Conjugated Polyelectrolytes

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Title:
Conjugated Polyelectrolytes Synthesis, Photophysics, Aggregation Studies and Sensor Applications
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english
Creator:
Zhu, Xuzhi
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University of Florida
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Gainesville, Fla.
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Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
Schanze, Kirk S
Committee Members:
Mcelwee-White, Lisa Ann
Smith, Ben W
Castellano, Ronald K
Brennan, Anthony B

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Subjects / Keywords:
cpes -- photophysics -- sensor -- synthesis
Chemistry -- Dissertations, Academic -- UF
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Chemistry thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Abstract:
Over the past several years, significant efforts have been devoted to synthesize new sets of conjugated polyelectrolytes (CPEs) and explore their application as chemical and biosensors for the detection and analysis of a variety of molecules of environmental and biological interests, including small molecules, ions and biological targets. In this dissertation, we focus on the design and synthesis of functional poly(phenylene ethynylene)s (PPEs) and the development of fluorescent sensors. In addition, we investigate the photophysical properties and the aggregation behaviors of PPEs to get deeper understanding and provide some guidelines for future PPE-based sensors. First, a OPE derivative, cationic alkylammonium-substituted oligo(phenylene-ethynylene) was designed and synthesized. A new graft-strategy for surface modifications of silica particles was designed based on click reaction. The grafting process was successful that the functional material (SiO2-OPEC1) was able to show fluorescence under luminescence and singlet oxygen production in oxygen-saturated deuterated methanol.Then, a new series of water-soluble PPEs with guanidinium side chains were synthesized and characterized. The photophysical properties indicated that this family of PPEs was aggregated in aqueous solution. A fluorescent “turn-off” sensor for PPi was developed based on GU-P1/surfactant complex, by taking advantage of the specified interaction between guanidinium and PPi, and the amplified quenching effects of PPEs. In order to relieve the aggregation of PPEs in aqueous solution, a novel family of PPEs was designed and synthesized. The introduction of methylene carboxylate side groups significantly suppressed the aggregation of PPEs in H2O, resulting in outstanding photophysical properties. In an application of dye-sensitized solar cell, the non-aggregated PPEs in solution led to non-aggregated PPEs chains on surface, confirmed by AFM images. A fluorescent sensor for mercury ions was developed based on P4/rhodamine system. Last, a new family of cationic methylene ammonium substituted PPEs was designed and synthesized. The excellent photophysical properties supported our promise that the methylene ammonium side groups can suppress the aggregation of PPEs, similar to methylene carboxylate groups. By taking advantage of their remarkable photophysical properties, a fluorescent sensor for ATP was developed and could be applied as a potential fluorescence assay for phosphatase (ALP) in the future.
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In the series University of Florida Digital Collections.
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Includes vita.
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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 Xuzhi Zhu.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: Schanze, Kirk S.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 C ONJUGATED POLYELECTROLYTES: SYNTHESIS, PHOTOPHYSICS, AGGREGATION STUDIES AND SENSOR APPLICATIONS By XUZHI ZHU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQ UIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Xuzhi Zhu

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3 To my family and my friends

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4 ACKNOWLEDGMENTS First of all, I would like to express my deep and sincere gratit ude to my advisor Professor Dr. Kirk S. Schanze for his sup port, advice and encouragement. My Ph. D. study and research could not have been finish ed without his guidance. He l ed me into the amazing area of conjugated polyelectrolytes and helped me t o lear n independent research and scientific writing. He is such a great supervisor with enthusiasm for science and patience to his students. Besides my advisor, I would like to thank the rest of my committees: Dr. McElwee White, Dr. Castellano, Dr. Brennan and Dr. Smith for their time, encouragement, insightful comments and suggestions. I warmly thank Dr. Parkinson from University of Wyoming for his nice advice on one of my projects. My deep gratitude also goes to all the former and current members from Dr. S chanze s group for all the help, support, advice and happy memories. Especially I want to thank Dr. Fude Feng and Dr. Chen Liao for all the discussion and advices on synthesis. They shared their broad knowledge and extensive experiences about Polymer Chemi stry and Organic Synthesis with me. I really want to thank Dr. Galyna Dubinina for the help on the dissertation She looked carefully and made a lot of valuable suggestion s Dr. Abby Shelton Dr. Anand Parthasarathy and Dr. Randi Price taught me how to use almost all the instruments in my lab and helped me whenever I had a problem I would like to thank Dr. Dongping Xie and Dr. Zhuo Chen for their help and support not only on research but also on life. They are my best friends here. I also want to thank Dr. Jie Yang for her help and advice on FCS experiments. Of course I would like to thank Dr. Jan Moritz Koene n Dr. Gyu Leem, Dr. Danlu Wu, Russell Winke l Zhen xing Pan, Hsien Yi Hsu, Subhadip Goswami, Ali Gundogan for their valuable

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5 advice and friendship. I n one of my projects, I collaborated with Dr. Alexander Nepomnyashchii in Dr. Parkinson s group. I would like to show my gratitude to him for his wonderful work in AFM images and photoelectroche mical experiments This dissertation would not have been possi ble without the love and support of my family. I want to express my deepest gratitude to my parents. They always encourage and support me to continue my study abroad. Fin al l y, I want to give my biggest thanks to my girlfriend Duo, without whose love and u nderstanding my work coul d not have been completed.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 16 ABSTRACT ................................ ................................ ................................ ................... 20 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 22 Conjugated Polyelectrolytes ................................ ................................ ................... 22 Synthesis of Poly(phenylene ethynylene)s ................................ ............................. 24 Pd Catalyzed Sonogashira Reaction ................................ ................................ 24 Direct Synthetic Approach for PPEs ................................ ................................ 25 Precursor Approach for PPEs ................................ ................................ .......... 27 Ampli fied Quenching Effects ................................ ................................ ................... 2 9 Stern Volmer Fluorescence Quenching ................................ ............................ 29 Molecular Wire Effects ................................ ................................ ...................... 30 Amplified Fluorescence Quenching in Conjugated Polyelectrolytes ................. 32 Side Group Effects on Aggregation of PPEs ................................ ........................... 33 Linear Side Group ................................ ................................ ............................ 34 Branched Side Group ................................ ................................ ....................... 36 Fluorescence Correlation Spectroscopy ................................ ................................ 38 Sensor Applications ................................ ................................ ................................ 41 Small Ion Sensing ................................ ................................ ............................ 41 Protein Sensing ................................ ................................ ................................ 44 DNA Sensing ................................ ................................ ................................ .... 45 Overview of This Dissertation ................................ ................................ ................. 47 2 SURFACE MODIFICATION OF SILICA PARTICLES USING A NOVEL WATER SOLUBLE OLIGO(PHENYLENE ETHYNYLENE) ................................ .... 50 Background ................................ ................................ ................................ ............. 50 Results and Discussion ................................ ................................ ........................... 51 Synthesis and S urface M odification ................................ ................................ 51 Oligomer synthesis ................................ ................................ .................... 51 Surface modification of silica particles ................................ ....................... 53 Characterization of S urface M odified S ilica P articles ................................ ....... 53 Infrared s pectroscopy ................................ ................................ ................ 53 Thermogravimetric a nalysis ................................ ................................ ....... 55

