In co mbination with above quenching study as well as CL turn-on assay for pe ptidase, the detection sensitivity of TCPO-CL system of CPE is not as good as that of PL system. The necessary of organic solvent, CH3CN, to dissolve TCPO is one of the main reasons to acc ount for this problem. It induces the decrease the quenching efficiency of the polymer and the change of the buffer condition which is important to enzyme activity. TCPO hydrolyses to a more or less extent in aqueous environment, which reduces its ability as CL reagent. Therefore, to find a substitute for TCPO with better water solubility and stability as CL reagent is advantageous to eliminate the need of organic solvent. An oxamide reagent with two sulfonate groups on the benzene rings, which is sp ecially designed for aqueous CL reagent, is a good example as the substitute. Another reason for th e low sensitivity may arise from the mixing method used in the CL process. Manually pipe tte-mixing is simple but less efficient. The approach to solve this problem and improve the performance of th e CL system is to couple it with flow injection or stop-flow which provi des rapid and reproducib le mixing, thus giving efficient CL monitoring, and allo ws rapid sample throughput. By improving the sensitivity of CL assay util izing these two methods, plus its inherent merit of simple and direct measurement, TCPO-C L approach opens a new path for luminescence CPEs in biosensor application.

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49 Experimental Chemicals PPE-CO2 (Figure 2-2) was synthesized according to literature methods and concentrated aqueous solution of them were diluted with wa ter or buffer solution to a final concentration ranging from 10 to 25 M. All chemicals were used as received, unless otherwise noted. Bis(2,4,6-trichlorophenyl) oxalate (TCPO) and imidazole (ImH) were purchased from Acros. 1,1-Oxalyldiimidazole (ODI) was obtained fr om Sigma-Aldrich. Hydrogen peroxide (H2O2), 30% solution in water was purchased from Fisher Chemical. The concentration of H2O2 was determined by titration with the solution of pota ssium permanganate prior to dilution. HPLCgrade acetonitrile (CH3CN) was obtained from Fisher and used 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 immediat ely before their use. TCPO solution was stored in dark prior to dilution and analysis. The quencher, copper (II) chloride (Cu2+) was obtained from Sigma-Aldrich. Stock solutions of Cu2+ in water were prepared before the que nching test and adjusted to 1.0 mM. The substrate for alkaline phosphatase sodium pyrophosphate (PPi), wa s purchased from J. T. Baker Chemical Company. The enzyme, alkaline phospha tase bovine intestinal mucosa (ALP) was purchased from Sigma-Aldrich. Buffe r 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 fluorescence assays. Prior to AL P CL assay, PPi and ALP were dissolved in Tris buffer solution (Tris-HCl, 10 mM, pH 7.5) and adju sted to 2.0 mM and 20 M as stock solution. The ALP assay was carried out in the same buffer solution.

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50 Chemiluminescence Measurements In a typical CL measurement, an aliquot of aqueous solution containing 10-25 M PPECO2, 10 40 mM H2O2 and 1.875 7.5 mM ImH was pipette d into the cuvette which was placed in spectrometer. An ali quot of 0.375 mM 1.5 mM TCPO CH3CN solution was pipetted into above solution in dark to initiate the CL reaction. Th e emitted light was detected simultaneously. The profile of CL 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, 1024 128 pixels). The profile of CL intensity vs time was measured at a specific wavelength on a JOBIN YVON-SPEX Industries Fluorolog-3 spectrofluorometer (Model FL3-21) with the lamp turned off. The emission wavelength to record time profiles was chosen from maximum wavelength in the PL spectrum measured under th e same condition but with the lamp turned on. The CL signal (intensity and duration time) were acquired as a function of the concentration of each reagent (PPECO2, H2O2, ImH and TCPO) and ratio of solvents (H2O/CH3CN). The CL intensity is the peak intensity in the CL vs. tim e profile, and the CL durat ion time refers to the range of time from addition of TCPO to the mome nt when the intensity does not fall anymore. The concentration of each reagent shown in the results and discussion se ction is the calculated concentration after mixing diffe rent ratio of aqueous and CH3CN solutions. Quenching Behavior The CL quenching test was done by a series of titration of PPECO2/H2O2/ImH aqueous solution by Cu2+. It was followed by addition of TCPO CH3CN solution and measurement of CL vs. time profile. The integrated ar ea of profile, which is proportiona l to CL intensity, was used as intensity of CL signal here. The PL quenching st udy in water was conducted in the same manner without addition of TCPO.

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51 Alkaline Phosphatase Chemiluminescence Assay with PPECO2 The ALP assay was carried out at 37 C. The initial CL intensity of PPECO2 was recorded first. The Cu2+ solution was then added to another PPECO2/H2O2/ImH mixture, the solution was incubated for 10 min, and the quenched CL inte nsity was measured. Subsequently the PPi solution was introduced to a nother freshly prepared PPECO2/H2O2/ImH/Cu2+ solution, this mixture was incubated for 10 min, and the recovered CL intensity was recorded. Finally, an aliquot of ALP solution was added to another fresh PPECO2/H2O2/ImH/Cu2+/PPi solution, and the CL profiles were measured as a functions of incubation time and concentration of ALP. The incubation time refers to the period after intr oduction of ALP into the aqueous solution but before mixing with TCPO CH3CN solution to initiate CL reaction. The integrated area of profile was used as intensity of CL signal.