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7 Electron m icroscopy c haracterization ................................ ........................ 56 Photophysical Properties ................................ ................................ .................. 59 Absorption and f luorescence p roperties of OPEC1 ................................ .... 59 Fluorescence and singlet oxygen emission spectrum of SiO 2 O PEC1 ...... 60 Fluorescence q uenching e xperiments ................................ ....................... 62 Summary ................................ ................................ ................................ ................ 64 Experimental ................................ ................................ ................................ ........... 64 Materials ................................ ................................ ................................ ........... 64 Instrumentation ................................ ................................ ................................ 65 General Methods of Fluorescence Quenching ................................ ................. 65 Synthe tic Procedures ................................ ................................ ....................... 66 Surface Modification of Silica Particles ................................ ............................. 69 3 CONJUGATED POLYELECTROLYTES WIT H GUANIDINIUM SIDE GROUPS : SYNTHESIS, PHOTOPHYSICS AND PYROPHOS PH ATE SENSING .................. 70 Background ................................ ................................ ................................ ............. 70 Results and D iscussion ................................ ................................ ........................... 72 Synthesis of PPEs with Guanidinium Side Groups ................................ ........... 72 Monomer synthesis ................................ ................................ .................... 72 Polyme r synthesis and characterization ................................ ..................... 73 Photophysical P roperties ................................ ................................ .................. 76 Absorption, fluorescence and fluorescence quantum yield ........................ 76 pH Effects on the absorption and fluorescence spectra ............................. 78 Fluorescence d ecay dynamics ................................ ................................ ... 79 Steady State Fluorescence Quenching of GU P1 and GU P2 in Methanol ...... 82 Application of GU P1 to PPi Sensing ................................ ............................... 87 S ummary ................................ ................................ ................................ ................ 91 Experimental ................................ ................................ ................................ ........... 92 Materials ................................ ................................ ................................ ........... 92 Instruments and General Methods ................................ ................................ ... 93 Synthetic Procedures ................................ ................................ ....................... 94 4 VARIABLE BAND GAP POLY(ARYLENE ETHYNYLENE)S FEATURING METHYLENE CARBOXYLATE SIDE CHAINS ................................ ....................... 97 Background ................................ ................................ ................................ ............. 97 Results and Discussion ................................ ................................ ........................... 99 Synthesis of PAEs with Methylene C arbox ylate S ide C hains ........................... 99 Monomer synthesis ................................ ................................ .................. 100 Polymer synthesis and characterization ................................ ................... 101 Photophysical P roperties ................................ ................................ ................ 104 Absorption and fluorescence spectroscopy ................................ ............. 104 pH Effects on the absorption and fluo rescence ................................ ........ 107 Fluorescence lifetime measurement ................................ ........................ 108 Fluorescence correlation spectroscopy ................................ .................... 113 Steady state fluorescence quenching with methyl viologen (MV 2+ ) .......... 115

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8 Application of P1 to M etal I on S ensing in A queous S olution .......................... 117 Application of P2 in Dye S ensitized Solar Cells ................................ .............. 121 Application of P4 in M e r cury (II) I on S ensing ................................ .................. 128 S ummary ................................ ................................ ................................ .............. 132 Experimental ................................ ................................ ................................ ......... 134 Materials ................................ ................................ ................................ ......... 134 Instrumentation ................................ ................................ ............................... 135 General M ethods for S urface and P hotoelectrochemical C haracterization .... 136 Synthe tic Procedure ................................ ................................ ....................... 137 5 HIGHLY FLUORESCENT CONJUGATED POLYELECTROLYTES FEATURING METHYLENE AMMONIUM SIDE GROUPS ................................ ... 143 Background ................................ ................................ ................................ ........... 143 Results and Discussion ................................ ................................ ......................... 144 Synthesis of PPEs with Cationic Methylene Ammonium Side Groups ........... 144 Monomer synthesis ................................ ................................ .................. 145 Polymer synthesis and characterization ................................ ................... 145 Fluorescence correlation spectroscopy ................................ .................... 147 Photophysical Properties ................................ ................................ ................ 149 Absorption, fluorescence and quantum yield ................................ ........... 149 Fluorescence lifetime measurement ................................ ........................ 150 Steady State Fluorescence Quenching Experiments ................................ ..... 153 Application to Adenosine Triphosphate Sensing ................................ ............ 155 S ummary ................................ ................................ ................................ .............. 158 Experimental ................................ ................................ ................................ ......... 159 Materials ................................ ................................ ................................ ......... 159 Instruments and General Methods ................................ ................................ 160 Synthetic Procedures ................................ ................................ ..................... 160 6 CONCLUSION ................................ ................................ ................................ ...... 164 Traditional PPE types CPEs ................................ ................................ ................. 164 Non oxygen PPE type CPEs ................................ ................................ ................ 165 Non aggregated PPEs ................................ ................................ .......................... 166 APPENDIX A NMR SPECTRA ................................ ................................ ................................ .... 167 B MASS SPECTRA ................................ ................................ ................................ .. 175 C FCS CALCULATION ................................ ................................ ............................. 176 LIST OF REFERENCES ................................ ................................ ............................. 179 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 187

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9 LIST OF TABLES Table page 3 1 GPC data of GU P1 Boc and GU P2 Boc ................................ ........................ 74 3 2 Photophysical data of GU P1 and GU P2 ................................ ......................... 78 3 3 Fluorescence lif etim e of GU P1 and GU P2 in MeOH and w ater (pH = 5). ........ 80 3 4 Stern Volmer constant and [Q 90 ] GU P1 in MeOH with different quenchers. ................................ ................................ ................................ .......... 84 3 5 Stern Volmer constan t and [Q 90 ] GU P 2 in MeOH with different quenchers. ................................ ................................ ................................ .......... 86 3 6 Diffusion time and hydrodynamic radius calculation. ................................ .......... 89 4 1 GPC analys i s for precursor polymers ( Pn E ) ................................ ................... 102 4 2 Phot ophysical data of PAEs ( P1 P4 ). ................................ ............................... 106 4 3 Fluorescence lifetime ( i ns) and relative amplitudes (RA %) for precursor polymer Pn E in CHCl 3 ................................ ................................ .................... 109 4 4 Fluorescence lifetime ( i ns) and relative amplitudes (RA, %) for Pn in basic MeOH and H 2 O (pH = 8.0). ................................ ................................ .............. 112 4 5 Diffusion time and hydrodynamic radius of PAEs in aqueous solution (pH = 8.0) ................................ ................................ ................................ .................. 114 5 1 Diffusion time and hydrodynamic radius of P P Es in aqueous solution. ............ 148 5 2 Photophysical data of P1 N and P2 N ................................ ............................. 150 5 3 F luorescence lifetime of P1 N and P2 N ................................ ......................... 152 5 4 Stern Volmer constant and [Q 90 ] for 2 polymer in H 2 O with AQS and K 4 Fe(CN) 6 ................................ ................................ ................................ ........ 154

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10 LIST OF FIGURES Figure page 1 1 Structures of different conjugated polymers (PPE, PPV, PT, PF, PPP). ............ 22 1 2 Structures of ionic conjugated polyelectrolytes. ................................ .................. 23 1 3 Structures of para meta and ortho PPE. ................................ ......................... 23 1 4 Mechanism of Sonogashira reaction. ................................ ................................ 25 1 5 Dire ct synthetic approach for PPE type CPEs. General direct approach; Synthesis of PPE SO 3 ; Synthesis of PPE Th NMe 3 ................................ .......... 26 1 6 Precursor approach for synthesis of PPE type CPEs. Gener al precursor approach; Synthesis of PPE CO 2 ................................ ................................ ...... 28 1 7 Structure of polymer, oligomer and quencher (MV 2+ group. ................................ ................................ ................................ ................. 31 1 8 Quenching mechanism of molecular wire effect in conjugated polymers. .......... 32 1 9 Absorption and fluorescence spectra of MPS PPV in water in the presence (dotted line) or absence (solid line) of 100 nM MV 2+ ................................ .......... 33 1 10 Absorption (left) and fluorescence (right) spectra of PPE SO 3 in MeOH (solid line), H 2 O (dashed line), and H 2 O/MeO H ( 1: 1) (dash dot line). .......................... 34 1 11 Normalized absorption and emission spectra of polymer O p in water. .............. 35 1 12 Absorption and fluore scence spectra of PPE OEG in various solvents. ............. 36 1 13 Structures of CPEs with polyionic side groups. R = CO 2 or NH 3 + ...................... 37 1 14 Absorption and fluorescence spectra of PPE NH 3 in methanol and water. [PPE NH 3 ................................ ................................ ............................. 38 1 15 Working principles of fluorescence correlation spectroscopy. ............................ 39 1 16 Setup of FCS system in our lab. ................................ ................................ ......... 40 1 17 Stern Volmer plots of PPE C O 2 ) with different metal ions (M 2+ ) in HEPES buffer s olution (0.01 M, pH 7.5). Photography of solutions of PPE CO 2 /M 2+ (5 /10 ) illuminated with a UV lamp. ................................ ........... 42 1 18 Proposed mechanism for PPi sensor based on PPE CO 2 /Cu 2+ complex. .......... 43

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11 1 19 Absorption and fluorescence spectra of PPE NH 3 in buffered solutions (pH = 6.5) with increasing PPi concentration. ................................ ............................... 43 1 20 Structures of polymer BpPPESO 3 10CPC and reaction scheme. Proposed mechanism of PLC turn off assay. ................................ ................................ ...... 45 1 21 DNA sens ing strategy based on PPE DNA and PPE DNA beacon .................. 46 2 1 Synthesis of OPEC1 ................................ ................................ .......................... 52 2 2 Surface modification strategy. ................................ ................................ ............ 53 2 3 Comparison of infrared spectra of silica particles: Unmodified silica particles ( SiO 2 OH ); alkyl azide modified silica particles ( SiO 2 N 3 ); OPEC1 grafted silica particles ( SiO 2 OPEC1 ). ................................ ................................ ............ 54 2 4 Thermogravimetric analysis of silica particles: unmodified silica particles SiO 2 OH ; azide modified silica particles SiO 2 N 3 ; OPEC1 grafted silica particles SiO 2 OPEC1 ................................ ................................ ....................... 55 2 5 Transmission e lectron m icroscop y images of silica particles: unmodified silica particles SiO 2 OH ; azide modified silica particles SiO 2 N 3 ; OPEC1 grafted silica particles SiO 2 OPEC1 ................................ ................................ .............. 57 2 6 Scanning e lectron m icroscop y images of silica particles: unmodified silica particles SiO 2 OH ; azide modified silica particles SiO 2 N 3 ; OPEC1 grafted silica particles SiO 2 O PEC1 ................................ ................................ .............. 58 2 7 Normalized absorption and emission spectra of OPEC1 in methanol (dash line) and water (solid line). ................................ ................................ .................. 59 2 8 Fluorescence spectra of SiO 2 O H and SiO 2 O PEC1 in methanol. Photographs of SiO 2 O H and SiO 2 O PEC1 in methanol under UV lamp irradiation. ................................ ................................ ................................ ........... 60 2 9 Singlet oxygen emission spectrum of SiO 2 O PEC1 in d euterated meth anol ..... 61 2 10 Fluorescence spectra of OPEC1 and SiO 2 OPEC1 upon addition of different quenchers in water: ................................ ................................ ............................ 62 2 11 Stern Volmer plo ts of OPEC1 and Si OPEC1 upon addition of AQS and K 4 Fe(CN) 6 in aqueous solution. ................................ ................................ .......... 63 3 1 Structures of the PPEs with guanidinium side groups. ................................ ....... 72 3 2 Synthesis route for monomer 5 ................................ ................................ .......... 73 3 3 Synthesis route for polymer GU P1 and GU P2 ................................ ................ 74

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12 3 4 1 H NMR spectra of monomer 5 ; GU P1 Boc ; GU P1 ................................ ........ 75 3 5 Normalized UV Vis absorption an d photoluminescence spectra of GU P1 Boc and GU P2 Boc in CHCl 3 ; G U P1 in MeOH and H 2 O ; GU P2 in MeOH and H 2 O ................................ ................................ ................................ ............. 77 3 6 Absorption and fluorescence spectra of GU P1 in H 2 O at different pH. .............. 78 3 7 Fluorescence lifetime of GU P1 in MeOH, GU P1 in H 2 O, GU P2 in MeOH and GU P2 in H 2 O. ................................ ................................ ............................. 81 3 8 Fluorescence spectra of GU P1 in MeOH upo n the addition of different quenchers. ................................ ................................ ................................ .......... 83 3 9 Stern Volmer Plot s of GU P1 ( 2 quenchers in MeOH. ................................ ................................ ........................... 84 3 10 Fluorescence spectra of GU P2 in MeOH upon addition of different quenchers. ................................ ................................ ................................ .......... 85 3 11 Stern Volmer Plot s of GU P2 different quenchers in MeOH. AQS; K 4 Fe(CN) 6 ; PPi; Pi ................................ ................................ ............................. 86 3 12 Fluorescence spectra and e mission change of GU P1 (3 ) in H 2 O (pH = 6.5) upon the addition of Triton X 100 ; fluorescence spectra of GU P1 /triton complex and emission change upon the addition of PPi ................................ ... 88 3 13 Fluore scence spectra of GU P1 /triton complex and emission change upon the addition of Pi ................................ ................................ ................................ 89 3 14 Normalized correlation curves for GU P1 (black), GU P1 /Triton X 100 (Red) and GU P1 /Triton X 100/ PPi (blue) in aqueous solutions ( pH = 6.5 ) ................ 90 3 15 Proposed PPi sensing mechanism. ................................ ................................ .... 91 4 1 Structures of poly(arylene ethynylene)s wi th methylene carboxylate side groups. ................................ ................................ ................................ ............... 99 4 2 Synthesis of monomer C1 ................................ ................................ ............... 100 4 3 Synthesis of PAEs through precursor route. ................................ ..................... 102 4 4 1 H NMR spectra of monomer C1 ; P1 E ; P1 ................................ ..................... 103 4 5 Normalized a bsorption and f luorescence spectra of P1 E P2 E P 3 E P4 E in CHCl 3 ................................ ................................ ................................ ........... 104 4 6 Normalized absorption and emission spectra of PAEs containing methylene carboxylate side chains in MeOH (solid line) and H 2 O (dash line). ................... 105

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13 4 7 Absorption and emission spectra of P1 in aqueous solutions as a function of pH. Absorption and emission s pectra of P2 in aqueous solutions as a function of pH. ................................ ................................ ................................ .. 107 4 8 Fluorescence lifetime at diff erent detection wavelengths: P1 in MeOH; P1 in H 2 O; P2 in MeOH; P2 in H 2 O; P3 in MeOH; P3 in H 2 O; P4 in MeOH; P4 in H 2 O. ................................ ................................ ................................ .................. 111 4 9 Normalized correlation curves for PAEs in aqueous solutions. ......................... 114 4 10 Fluorescence spectra of PAEs upon the addition of MV 2+ quen cher. ............... 116 4 1 1 Stern Volmer plots of PAEs upon the addition of MV 2+ quencher. .................... 117 4 1 2 Fluorescenc e spectra of P1 in H 2 O (pH = 8.0) upon addition of different metal ions. ................................ ................................ ................................ .................. 118 4 1 3 Stern Volmer plots of P1 with different metal ions in aqueous solution (pH = 8.0 ) C omparison of K s v values for different metal ions. ................................ .... 119 4 1 4 Diffusion time of P1 in the presence of different metal ions in H 2 O (pH = 8.0) obtained by the fluorescence correlation spectroscopy using the fluore scein standard. ................................ ................................ ................................ .......... 120 4 15 Synthesis of P2 H ................................ ................................ ............................ 122 4 1 6 Normalized absorption (A) and fluorescence spectra (B) o f P2 in MeOH P2 in H 2 O and P2 H in DMF ................................ ................................ ................. 123 4 1 7 Non contact tapping mode AFM images of P2 H deposited on Zn O (0001) surface from DMF solution s of diffe rent concentrations: 0, 6 and 60 g/m L. C ros s se ction analysis for the red line ................................ ............................. 124 4 1 8 Distribution of the particles with different heights obtained from Figure 4 1 7 B Distribution of the polymer chains over calculated radius. ................................ 126 4 1 9 IPCE spectra for a ZnO electrode dipped into various concentration of P2 H in DMF solution IPCE values as a function of th e dipping solution concentration ................................ ................................ ................................ ... 127 4 20 Structures of P4 and S Rho ................................ ................................ ............ 129 4 2 1 Normalized fluorescence spectrum of P4 (solid line) and absorption spect rum of S Rho Hg 2+ (dashed line) ................................ ................................ ............ 129 4 22 Fluorescence spectra of P4 and P4 / S Rho upon the addition of Hg 2+ (300 nM) in H 2 O /DMSO (99/1, v/v) F luorescence spectra of P4 / S Rho upon the addition of v arious concentration of Hg 2+ in H 2 O /DMSO (99/1, v/v). ............... 130