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52 CHAPTER 3 ALKALINE PHOSPHATASE SENSORS USING FLUORESCENT DENDRITIC POLYMER Introduction Dendritic polymers form a special class of macromolecules composed of molecular chains that branched out from a common center. There are four major subclasse s of dendritic polymers: random hyperbranched polymeric architectures: dendrigraft polym ers, dendrons, and dendrimers. (Figure 3-1)74 Usually there is no entanglement betw een dendrimer molecules. The unique physical and chemical properties of these materi als has led to a wide range of applications: adhesives and coatings, chemical sensors, me dical diagnostics, drug-delivery systems, highperformance polymers, catalysts, building blocks of supermolecules, separation agents and etc. Figure 3-1. Four major subclasses of dendri tic polymers. (a) Radom hyperbranched. (b) Dendrigrafts. (c) Dendrons. (d) Dendrimers Reprinted with permission from Tomalia et al.74 This chapter focuses on the dendritic CPEs, which belong to dendrigrafts subclasses and their application in biosensors. Since the CPEs te nd to aggregate in good solv ents such as water, addition of dendritic side-groups to the conjugated backbone help s keep the molecules apart, leading to significantly enhanced luminescence qua ntum efficiencies. Some research shows that the dendritic side-group architec ture may accommodate a variety of moieties that could perhaps enhance transport properties and alter the mechani cal characteristics of the polymer. In dendritic CPEs, the conjugated backbone defines the key el ectronic properties such as the absorption and emission wavelength, while the surface groups contro l processing properties, such as solubility.

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53 The disruption of CPE aggregates in aqueous solution by introduction of dendritic groups has been reported in the literatures.74,75 Due to the influence of inte rmolecular interactions on both charge transport and light emission, the molecula r engineering of conjuga ted dendrimers allows control of the spacing of polymer assemblies and can be used to make highly efficient solutionprocessed LEDs. In the Schanze research group, Zhao recently synthesized PPE-dNH3Cl (Figure 3-2) and examined its optical properties under varying cond itions, such as solvent, pH, and ionic strength. As mentioned above, the polymers optical proper ties are mainly determin ed by the structure of the conjugated backbone. Compared to similar conjugated polymers carry ing linear side-groups, the absorption maxima of dendriti c polymers are blue-shifted, due to the more twisted backbone conformation caused by the increas ed electronic repulsion between the charged dendritic groups. The maximum fluorescence emission wavelength for PPE-dNH3Cl has negligible shift compared to its linear counterpart. O O CONH CONH n H3N NH3 NH3 NH3 NH3 H3N 6Cl Figure 3-2. The structure of PPE-dNH3Cl. Zhao also checked the solvent effects on ab sorption and fluorescence of this dendritic polymer by varying the MeOH/H2O ratios in the solvent mixt ures. The absorption spectra showed little change, while the fluorescence decreas ed in intensity. But compared to their linear counterparts, their fluorescence is still relatively efficient and re tains high quantum efficiency in

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54 water. The results suggest that the interchain aggregation of dendritic CPEs in the aqueous solutions is attenuated by attaching the bulky ionic groups. Figure 3-3. Absorption (a) and fl uorescence (b) spectra of PPE-dNH3Cl in water as a function of pH. [PPE-dNH3Cl] = 5M, pH range from 4.5 to 10.5 in 1.0 pH unit intervals. These experiments were carried out by Xiaoyong Zhao in the Schanze group. Figure 3-3 shows the absorption and emission spectra of PPE-dNH3Cl in water as a function of pH. When the solution is acidic, the neighboring phenylene ethynylene groups are twisted in the polymer to minimize the electr ostatic repulsion between the positively charged ammonium side groups. With increasing pH, th e ammonium groups are deprotonated, which leads to the planarizatio n of the conjugated backbone The fluorescence of PPE-dNH3Cl is quenched with increasing pH, which i ndicates that aggr egation of PPE-dNH3Cl is occurring. Meanwhile, the red-shifted, broad band dominating the fluorescence spectrum is believed to arise from the stacking of the polymer chains. The interactions between cationic dendritic conjugated polymers and small negative ions including PPi and Pi were also investigated. Figure 3-4 show s the absorption and fluorescence spectra of PPE-dNH3Cl upon titration of PPi and Pi. As PPi is added, the absorption maxium is red-shifted concomitant with the emergence of a new low-energy band, and the fluorescence intensity is quenched. It is believed that PPi induces aggregation of polymer chains by

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55 neutralizing the positive charges on the ammonium side groups. Ho wever, when the negative Pi is added, negligible effects are observed. Figure 3-4. The absorption (a) and fluorescence (b) spectra of PPE-dNH3Cl upon titration of PPi. The absorption (c) and fluorescence (d) spectra of PPE-dNH3Cl upon titration of Pi. [PPE-dNH3Cl] = 10 M, in MES (10mM, pH 6.5) buffer. These experiments were carried out by Zhao Xiaoyong in the Scha nze research group and repeated by the present author. By taking advantage of the dist inct fluorescent response of PPE-dNH3Cl with addition of PPi and Pi, we developed a fluorescent turn-on assay for ALP using catio nic dendritic CPE. The enzymatic assay employs PPE-dNH3Cl as an amplified fluorescent transducer to monitor the PPi activity of alkaline phosp hatase under physiological conditions in real-time. The new ALP assay based on the quenching-unquenching mechanism is more convenient and sensitive than the former assays for alkaline phosphatase. Results and Discussion Overview of Alkaline Phosphatase/PPi assay Although a sensitive real-time ALP assay has previously been developed in our group,27 the method requires Cu2+ as a signal transducer between CPE and the substrate, since there is no a b c d

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56 direct correlation between the fluorophore and substrate. The fluorescence of CPE is first quenched by Cu2+ via a charge-transfer mechanism, and the fluorescence is subsequently recovered by PPi, since PPi sequesters the Cu2+ by complexation. In order to derive a relationship between fluorescen ce intensity and PPi concentra tion, the concentration of Cu2+ has to be established. An arbitrary logarithmic calibration plot is em ployed to convert the fluorescence signal into substrate concentration, which eventually introduces errors and affects the precision of the assay method. In this chapter, we describe a real-time ALP assay using PPE-dNH3Cl, which displays a direct fluorescent response to PPi but no fluorescent response to Pi. Since PPi is a substrate for ALP, which catalyzes the hydrolysis of PPi to Pi, this property of PPE-dNH3Cl provides a unique platform for a turn-on assay via a quenching-unqu enching mechanism. The mechanism of this assay is illustrated in Figure 3-5. The PPE-dNH3Cl features dendritic ammonium side groups, which induce three-dimensional separation of the conjugated backbone and enhances the luminescence quantum efficiency. The random-c oil conformation dominates the photophysics of the polymer. With the addition of PPi, the repu lsive interaction between neighboring polymer chains is reduced by neutralizing the positiv e charges in the sidegroups. Furthermore, pyrophosphate may complex with th e dendritic sidegroups from different polymer chains, which brings the neighboring polymer chains into clos e proximity and promotes the planarization of polymer aggregates. So the fluorescence of PPE-dNH3Cl is quenched by addition of a small amount of PPi. Then, ALP is added to the poly mer solutions. ALP catalyzes the hydrolysis of PPi to Pi, which exists in the form of HPO4 2and H2PO4 in the buffered solution (pH 7.5). Pi is unable to induce aggregation of PPE-dNH3Cl, so the polymer assemblies are disrupted and the fluorescence is recovered. As catalyses proceeds, the amount of PPi available to complex with

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57 dendritic sidegroups decreases, and t hus the fluorescence intensity of PPE-dNH3Cl increases with time. With a calibration pl ot of fluorescence of PPE-dNH3Cl versus PPi concentration, the amount of substrate PPi can be monitored by tim e-resolved measurement of the fluorescence. This allows determination of the kinetic para meters and inhibition studies of ALP activity. Figure 3-5. Mechanism of ALP turn-on sensor using PPE-dNH3Cl. In preliminary research, MES (10 mM, pH 6.5) was used as the buffer system to study the photophysical properties of PPE-dNH3Cl. As shown in Figure 3-4, the optical properties of PPE-dNH3Cl are strongly affected by pH in aqueous solution. In acidic solution, the bulky side groups in PPE-dNH3Cl are positively charged, which promotes the three-dimensional separation of polymer chains by electrophobic repulsion. Howeve r, with increasing pH the ammonium groups are deprotonated which reduces the repulsive forces between ne ighboring chains so the polymers tend to aggregate. Therefore, the absorption is red-shifted and the fluorescence is quenched. In order to apply PPE-dNH3Cl in an ALP assay, two conditions ar e prerequisite. Fi rst, the initial conformation of polymers has to be random-co il to maintain relatively high fluorescence intensity before it is quenched by PPi. Second, the assay has to be usable under physiological conditions. The pyrophosphatase activity has been reported to show an upward trend with the increase of pH up to 9.2.67 In order to compromise these tw o requirements, MES buffer at pH 6.5 is used. At pH 6.5, the polymers retain high quantum yields, and the fluorescence spectra