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14 4 2 3 Stern Volmer plots of P4 / S Rho upon the addition of different metal ions in H 2 O /DMSO (99/1, v/v); fluoresc ence intensity changes of P4 / S Rho upon the addition of different metal ions (300 nM) in H 2 O /DMSO (99/1, v/v) .......... 131 4 2 4 Proposed sensing mechanism for Hg 2 + ................................ ........................... 132 5 1 Structures of P1 N and P2 N ................................ ................................ ........... 144 5 2 Synthetic route for the monomer N1 ................................ ................................ 145 5 3 Synthesis rou te for P1 N and P2 N ................................ ................................ 146 5 4 1 H NMR spectra (500 MHz) of monomer N1 ; P1 N ; P2 N in D 2 O. .................... 147 5 5 Normalized absorption and fluorescence spectra of P1 N and P2 N in MeOH (solid line) and H 2 O (dash line). ................................ ................................ ........ 149 5 6 Fluorescence lifetime at different wavelengths: P1 N in MeOH; P1 N in H 2 O; P2 N in MeOH; P2 N in H 2 O. ................................ ................................ ............ 151 5 7 Fluorescence spectra of PPEs in H 2 O upon addition of quenchers. P1 N by AQS; P1 N by K 4 Fe(CN) 6 ; P2 N by AQS; P2 N by K 4 Fe(CN) 6 ........................ 153 5 8 Stern Volmer plots of P1 N and P2 N with various concentrations of the quenchers in H 2 O. P1 N by AQS ; P1 N by K 4 Fe(CN) 6 ; P2 N by AQS; P2 N by K 4 Fe(CN) 6 ................................ ................................ ................................ .. 154 5 9 Dephosphorylation of adenosine triphosphate (ATP) by alkaline phosphatase (ALP). ................................ ................................ ................................ ............... 156 5 10 Fluorescence spectra of P1 N (2 ) in MES buffer (10 mM, pH = 6.5) upon addition of ATP ADP AMP PPi and Pi ................................ .......................... 156 5 11 Fluorescence spectra of P1 N (2 ) in MES buffer (10 mM, pH = 6.5) upon additi on of 10 of different quenchers. Stern Volmer plots of P1 N in MES buffer upon addition of different quenchers. ................................ ..................... 157 A 1 1 H NMR spectrum (500 MHz, CDCl 3 ) of compound 5 ( C hapter 2). .................. 167 A 2 1 H NMR spectrum (500 MHz, DMSO d 6 ) of OPEC1 ( C hapter 2). ..................... 167 A 3 1 H NMR spectrum (500 MHz, CDCl 3 ) of GU P1 Boc ( C hapter 3). ................... 168 A 4 1 H NMR spectrum (500 MHz, DMSO d 6 ) of GU P1 ( C hapter 3). ...................... 168 A 5 1 H NMR spectrum (500 MHz, CDCl 3 ) of GU P2 Boc ( C hapter 3). ................... 169 A 6 1 H NMR spectrum (500 MHz, DMSO d 6 ) of GU P2 ( C hapter 3). ...................... 169

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15 A 7 1 H NMR spectrum (500 MHz, CDCl 3 ) of P1 E ( C hapter 4). .............................. 170 A 8 1 H NMR spectrum (500 MHz, CD 3 OD) of P1 ( C hapter 4). ................................ 170 A 9 1 H NMR spectrum (500 MHz, CDCl 3 ) of P2 E ( C hapter 4). .............................. 171 A 10 1 H NMR spectrum (500 MHz, CD 3 OD) of P2 ( C hapter 4). ................................ 171 A 11 1 H NMR spectrum (500 MHz, CDCl 3 ) of P3 E ( C hapter 4). .............................. 172 A 12 1 H NMR spectrum (500 MHz, CD 3 OD) of P3 ( C hapter 4). ................................ 172 A 13 1 H NMR spectrum (500 MHz, CDCl 3 ) of P4 E ( C hapter 4). .............................. 1 73 A 14 1 H NMR spectrum (500 MHz, CD 3 OD) of P4 ( C hapter 4). ................................ 173 A 15 1 H NMR spectrum (500 MHz, D 2 O, 50 o C) of P1 N ( C hapter 5). ...................... 174 A 16 1 H NMR spectrum (500 MHz, D 2 O, 50 o C) of P2 N ( C hapter 5). ..................... 174 B 1 Mass spectrum of Compound 5 ( C hapter 2). ................................ .................... 175 B 2 Mass spectrum of OPEC1 ( C hapter 2). ................................ ............................ 175

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16 LIST OF ABBREVIATIONS 3D Three dimensions 10CPC P hospholipid A DP Adenosine diphosphate A FM Atomic force microscopy A LP Alkaline phosphate A MP Ade nosine monophosphate AQS 9.10 A nthraquinone 2,6 disu lfonic acid disodium salt A TP Adenosine triphosphate BOC tert Butyloxycarbonyl BpPPESO 3 Sulfonated poly(phenylene ethynylene co p y ridi ne ethynylene) CO 2 Carboxylate CPE Conjugated polyelectrolyte D Diff usion coefficient DABCY 4 (4 Dimethylamino)phenyl azo) benzoid acid DLS Dynamic light scattering DN A Deoxyribonucleic acid DSSC Dye sensitized solar cell FCS Fluorescence correlation spectroscopy FET Field effect transistor FRET F rster resonance energy tr ansfer FTIR Fourier transform infrared spectroscopy G ( ) Autocorrelation function GPC Gel permeation chromatography GU P1 Poly( phenylene ethynylene) with guanidinium side chains

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17 GU P2 Homopolymer of poly(phenylene ethynylene) with guanidinium side chains HEX Hexachlorofluorescein HOMO Highest occup ied molecular orbital I Fluorescence intensity K sv Stern Volmer constant LED Light emitting device LUMO Lowest unoccupied molecular orbital MES 2 (N morpholino) ethanesulfonic acid MPS PPV Sulfonated poly(phenylene vinylene) MV 2+ Methyl viologen MW Molecu lar weight M n Number average molecular weight M w Weight average molecular weight NR 3 + Quaternary ammonium O p Homopolymer of poly(phenylene ethynylene) with carboxylate side chains OPE Oligo(phenylene ethynylene) OPEC1 Cationic oligo(phenylene ethynylene) with trimethylsilyl acetylene P1 P oly(phenylene ethynylene) with methylene carboxylate side chains P2 P oly(phenylene ethynylene co thiophene ethynylene) with methylene carboxylate side chains P2 H P oly(phenylene ethynylene co thiophene ethynylene) with met hylene carboxylic acid side chains P3 P oly(phenylene ethynylene co ethylenedioxythiophene ethynylene) with methylene carboxylate side chains P4 P oly(phenylene ethynylene co tetrafluorophenylene ethynylene) with methylene carboxylate side chains