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58 correspond to non-aggregated conformation. But the pyrophosphatase activity of ALP is not affected adversely. Quenching Studies of PPE-dNH3Cl by PPi According to previous research conducted by our group, PPE-dNH3Cl can be selectively quenched by negative ions with charges more than three, including ATP, ADP and PPi, while it cannot be quenched by those negative ions char ges lower than three, including AMP and Pi. Similar results have been obtained for the polyth iophene derivative, whic h has been applied in the detection of ATP.76 As described above, the PPE-dNH3Cl by itself is in a random-coil conformation with twisted c onjugated backbone due to the electron repulsion caused by the positively charged bulky side groups. The negative charges in ATP, ADP or PPi promote the formation of a -stacked supramolecular complex, the higher the negative charge in the molecule, the more fluorescence quenching can be achieved for the same concentration. The adenosine in the biological molecules such as ATP and ADP may enhancve the -stacked aggregates of polymer chains. So the fluorescence quenching m echanism is suggested to be anion-induced aggregation of cationic conjugated polymers. Addition of PPi (c = 0 10 M) into the 10 M PPE-dNH3Cl solution in MES buffer (10 mM, pH 6.5) results in the quenching of fluor escence (Figure 3-6). The ratio between initial fluorescence (I0) and quenched fluorescence (Iq) as a function of quenche r concentration ([PPi]) affords the Stern-Volmer plot, which disp lays a superlinear sh ape (Figure 3-6B). Similar to other CPE/quencher systems, the pl ot is linear at lower quencher concentration, but it curves upward at higher que ncher concentrations. The SternVolmer constant (Ksv) can be derived from the linear region of th e superlinear correlation as 2.7 105 M-1. In order to carry out kinetic studies of pyrophosphatase activity of ALP, a calibration plot for determination of [PPi] from the fluorescence intensity is needed. Th e linear region of the Stern-Volmer plot (up to

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59 4 mM PPi), affords a correlation coefficient R2 = 0.9886, and the intercept is very close to the theoretical value of 1.00, indicating a reasonable fit to derive the kinetic parameters. The following equation is derived from the Stern-Volmer equation and can be used to calculate PPi concentrations from the fluorescence intensities directly: (2-1) Figure 3-6. (a) The fluorescence changes upon titra tion of PPi into a solution of 10 M PPE-dNH3Cl in 10 mM MES buffe r (pH 6.5) at 37 C, ex = 380 nm. (b) Stern-Volmer plot of fluorescence quenching of PPE-dNH3Cl by PPi. Inset: Calibration curve for the fluorescence quenching by PPi. In Equation 2-1, [Q]0 and [Q]t represent the initial quencher concentration and the quencher concentration during the enzymatic r eaction at time t, respectively; I0 represents the fluorescence [Q]t = [Q]0 (b) (a)

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60 intensity before the qu encher is added; IQ represents the fluorescence intensity quenched by initial concentration of quencher before the addition of enzyme; It represents the fluorescence intensity during the enzyma tic reaction at time t. Real Time ALP Turn-on Assay To guarantee the feasibility of this ALP a ssay, a series of control experiments were performed to examine the effect of the chemi cals involved in the assay on the fluorescence of PPE-dNH3Cl (Figure 3-4). The hydrolysis product, phosphate, has negligible effect on the fluorescence of PPE-dNH3Cl. The enzyme ALP also has no effect on the fluorescence of PPEdNH3Cl. These control experiments rule out the possibility that the fluorescence intensity changes arise from any interference by these components in the solution. Figure 3-7. Fluorescence changes observed in the ALP turn-on assay. Increase of fluorescence intensity at 430 nm recorded every 10 s ec during the real-time ALP turn-on assay with varying concentrations of ALP. Conditions: [PPE-dNH3Cl] = 10 M, [PPi] = 20 M in the MES buffer (10 mM, pH 6.5) at 37 C, ex = 380 nm, em = 431 nm. In practice, the ALP turn-on assay is conducted by first measure the in itial fluorescence of 10 M PPE-dNH3Cl to obtain I0. Then the fluorescence is quenched using 20 M PPi, and the quenched fluorescence intensity, IQ, is measured. After addition of ALP solution, the fluorescence keeps rising with the hydrolysis of PPi, since the polymer aggregates are

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61 dissembled in the absence of PPi. The fluorescence intensity, It, can be monitored by timeresolved measurement, which affords the enzymatic reaction processing curve (Figure 3-7). The hydrolysis rate increases with increasing enzyme concentration. Since the fluorescence intensity increases linearly with time when the enzyme concentrations are in the range of 10-100 nM, which means the rate of reaction is constant over the time range of the measurement for these concentrations. Figure 3-8. Decrease of [PPi] dur ing the enzymatic reaction in the ALP turn-on assay with varying ALP concentrations. Conditions: [PPE-dNH3Cl] = 10 M, [PPi]0 = 4 M, in MES buffer (10 mM, pH 6.5), at 37 C, ex = 380 nm, em = 431 nm. To study the kinetics of ALP hydrolysis by this assay, the initial reaction rate is determined by first converting the fluorescence intensity, It, into substrate concentrations using the calibration plot. Since the calibration curve is lin ear only when the PPi concentrations are below 4 M, the initial substrate concentration is set to be 4 M in the kinetic experiments. Then the processing curve can be transformed into the pl ot of PPi concentrati on as a function of time using Equation 2-1. The plots in Figure 3-8 are linear in the first 60 seconds. The slopes of these lines afford the values of the initial velocity of the enzyma tic reaction with different ALP concentrations.