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18 P1 N P oly(p henylene ethynylene) with methylene ammonium side chains P2 N P oly(phenylene ethynylene co thiophene ethynylene) with methylene ammonium side chains PAE Poly(arylene ethynylene) PDI Polydispersity PF Polyfluorene Pi Phosphate PLC Phosphatase C PNA P eptide nucleic acid PO 3 2 Phosphate PPi Pyrophosphate PPE P oly(phenylene ethynylene) PPE CO 2 Carboxylated poly(phenylene ethynylene) PPE d CO 2 Anionic poly(phenylene ethynylene) with dendric carboxylate side chains PPE NH 3 Cationic po ly(phenylene ethynylene) with dendric ammonium side chains PPE OEG Poly(phenylene ethynylene) with oligo(ethylene glycol) side chains PPE Th NMe 3 Cationic poly(phenylene ethynylene co thiophene ethynylene) with trimethyl ammonium side chains PPE SO 3 Sulfon ated poly(phenylene ethynylene) PPP Poly( phenylene phenylene ) PPV P oly(phenylene vinylene) PT P olythiophene R H Hydrodynamic radius RN A Ribonucleic acid SEM Scanning electron microscopy

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19 SiO 2 OH Blank silica particles SiO 2 N 3 Azide modified silica particles SiO 2 OPEC1 OPEC1 modified silica particles SS DN A Single strand DNA SV Stern Volmer S Rho R hodamine B thiolactone TCSPC T ime correlated single photon counting TEM Transmission electron microscopy TGA Thermogravimetric analysis THF Tetrahydrofuran TMS Tri methylsilyl TNT Trinitrotoluene V ef f Effective detection volume F Fluorescence quantum yield ma x Wavelength of maximum emission peak Viscosity of the solvent Diffusion time or lifetime k Boltzmann s constant Structure parameter r Transversal or waist radius of confocal volume z Longitudinal radius of confocal volume

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20 Abstract of Disse rtation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy C ONJUGATED POLYELECTROLYTES: SYNTHESIS, PHOTOPHYSICS, AGGREGATION STUDIES AND SENSOR AP PLICATIONS By Xuzhi Zhu Aug ust 2013 Chair: Kirk S. Schanze Major: Chemistry Over the past several years, significant effort s ha ve been devoted to synthesize new set s of conjugated polyelectrolytes (CPEs) and explore the ir application as chemical and bios ensors for the detection and analysis of a variety of molecules of environmental and biological interests, including small molecules, ions and biological targets. In this dissertation, we focus on the design and synthesis of functional poly(phenylene ethyn ylene)s (PPEs) and the development of fluorescent sensors. In addition, we investigate the photophysical properties and the aggregation behaviors of PPEs to get deeper understanding and provide some guidelines for future PPE based sensors. First, a n OPE derivative, cationic alkylammonium substituted oligo (phenylene ethynylene) wa s designed and synthesized A new graft strategy for surface modifications of silica particles was designed based on click reaction. The grafting process was successful that the f unctional material ( SiO 2 OPEC1 ) was able to show fluorescence under luminescence and singlet oxygen production in oxygen saturated deuterated methanol

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21 Then, a new series of water soluble PPEs with guanidinium side chains were synthesized and characterized The photophysical properties i ndicat ed that this family of PPEs was aggregated in aqueous solution. A fluorescent turn off sensor for PPi was developed based on G U P1 /surfactant complex by taking advan ta ge of the specified interaction between guanidin ium and PPi, and the amplified quenching effects of PPEs. In order to relieve the aggregation of PPEs in aqueous sol ution, a novel family of PPEs was designed and synthesized. The introduction of methylene carboxylate side groups significantly suppress ed the aggregation of PPEs in H 2 O, resulting in outstanding photophysical properties. In an application of dye sensitized solar cell, the non aggregat ed PPEs in solution le d to non aggregated PPEs chains on surface, confirmed by AFM images. A fluorescent sens or for m ercury ions was developed based on P4 /r hodamine system. Last, a new family of cationic methylene ammonium substituted PPEs was designed and synthesized. The excellent photophysical properties support ed our promise that the methylene ammonium side groups can suppress the aggregation of PPEs, similar to methylene carboxylate groups. By taking advantage of th eir remarkable photophysical properties, a fluorescent sensor for ATP was developed and could be applied as a potential fluorescence assay for ph osphatase (ALP) in the future.

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22 CHAPTER 1 INTRODUCTION Conjugated Polyelectrolytes In the past decades, conjugated polymers in cluding poly(phenylene ethynylene) (PPE), poly(phenylene vinylene) (PPV), polythiophene (PT), polyfluor e ne (PF) and poly( para p henylene) (PPP) (Figure 1 1) have been extensively studied. Many applications have been developed owing to their unique properties such as high conductivity, charge transport and electron polarization. 1 5 Figure 1 1. Structures of different conjugated polymers (PPE, PPV, PT, PF, PPP) Conjugated polyelectrolytes (CPEs) are con jugated polymers with ionic functional group s such as su l f onate (SO 3 ), carboxylate (CO 2 ), phosphate (PO 3 2 ) and ammonium (NR 3 + ) Some examples are shown in Figure 1 2 The conjugated backbone defines the optical and electronic characteristics of the polymers The pendant ionic solubilizing groups provide the polymers with solubility in polar solvents, including methanol and water. 6 7 Since the first anionic PPV type CPE developed by Whitten, Wudl and co workers in 1999 8 9 extensive studi es have been performed by many scientist s over the world to develop a variety of applications such as organic light emitting diodes (OLEDs) 10 12 field effect transistors (FETs) 13 dye sensitized solar cells (DSSCs) 14 16 antibacterial materials 17 23 and chemo and biosensors 24 25 Poly(phenylen e ethynylene)s (PPEs) comprise one of the most important types of CPE s and receive considerable attention s due to their remarkable fluorescence properties and facile synthesis based on palladium catalyzed Sonogashira cross

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23 coupling reaction. 7 26 28 PPEs possess the unique electronic and optical properties which arise from the conjugated polymer backbone and good solubility in water. For example, the inte rplay between the hydrophilic ionic side groups and the hydrophobic conjugated polymer backbone afford s PPEs with amphiphilic character and ability to form supra structures. As a result, many studies have explored the self assembly of PPEs in solution to form nanoscale colloidal aggregates as well as sol id liquid interfaces to form self assembled layer by layer (LbL) films. 7 29 30 In addition, the ionic side groups enable PPEs to bind strongly to ionic species by elec trostatic interactions. M any fluorescent sensors h av e been dev eloped based on PPEs, such as trinitrotoluene sensors 31 34 mercury sensors 35 36 DNA sensors 37 39 and enzym e assays 18 40 41 Figure 1 2. Structures of ionic conjugated polyelectr olytes. Figure 1 3. Structures of para meta and ortho PPE. Based on the ma i n chain conformation PPEs can be divided into three categories : para meta and ortho poly(phenylene ethynylene)s (Figure 1 3). While meta and ortho PPEs exist as helical conformation s 42 43 para PPE s adopt a linear rigid rod structure 44 45 The PPEs discussed in this dissertation are mainly para PPEs.

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24 Synthesis of Poly(phenylene ethynylene)s Pd C atalyzed Sonogashira Reaction As well known in literatures the most common synthetic method to prepare pol y(phenylene ethynylene)s i s Pd catalyzed Sonogashir a cross coupling reaction between terminal alkyne s and terminal iodid e s or bromide s. The use of CuI as a co ca talyst enables the reaction to occur at a lower temperature or eve n at room temperature. In addition, the Sonogashira reaction can be carried out in both organic solvents and aqueous solutions, which provide s a way to prepare the desired compounds with a variety of functional groups in mild and compatible conditions The mec hanism of Sonogashira reaction ha s not been clearly understood yet and the general accepte d mechanism pathway includes a palladium cycle and a copper cycle as shown in Figure 1 4. 46 In the palladium cycle, the active catalyst, 14 electron Pd 0 L 4 is either commercially available as Pd(PPh 3 ) 4 or generated from a palladium (II) source such as Pd(PPh 3 ) 2 Cl 2 by reduction. Then the oxidative addition happens between the aryl iodide or bromide with Pd(0) center. The next step in the Pd cycl e would connect with the cycle of copper co catalyst. Then a usually rate determining transmetal l ation from copper acetylide to Pd center generates the R 1 Pd( C C R 2 )L 2 specie. The final coupled alkyne is produced by reductive elimination after trans/ cis is omerization and the catalyst is regenerated. The second copper cycle is still poorly understood. It is suggested that the presence of base (usually amine) results in the formation of a Cu alkyne complex, which makes the terminal proton on the alkyne more a cidic The abstraction of the proton by the amine leads to the formation of copper acetylide.

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25 Figure 1 4. Mechanism of Sonogashira reaction. Direct Synthetic Approach for PPEs PPE type conjugated polyelectrolytes are generally prepared by the same metho ds used to synthesize the non ionic PPEs. The most important distinction is the timing and method for incorporation of the ionic units. In general, two different well known approaches have been used in the literatures. The first, which we refer to as the direct approach involves the direct polymerization of ionic monomers to afford the PPE type conjugated polyelectrolytes (Figure 1 5A). This method has some advantages, and the polyelectrolyte is prepared directly from the ionic monomers. However, a big di sadvantage of the approach is that it is much more difficult to apply gel permeation

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26 chromatography (GPC) to determine the relative molecular weight of the resulting CPE sample. M olecular weight determination of water soluble, amphiphilic polymers by GPC i s difficult because it requires special columns and instrumentation compatible with the aqueous mobile phase. In addition, the tendency of CPEs to aggregate in aqueous solution can further complicate the molecular weight and polydispersity analysis. Figure 1 5. Direct synthetic approach for PPE type CPE s (A) General direct approach; (B) Synthesis of PPE SO 3 ; (C) Synthesis of PPE Th NMe 3 Most early studies of conjugated polyelectrolytes and their synthesis relied on prepa rations that followed the direct approach (Figure 1 5) He re we provide some examples of PPEs that were prepared by the direct route. Pinto, Tan and Schanze were

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27 the first who report ed the synthesis of an anionic CPE featuring a PPE backbone (PPE SO 3, Figu re 1 5B). 6 By using a direct approach, the anionic alternating polymer PPE SO 3 was pr epared in a Sonogashira coupling reaction between a bis sulfonate 1,4 diiodobenzene monomer and 1,4 diethynylbenzene. The reaction was carried out in aqueous/DMF solution, with a Pd/Cu cataly st system in the presence of diisopropyl amine as a base. The rela tive molecular weight of PPE SO 3 was determined by intrinsic viscometer and it was found that the behavior of the polymer was corresponding to ~ 200 repeat units. 47 In a further study, S chanze and co workers reported a direct approach to prepare the cationic poly(arylene ethynylene) (PPE Th NMe 3 ) which feature d a backbone that alternate d thi ophene ethynylene and phenylene ethynylene repeat units. 18 The polymerization of the cationic trimethylammonium substituted 1,4 diiodobenzene with 2,5 diethynylthiophene was carried out in aqueous/DMF solution via Sonogashira reactio n. The resulting polymer PPE Th NMe 3 was purified by dialysis using an 8 kD molecular weight cut off membrane. The molecular weight of the cationic polymer was not determined. Precursor Approach for PPEs The second approach which has been widely used was referred to as the precursor approach (Figure 1 6). 24 48 50 In this approach the monomers used in the polymer ization are uncharged because the ionic units are protected or masked; thus polymerization leads to a conjugated polymer precursor which is soluble in organic solvents. In the next step, t he ionic groups are de protected by base ass isted hydrolysis for the esters or acid assisted hydrolysis of the BOC groups Despite the extra synthetic step, the precursor approach has several distinct advantages. First, the precursor polymer is uncharged and soluble in organic solvents, which allows determin ations of

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28 the m olecular weight and polydispersity by standard GPC. Second, high resolution 1 H and 13 C NMR spectroscopy can be applied for structural and end group analysis because of the better solubility of th e precursor in organic solvents. The previous experience show ed that 1 H NMR signals of PPEs in aqueous solvents typically appeared as broad lines, due to the slow rotational and translational diffusion of the chains and the possible aggregation. Figure 1 6. Precursor approach for sy nthesis of PPE type CPEs. (A) General precursor approach ; (B) Synthesis of PPE CO 2 A v ariety of PPEs ha ve been prepared by the precursor route. In most cases, the precursors feature ester (for anionic) or alkyl bromide (for cationic) functionality that ca n be easily converted to the ionic form in high yield by the follow up reaction A prototypical example of the use of the precursor approach to synthesize PPE type CPE is shown in Figure 1 6B. The polymer PPE CO 2 is prepared by Sonogashira polymerization o f a dodecyl ester protected 2,5 dic arboxy 1,4 diiodobenzene monomer with 1,4 diethynylbenzene. The precursor polymer is very so luble in organic solvents, due to the presence of the long dodecyl chains. The molecular weight and

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29 polydispersity are characteri zed by GPC. The s ubsequent base assisted hydrolysis of the ester groups using tetrabutylammonium hydroxide (Bu 4 OH) gives rise to the water soluble PPE CO 2 In a detailed study, Zhao and Schanze reported the synthesis of a series of PPE CO 2 with different m olecular weight using an end cap strategy. 50 The series of end capped ester polymers were analyzed by GPC T he molecular weight s o btained from the GPC analysis were compared to those obtained by 1 H NMR spectra analysis. It was found that the GPC method systematically overestimates the degree of poly merization by a factor of ~ 1.5 compared to N MR method. 50 Amplified Quenching Effects Most of interest related prope rties and applicat ions of PPEs are associated with the observation of efficient fluoresce nce quenching at low quencher concentration which is also referred to as super quenching or amplified quenching 51 52 The increased sens itivity arises from the ability of a conjugated polymer to serve as a highly efficient transport medium. C onjugated polymers transport excited states, whi ch are referred to as quasiparticles called excitons. Excitons in the conjugated polymer are highly mobile and can diffuse throughout the polymer chains. Before discussing the amplified quenching effects in more detail s it is necessary to brief ly review the mechanisms for the fluorescence quenching. 53 Stern Volmer Fluorescence Quenching ( 1 1 ) ( 1 2 ) ( 1 3 )

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30 In Equations 1 1 and 1 2, F* is an excited state chromophore, Q is a quencher molecule, k q is the bimolecular quenching rate constant, and K a is the association constant for the ground state complex formation [F, Q]. Treatment of the fluorescence quenching da ta with the Stern Volmer method yields to the Equation 1 3, where I 0 is the fluorescence intensity without a quencher, I is the fluorescence intensity in the presence of a quencher, and K sv is the Stern Volmer quenching constant. Fluore scence quenching can occur by t w o different mechanisms, namely dynamic quenching and static quenching. The dynamic quenching (Equation 1 1) is a diffusive process in which the excited state chromophore encounters the quencher molecule and the fluorescence is quenche d In the static quenching mechanism, the quencher is bo u nd to the chromophore O nce generated, the excited state is immediately and quantitatively quenched (Equation 1 2) In the case of dynamic quenching, K sv is equal to k q 0 where 0 is the fluorescence lifetime of F*. On the other hand, K sv = K a if quenching is dominated by the static mechanism. The fluoresc ence lifetime is independent on the quencher concentration. In static quenching or dynamic quenching, the Stern Vol mer plots of I 0 / I versus [ Q ] should be linear according to Equation 1 3. However, in most cases, the Stern Volmer plots are curved upward. This can be explained by a lot of complex processe s, such as variation in the association constant with quencher conc entration, mixed dynamic and static quenching mechanism, and chromophore aggregation. Molecular Wire Effects by Swager and co workers in 1995 51 To study the amplified quenching effects, fluorescence quenching of a cycl ophane containing poly(phenylene ethynylene) and an