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62 Kinetic Studies of ALP Turn-on Assay for Pyrophosphatase Activities To derive the kinetic parameters from the processing curves above, the plots of fluorescence changes as a function of time are conve rted to the plots Ln [PPi] vs. time for five different enzyme concentrations ranging from 50 nM to 400 nM, when the substrate concentration is kept at a c onstant value 4 M (Figure 3-9). The kinetic parameter, Vmax/KM can be calculated from the slopes of the pl ots. (See Experimental part) These Vmax/KM values are plotted as a function of ALP concentrations, which affords a linear relationship, because Vmax should be directly proportional to enzyme concentration (Figure 3-9 inset). The slope of this linear plot affords a value of enzyme efficiency, kcat/ KM = 2.18 104 M-1s-1, which is in reasonable agreement with the kcat/ KM value (3.13 103 M-1s-1) obtained by Liu et al. using a PPE-CO2 -/Cu2+/PPi sensor. The observed kcat/ KM is also within the range of kcat/ KM (1.6 102 ~ 8.9 104 M-1s-1) obtained from a different ALP assa y systems in a previous literature.77 This experiment shows the PPE-dNH3Cl/PPi assay is able to detect the pyrophosphatase activity of ALP in the nanomolar range of en zyme concentration. The assay allows kinetic studies by monitoring the rapid fluorescence re sponses. The detection limit of ALP enzyme calculated from the plot of Vmax/KM vs. [ALP] is about 29 nM. However, the detection limit can be even lower when higher substrate concentration is used. (Figure 3-7) Additionally, a series of assa ys were conducted to monitor the hydrolysis of pyrophosphate with different initial substrate concentrations, in which the ALP concentration was maintained at 100 nM. The initial substrate con centrations were varied over the range of 1 M 4 M. Figure 3-10 shows plots of the changes of product concen tration [Pi] (obtained by difference, [PPi]0 to [PPi]t) as a function of reaction time with different initial substrate concentrations. Although the substrate concentrations are low and the concentr ation range is narrow, the fluorescence response curve in different samples can still be resolv ed within 200 seconds. The slopes of these plots

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63 increase with substrate concentrations as expect ed, because the initial su bstrate concentrations are far below the Km values reported in the previous literatures.68,78-81 Time (s) 0100200300 LN[PPi] -16 -15 -14 -13 -12 ALP (nM)0 50 100 200 300 400 ALP (nM) 0100200300400 V max /K m*10 -3 (s -1 ) 0 2 4 6 8 10 Figure 3-9. Natural logarithm of th e concentration of substrate fo r ALP activity as a function of reaction time plotted for different enzyme concentrations. Inset: Vmax/KM as a function of enzyme concentration. Conditions: [PPE-dNH3Cl] = 10 M, [PPi]0 = 4 M, in MES buffer (10 mM, pH 6.5), at 37 C, ex = 380 nm, em = 431 nm. Time (s) 050100150200 [Pi] (uM) 0.0 0.5 1.0 1.5 2.0 0 uM PPi 1 uM PPi 2 uM PPi 3 uM PPi 4 uM PPi Figure 3-10. Concentration of the hydrolysis produc t Pi as a function of time at various initial substrate concentrations in ALP catal yzed reaction system Conditions: [PPE-dNH3Cl] = 10 M, [ALP] = 100 nM, in MES buffer (10 mM, pH 6.5), at 37 C.

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64 Inhibition Studies of the ALP Activity The PPE-dNH3Cl/PPi system was also used to investigate the inhibition of the ALP activity. Since inorganic phosphate has been reported to be an inhibitor of pyrophosphatase activity,82-84 phosphate can be added into the enzyme systems to reduce the hydrolysis rate of the PPi. Results demonstrate that the initial react ion rate decreases with the risi ng of phosphate concentrations. [Pi] (uM) -50510 1/V 0 (s/uM) 0 200 400 600 3uM PPi 4uM PPi Ki = 4.67 uM Figure 3-11. Inhibition of ALP activity by inorganic phosphate. Conditions: [PPE-dNH3Cl] = 10 M, [ALP] = 100 nM, in MES buffer (10 mM, pH 6.5), at 37 C, ex = 380 nm, em = 431 nm, with the presence of two substrate concentrations: 3 M PPi (black dot) and 4 M PPi (red dot). The data represen ted in the Dixon plot, displaying a 1/v0 vs. [I] dependence for each [PPi]. To derive the inhibition constant Ki in this assay, a series of phosphate samples with increasing concentrations were added to the ALP assay systems with consta nt ALP concentration. The reciprocal of initia l reaction rate was plotte d as a function of phosphate concentration at two different initial substrate con centrations. (Figure 3-11) These two plots, called Dixon plots, correspond to two different substr ate concentrations, respectively ; converge in the left upper quadrant. This proves phosphate is a competitive inhibitor of pyrophosphatase activity of ALP, which means phosphate competitively binds to th e same active site of ALP as pyrophosphate. The intersection point of th ese affords a value of Ki to be 4.67 M, which is comparable to the

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65 value obtained in a former ALP assay using p -nitrophenylphosphate as substrate (5.3 M).85 This means the pyrophosphatase activity in this ALP a ssay system is very sensitive to phosphate inhibition. Therefore, we suggest that the pyr ophosphates activity is inhibited by the product formation. This explains the plot s of fluorescence response vs. tim e are initially linear, but they level off with the extended incubation time, because of the build-up of product Pi. Selectivity of PPE-dNH3Cl/PPi Turn-on Assay for ALP The selectivity of the PPE-dNH3Cl/PPi assay for ALP activity was evaluated by comparing the fluorescence responses to ALP to those with other proteins (Peptidase, BSA, Peroxidase, Glucose oxidase, Phospholipase, and hexokinase). Since none of these proteins has a specific interaction with PPi, no fluorescence response is expected to be observed from these systems. I 60min /I Q 0 20 40 60 80 100 20uM PPi 20uM PPi/200nM ALP 20uM PPi/200nM Peptidase 20uM PPi/200nM BSA 20uM PPi/200nM Peroxidase 20uM PPi/200nM Glucose oxidase 20uM PPi/200nM Phospholipase 20uM PPi/200nM hexokinase PPi ALP PTD BSA HRPGOx PLA 2 HK Figure 3-12. The fluorescence responses of PPE-dNH3Cl/PPi (10 M/20 M) to various proteins with concentration of 200 nM in ME S buffer (10 mM, pH 6.5), at 37 C. ex = 380 nm, em = 431 nm. The PPE-dNH3Cl/PPi (10 M/20 M) mixture in MES buffer (10 mM, pH 6.5) has low fluorescence intensity due to the quenching of fluorescence of PPE-dNH3Cl by PPi. The proteins (200 nM) listed above were added to PPE-dNH3Cl/PPi mixtures, and then incubated at 37 C for 60 minutes. The ratios of fluorescence intensity after incubation to that before incubation are

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66 shown in Figure 3-12. As expected, only ALP is ab le to turn-on the fluorescence intensity, while the other proteins induce only small fluorescen ce changes, which may arise from nonspecific interactions. The fluorescent respons e is 20~90 fold larger in PPE-dNH3Cl/PPi/ALP system than in other protein systems. This shows that the PPE-dNH3Cl/PPi is highly selective to ALP because of the specific pyrophosphatase activity of ALP. Discussion This ALP turn-on assay for pyrophosphotase acti vity is based on the amplified quenching of the fluorescence of PPE-dNH3Cl by pyrophosphate. The distinct fluorescent responses of PPE-dNH3Cl to pyrophosphate and phosphate allow us to monitor the hydrolysis of pyrophosphate to phosphate catalyzed by ALP. Although this is not the first ALP assay using a conjugated polyelectrolyte, this assay is superior to the former AL P turn-off assay using PPE-CO2 -, which was also developed by our group. First, in the new assay, the quencher of the fluorophore is also the substrate of ALP enzyme. This highly sensitive sensor relies on the sensitive fluorescent response of the PPE-dNH3Cl to the perturbation from small amount pyrophosphate. Therefore, only the substrate, enzyme and the fluorophore are involved in the new assay. This makes this assay easier and more efficient because fewer reagents are needed, which means the assay is more amenable to being applied in a high-throughput screeni ng (HTS) technology. Second, the Stern-Volmer equation can be directly used as a calibration plot to convert the fluorescence intensity into substrate concentrations. This simplifies the assay by eliminating the cumbersome calibration steps. This not only makes the kinetic studies more convenient but also avoids the errors introduced in the calibration process. Third, the PPE-dNH3Cl/PPi system is simpler than the previous assay. The chances of inducing interfer ence from nonspecific interactions are reduced to some extent. This greatly improves the select ivity of this assay to ALP over other proteins,

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67 with 20~90 fold increase of intensity. The fluorescence of dendritic cationic PPE-dNH3Cl also shows amplified quenching response to other biol ogical anions including ATP and ADP, while no response to AMP. The distinct fluorescence responses of PPE-dNH3Cl to different anions provide a unique platform to de velop more assays for other enzy mes which use these anions as substrates. This ALP turn-on assay paves the way for broader application of CPEs into enzymatic activities assays. Although there are many advantages of this ALP turn-on assay, some limitations are still yet to be addressed. The Stern-Volm er plot is only linear at low substrate concentrations, so the calibration plot is valid only over a narrow PPi concentration range (0-4 M). The sensitivity of this assay is undoubtedly affected by this limita tion because fewer reaction processing plots can be collected in order to guaran tee the resolution. Also, because the fluorescence intensity of PPE-dNH3Cl is sensitive to pH, the assay is performe d at pH 6.5, which is not the optimum pH for enzyme activity. In order to improve the ALP assay, more CPEs being designed to maintain high fluorescence quantum yields at higher pHs. Experimental Materials All stock solutions were prepared with water th at was distilled and th en purified by using a Millipore purifica tion system. PPE-dNH3Cl was synthesized by Zhao Xiaoyong in our group. MES buffer solution was prepared with reagent-gr ade material from Fisher. The concentrated solution of PPE-dNH3Cl is diluted with MES buffer soluti on to a final concentration of 10 M. All chemicals were used as received, unl ess otherwise noted. Sodium pyrophosphate (Na4P2O7) was purchased from J.T.Baker Chemical Co mpany, and sodium phosphate tribasic (Na3PO4) was obtained from Fisher Scientific. Each reagent was dissolved in water and adjusted to 50 mM as stock solution. Alkaline Phosphatase from bovine intestinal mucosa (ALP) and the other control