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31 oligo(phenylene ethynylene) by methyl viologen (MV 2+ ) was compared M ethyl viologen (MV 2+ ) is a well known electron transfer quencher and can bind to the cyclophane unit, thus the fluorescence of the p oly mer was efficiently quenched. This study also showed that t he fluorescence of the polymer wa s quenched about 60 times more efficiently compared to the oligomer (Figure 1 7). Figure 1 7. Structure of polymer, oligomer and q uencher (MV 2+ ) studied by Swager s group. 51 In the mono receptor system (oligomer), the fluorescence i s quenched only for the receptor forming complex with methyl viologen. In contrast, the fluorescence of the entire polymer chain will be quenched once one or several units are occupied by the quenc her. The amplified quenching effect in the conjugated polymers is attributed to the molecular wire effect via excito n delocalization and transport by the polymer chain ( Figure 1 8). Upon excitation an exciton (a bound electron hole pair) is generated on t he polymer backbone. The conjugated polymer acts as a conduit wire for the exciton, allowing it to migrate rapidly along the chain. When the exciton reaches a repeat unit that is occupied by a quencher, it is quenched. Because of the extremely efficient

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32 ex c iton migration, a single quencher bound to one receptor unit can quench many repeat units in the polymer chain, leading to the amplified response to the target analyte. Figure 1 8. Quenching mechanism of molecular wire effect in conjugated polymers. Re printed with permission from Zhou et al 51 Amplified Fluorescence Quenching in Conjugated Polyelectrolytes The amplified quenching effect in CPEs was first reported by Whitten and co workers in the investigation of the fluorescence quenching of MPS PPV by MV 2+ (Figure 1 9 A ). 9 T he fluorescence of MPS PPV solution (10 M ) was quenched by MV 2+ (100 nM) very efficient ly with a n extremely large K sv value ~ 1.7 x 10 7 M 1 (Figure 1 9B ). The negatively charged polymer formed complex with MV 2+ driven by the electrostatic interaction. The quenching effects were amplified by th e ability of this CPE to allow excitons diffuse rapidly and efficiently within the polymer chains. In addition, the distinct

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33 red shift in absorption spectrum indicated that other mechanism may also be present such as quencher induced aggregation of the pol ymer chains. 9 Figure 1 9. (A) Structures of MPS PPV and quencher (MV 2+ ). (B) Absorption and fluorescence spectra of MPS PPV in water in the presence (dotted line ) or absence (solid line) of 100 nM MV 2+ Reprinted with permission from Chen et al 9 Side Group Effects on Aggregation of PPEs The photophysical properties of PPE s have been extensively studied in different solvents. In general, their optical properties are determined by the chemical and electronic structure of the conjugated backbone. Similar absorption and fluorescence spectra are usually obtained for PPEs, becau se they have the same phenylene ethynylene backbone. However, their photophysical properties can be strongly dependent on the solvent, because of the hydrophobic backb one and hydrophilic side groups. PPEs with ionic side groups such as sulfonate (SO 3 ) ca rboxylate (CO 2 ) or alkyl ammonium (NR 3 + ) are molecularly dissolved in methanol. However, PPEs exist as aggregates in water solution, due to the hydrophobic interaction and stacking effect. 6 54 Although the aggregation sometimes brings positive effects including

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34 enhanced response f or analyte sensing, it also results in poor solubility, low fluorescence quantum yield, broad and weak emission Therefore, significant efforts have been put forward to reduce the aggr egation of PPEs by changing different side groups Linear Side Group Ta n and Schanze reported the first water soluble PPE with anionic side groups (PPE SO 3 Figure 1 5B) in 2002. 6 In order to obtain the conformational information, t he absorption and fluorescence properties of PPE SO 3 were investigated in water, methanol and water/methanol (1:1) mixture. Figure 1 10 Absorption (left) and fluorescence (righ t) spectra of PPE SO 3 in MeOH (solid line), H 2 O (dashed line), and H 2 O/MeOH ( 1 : 1) (dash dot line). Fluorescence spectra are area normalized to reflect relative quantum yields Reprinted with permission from Tan et al 6 As shown in Figure 1 10, the absorption spectrum gradually shift ed to the long er wavelength as the ratio of water increas ed. The solvent effect wa s more pronouned in the fluorescence spectra. In pure MeOH, the fluorescence of PPE SO 3 wa s sharp,

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35 structured with a maximum ~ 450 nm. Upon the introduction of water, the fluorescence intensity decreased significantly and a broad ex c imer like band show ed up at a longer wavelength ~ 550 nm. In MeOH, PPE SO 3 exist ed as molecularly dissovled p olymer ch a i ns with photop h ysical properties similar to non ionic PPEs in organic solvents like THF or CHCl 3 55 However, PPE SO 3 wa s believed to aggregate in aqueous solution, driven by the hydrophobic interaction and interaction between adjacent polymer chains. 56 58 The dec r eased fluorescence intensity and the red shift to longer emission wavelength wer e attributed to the formation of aggregates which ha d lower energ ies and longer radiative lifetime s In addition, the fluorescen ce quantum yield wa s very low ~ 0.04 in aqueous solutions Similar aggregation behavior s a re observed for PPEs with linear cationic or anionic side groups. 47 Figure 1 11. Normalized absorption and emission spectra of polymer O p in water. Reprinted with permission from Kim et al 59 In order to reduce the aggregation, S c hanze and co workers reported a homopolymer O p with two linear carboxylate side groups per benzene. 60 61 By increasing the functional group density, the aggregation tendency in aqueous solution seem ed to be reduced, re sult ing in a emission si m i l ar to that i n MeOH. In addition ,the

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36 fluorescence quantum yield wa s improved to ~ 0.08. This work was repeated by Bunz and co workers in 2005 and a similar res ult was observed (Figure 1 11). 59 Branched Side Group Aggregation of PPEs in aqueous solution can be avoided by incorporating branched side groups. Hecht and co workers reporte d a poly(phenylene ethynylene) featuring branched oligo ( ethylene glycol ) side chains (PPE OEG Figure 1 12 ) with a surprising ly high quantum yield in aqueous solution ~ 0.43. 62 Figure 1 12. Absorption and fluorescence spectra of PPE OEG in various solvents. Absorption spectra are scaled to the same optical density, while emission spectra are corrected according to quantum yie ld. Reprinted with permission from Khan et al 62 This non ionic PPE wa s soluble in both organic solvent s, including CHCl 3 CH 3 CN, and polar solvents like MeOH and H 2 O. The fluorescence of PPE OEG in aqueous solution wa s similar to those in organic solvent s with a slightly l ower fluorescence quant um yield. By introduction of the branched oligo ( ethylene glycol ) side chains, aggregation of PPE OEG wa s efficiently suppressed. Based on this discovery,