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68 proteins including peptidase from porcine inte stinal mucosa (PTD), bovine serum albumin (BSA), peroxidase from horseradish Type I (H RP), gluocose oxidase from Aspergillus niger (GOx), phospholipase A2 from bovine pancreas (PLA2) and hexokinase (HK) were purchased from Sigma-Aldrich. These proteins were dissolv ed in MES buffer and adjusted to 20 M as stock solution. The enzyme solutions were fres hly prepared immediately before their use in assays. All the assays were conducted in the same buffer solution. Instrumentation Fluorescence spectra were re corded on a spectrofluorometer from Photon Technology International and corrected by us ing 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. Fluorescence Assays The enzyme assays were carried out in ME S buffer (10 mM, pH 6.5) at 37 C. All the fluorescent intensities were r ecorded with excitation wavelength of 380 nm and emission wavelength of 431 nm. A 2-ml aliquot of polymer solution was placed in a cuvette, and the initial fluorescence intensity (I0) was measured after the sample was allowed to equilibrate thermally. The substrate was then added, and after incubating for 10 min, the fluorescence intensity (IQ) was again measured. For end-point assays an aliquot of the enzyme solution or protein solution was then added and then inc ubated for 60 min, the fluor escence intensity was recorded. For real-time assays, after the PPE-dNH3Cl/PPi solution was incubated for 10 min, the fluorescence intensity was first record ed at 10-s intervals as a blank (Ibt). The fluorescence intensity decreased with time due to the phot obleaching of polymer. Then another freshly prepared PPE-dNH3Cl/PPi solution was incubated for 10 mi n, an aliquot of enzyme solution was

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69 added to the mixture, instantly the fluorescen ce intensity was recorded at 10-s intervals (It). The signal It was corrected by Ibt for photobleaching effect. Calculation of Kinetic Parameters and the Inhibition Constants The corrected fluorescence intensities, were converted to substrat e concentrations [Q]t using Equation 2-1. The real-time fluorescence pl ots were then transformed into enzymatic reaction processing curves. The [PPi]t vs time plots were initially lin ear and then leveled off. The initial velocities were calculated from the sl opes of the linear regions in the plots. The Michaelis-Menten equation is usually used to derive kinetic parameters and expressed as (2-2) where v0 is initial rate of reaction, [S]0 is the initial substrate concentration, Vmax is the maximum rate of reaction, Km is the Michaelis-Menten constant. In this assay, the initial substrate concentrations are far below the reported Km values. So this equation can be transformed into = (2-3) where [E]0 is the initial en zyme concentration, kcat is catalytic constant or turnover number, and kcat/ Km is called as specificity constant. Accordin g to Equation 2-3, the specificity constant kcat/ Km can be obtained from the slope of a plot Vmax/ Km vs. [E]0. Meanwhile, the initial velocity can be expressed by Equation 2-4: (2-4) We can infer that Vmax/ Km can be calculated from the slope of the natural logarithm of [PPi]t vs time plot. So from the natural logarithm of [PPi]t vs time plots at different initial enzyme concentrations, we derived the Vmax/ Km for different enzyme concentrations, which affords a linear plot of Vmax/ Km vs. [E]0. From the slope of this plot we derived the specificity constant kcat/ Km for ALP enzyme. v0 =

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70 To study the competitive inhibition of ALP activity by phosphate, the inhibition constant of ALP activity Ki was calculated by Dixon plots. The reac tion velocity was measured at a fixed concentration (3 M or 4 M) of substrate but at a variety of inhibitor concentrations ranging from 0-10 M. Two plots of the reciprocal of velo city against inhibitor concentrations are plotted using two different substrate con centrations. A vertical line from the intersection point of these two plots to the inhibitor axis gives Ki.

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71 CHAPTER 4 CONCLUSION In the previous chapters, the development and optimization of two optical biosensors for alkaline phosphatase activity us ing functionalized poly(para-phe nylene ethynylene) (PPE) have been presented. Both of the ALP assays ar e based on the quenching-unquenching mechanism, which takes advantage of the amplified fluores cence responses of conj ugated polyelectrolytes (CPE) to small molecular quenche rs. Therefore, they feature bot h good sensitivity and specificity for the enzymatic activity assay. The ALP sensor described in Chapter 2 uses an imidazolecatalyzed peroxyoxalate chemilu inescence reaction to excite the fluorophore poly(phenylene ethynylene) carboxylate (PPE-CO2 -). This method displays the advantages of simple instrumentation with no need for a light source. Chapter 3 described a fluorescent ALP assay using poly(phenylene ethynylene) with ch arged amino dendritic sidegroups (PPE-dNH3 +) to monitor the rapid fluorescence responses caused by the hydrolysis of pyrophos phate in real time. These results allowed the de rivation of kinetic paramete rs and inhibition studies. Chemiluminescent Conjugated Polyelectrolyte The chemiluminescence system is base d on imidazole-catalyzed peroxyoxalate chemiluminescence reaction which consists of bi s(2,4,6-trichlorophenyl) oxa late (TCPO) as CL reagent, H2O2 as the oxalate, ImH as the catalyst and PPE-CO2 as fluorophore in buffer/CH3CN(50/50) solvent. The chemiluminescence of PPE-CO2 shows an amplified quenching response to Cu2+, because Cu2+ induces the aggregation of conjugated backbones. The sensitivity of the chemiluminescent quenching of PPE-CO2 is even higher than that of fluorescent quenching of the same polymer under the same conditions. The quenched chemiluminescenc of PPE-CO2 can be recovered by PPi since PPi sequesters Cu2+ from the polymer aggregates by forming a complex with Cu2+. Upon addition of ALP into the PPE-CO2 -

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72 /Cu2+/PPi system, the chemiluminescence intensity is quenched again, because ALP hydrolyzes PPi into Pi, which is not able to complex with Cu2+. Therefore, the Cu2+ is released from the complex with PPi and forms aggregates with polymer chains, resulting in decreased chemiluminescence intensity with incubation time. Also, the ratio of initial chemiluminescence intensity from PPE-CO2 over the quenched chemiluminescence intensity displays a linear relationship with the concentr ation of ALP concentration. This means the chemiluminescent conjugated polyelectrolyte prov ides a platform for quantitati ve analysis of ALP enzyme. This CL-based endpoint sensor using CPE utilizes simple in instrumentation while maintaining good sensitivity. However, the chemiluminescence intensity of conjugated polyelectrolyte is sensitive to the solv ent system, being highest in TrisHCl/CH3CN, followed by water/CH3CN and then HEPEs/CH3CN. CH3CN is a necessity in the system in order to stabilize the CL reagent. To extend the application of ch emiluminescent conjugated polyelectrolytes into enzymatic sensors, we hope to improve th e CL system by using more water stable chemiluminescent reagents and introducing more efficient sampling technology such as flow injection. Fluorescent Real-Time ALP Assay The PPE-dNH3Cl-based turn-on assay for pyrophosphatase activity of ALP is based on the direct interaction between the cationic dendritic polymer PPE-dNH3Cl and small negative PPi ions. The dendritic amino pr otonated side groups in PPE-dNH3Cl promote the three-dimensional separation of the neighboring polymer chai ns by electrophobic repulsions. Thus, the PPE-dNH3Cl is in random-coil conformation with high quantum yield. However, the fluorescence of PPE-dNH3Cl is highly sensitive to small amounts of an ions with more negative charges such as ATP, ADP and PPi, while insensitive to those w ith less negative charges such as AMP and Pi. The PPi is believed to form aggregates with PPE-dNH3Cl inducing the planarization of the

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73 polymer backbones by complexation with the ami no dendritic groups and re sulting neutralization of the positive charges from the side groups. Therefore, the fluorescence intensity of PPE-dNH3Cl is strongly quenched by PPi, affording a s uperlinear Stern-Volmer plot. The quencher PPi is also the substrate of th e ALP for pyrophosphatase activity. So the hydrolysis of PPi into Pi catalyzed by ALP can be monitored by r ecording the fluorescent responses of PPE-dNH3Cl/PPi upon addition of ALP in real time. Since the linear range of the Ster n-Volmer plot can be used to convert the fluorescence intensity into the PPi concentrations, the time-resolved fluorescence spectra are conveniently transformed into enzy matic reaction processi ng curves. These plots allow the derivation of kinetic parameters and the competitive inhibition by phosphate. This real-time turn-on ALP assay using ca tionic dendritic CPE is easy to perform, convenient for kinetic studies, hi ghly selective to ALP, free of non-specific interference and amenable to High-throughput screening (HTS) te chnology as well as biol ogical applications. Also, the ALP assay can be extended to other enzymatic assays, because PPE-dNH3Cl shows distinct responses to other bi ological anions, such as ATP, ADP and AMP. However, several limitations are yet to be addressed: first, the narrow substrate concentration ranges affect the sensitivity of the assay; and second, the fluorescence of PPE-dNH3Cl is sensitive to pH. The optical properties of dendritic CPEs are expe cted to be improved to further advance their applications to biological sensors.

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BIOGRAPHICAL SKETCH Lijuan Huang was born in 1983, in Jiangxi pr ovince, China, and she spent her childhood and youth there. In 1999, she left her hometown and began her undergraduate studies in Wuhan University in Hubei province, China, and obtai ned her B.S. degree in chemistry in 2003. She continued her study in Shanghai Institute of Orga nic Chemistry, Chinese Academia of Sciences and was awarded the M.S. degree in biochemi stry in 2006. After that, she was accepted by the Chemistry Department at the University of Fl orida (Gainesville) and joined the Schanze group for research on biosensors using conjugated polymers until now.