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37 PPEs with branched oligo ( ethylene glycol ) side chains have been synthesized and used in the area of DN A detection. 39 63 64 Figure 1 13. Structures of CPEs with p olyionic side groups. R = CO 2 or NH 3 + Reprinted with permission from Lee et al 48 Recently, Schanze and co workers reported a set of poly(arylene ethy ny lene)s featuring branched polyionic side groups. 24 48 The new series of CPEs consist ed of different arylene ethynylene backbone including phenyl and 2,1,3 benzothiadiazole (Figure 1 13) T hese bulky and hi ghly charged functional groups increas e d the electrostatic repulsion between adjace nt polymer chains and twist ed the backbone effectively decreasing the hydrophobic interaction and stacking interaction T he presence of these large ionic groups also significantly enhance d the solubility of CPE s in aqueous solution. Figure 1 14 B shows the absorption and fluorescence spectra of PPE NH 3 in methanol and water. PPE NH 3 showed a negligi ble change in absorption spectrum with a maximum at 405 nm in water, the same as that in methanol. The fluorescence quantum yield was lower ~ 0.13 in aqueous solution In summary significant efforts have been made to reduce the aggregation of PPE in aque ous solution by introducing bulky and highly charged side groups. Most

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38 PPEs with branched side groups maintain the structured emission with an improved emission quantum yield in aqueous solution. However, the synthesis efforts needed for the branched side groups are usually tough and time consuming, which limit the application. Figure 1 14. (A) Structure of PPE NH 3 (B) Absorptio n and fluorescence spectra of PPE NH 3 in methanol and water. [PPE NH 3 Reprinted with permission from Zhao et al 24 Fluorescence Correlation Spectroscopy In order to obtain the direct information of polymer size, dynamic light scattering (DLS) was initially applied. However, und er the experiment conditions like mi lli molar concentration, PPEs tend to form aggregates in aqueous solution which makes the results complicated and not trustable In 1972, f luorescence correlation spectroscopy (FCS) was first repor ted by Webb and co workers in a study of DNA drug intercalation. 65 Similar to the dynamic light scattering (DLS) technique, FCS is based on the statistical analysis of spontaneous fluorescence fluctuations This method is very

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39 sensitive to polymers or materials with fluorescence at very low concentrati ons such as micromolar even nanomolar concentrations 66 FCS can pr ovide useful informati on such as diffusion constants hydrodynamic radi us and conformational change s. In addition, it is an ideal approach to investigate the thermodynamic s and kinetic s of molecular interactions. 67 69 Figure 1 1 5. Working principles of fluorescence correlation spectroscopy. FCS analysis calculates a correlation function from the time dependent intensity fluctuations of fluorescent particles observed by confocal micros copy. As shown in

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40 Figure 1 1 5, the detector records the emission fluctuations from fluorescent part icles moving in and out of a fe m toliter confocal volume formed by a focused laser beam. Figure 1 1 6 Setup of FCS system in our lab The fluorescence inte nsity fluctuates, due to Brownian motion which can provide the useful information including conformational change and molecular weight change 70 71 After an auto correlation function G( ) is applied the data are transformed in to a correlation curve. Two major results can be obta ined from this correlation curve: the diffu sion time which is defined by the temporal autocorrelation ; and the average number of fluorescent particles in t he detection volume which is calculated from the variance of the intensity L arge r molecule s usually diffuse slowly thus feature longer diffusion time, resulting in a cor relation curve at longer time. In addition, the values of G( ) decrease as the number of particles in the volume increase Therefore FCS has been used extensively to determine sample concentrations diffusion coefficient s and

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41 rate const ant s related to rotation and translation and some other important parameters i n biophysics and chemistry. 72 Sensor Applications Over the past several years, the use of PPEs as chemo or biosensor has been the subject of considerable res earch interest. Numerous sensor systems based on PPEs have been developed for various analytes, including metal ions, biomolecules 24 25 proteins, 60 enzymes 18 41 73 and nucleic acids. 38 39 74 Compared to the conventional sensory methods, the fluoresc ence sensors based on PPEs have several advantages First, PPEs are water soluble and bio compatible. Second, the amplified quenching effects of PPEs provide the sensors with hi gh sensitivity. Third, the meas urement of fluorescence is easy and convenient In general, PPE based fluorescent sensors can operate either in turn off or turn on modes. In the turn off mode, the polymer is fluoresce nt without quencher, and upon addit ion of the analyte, the polymer fluorescence is quenched. By contrast, in the turn on mode, the addition of the analyte recovers the flu orescence of the polymer. Most P PE based fluorescence sensors utilize one of the following mechanisms : pho to induced ele ctron transfer, F r ster ene rgy transfer (FRET) and conformational change (including analyte induced aggregation quenching mechanism). Since these three mechanism s are not independent, some sensors systems utilize more than one mechanism. Small Ion Sensing Many fluorescence sensors based on PPEs have been developed for small ions including Hg 2+ an d pyrophosphate (PPi). In a study reported by Schanze and co w orkers in 2008, it was found that the fluorescence of the polymer PPE CO 2 (Figure 1 2)

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42 was selectivel y quenched by Cu 2+ in aqueous solution with a K sv ~ 2.5 x 10 6 M 1 25 Several other divalent metal ions including Ca 2+ Mn 2+ Co 2+ Ni 2+ Zn 2+ and Hg 2+ we re tested and none of them was able to induce significant fluorescence quenching as shown in Figure 1 1 7 A Figure 1 17 (A) Stern Volmer plots of PPE C O 2 ) with different metal ions (M 2+ ) in HEPES buffer solution (0.01 M pH 7.5 ). (B) Photography of solution s of PPE CO 2 /M 2+ (5 /10 ) illuminated with a UV lamp. Reprinted with permission from Zhao et al 25 A photography of PPE CO 2 (5 M ) with different m etal ions (10 M ) under the illumination of a UV lamp is shown in Figure 1 17B. Clearly, the polymer solution containing Cu 2+ was dark, which indicated that the fluorescence of PPE CO 2 was quenched. It was also found that the quenched fluorescence of PPE C O 2 can be recovered upon addition of small ion pyrophosphate (PPi). 25 F igure 1 18 shows the

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43 proposed sensing mechanism for PPi based on PPE/Cu 2+ complex. The fluorescence was first quenched by the introduction of Cu 2+ via the photo induced electron transfer mec hanism Upon addition of PPi, which can chelate Cu 2+ from polymer chains, the fluorescence of the polymer was recovered Therefore, a turn on fluorescence sensor for PPi was successfully developed. Figure 1 18. Proposed mechanism for PPi sensor based on PPE CO 2 /Cu 2+ complex. Reprinted with permission from Zhao et al 25 Figure 1 19. Absorption (A) and fluorescence (B) spectra of PPE NH 3 in buffered solutions (pH = 6.5) with increasing PPi concentration. [PPE NH 3 ] = 10 Reprinted with permission from Zhao et al 24

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44 A di rect detection of PPi in aqueous solution using PPE NH 3 (Figure 1 14) was reported by Schanze and co workers in 2010. 24 PPE NH 3 wa s molecular ly dissolved in aqueous s olution with blue fluorescence by incorporation of branched ammonium side groups. It was found that pyrophosphate can induce the aggregation of PPE NH 3 and quench the fluorescence. As shown in Figure 1 19, upon addition of PPi, the absorption spectra gradually red shifted and a shoulder at long er wavelength showed up, indi cat ing the formation of aggregation. The fluorescence intensity of PPE NH 3 at ~ 430 nm decreased and an excimer like band at 540 nm appeared as the PPi concentration increased. Protein Sensing Protein s are one of the most important biological molecules, which have a variety of physiological and biological functions such as molecular recognition, gene expression, and reaction catalysis as enzymes. 52 Therefore, thousands of protein sensors have been developed for detection and analysis in medical and biological resea rch. In a study re ported by Schanze and co workers in 2008, a fluorescence turn off a ssay for phospholipase C (PLC) wa s developed based on the reversible interaction between the natur al substrate, phosphatidylcholin e and a water soluble BpPPESO 3 73 As shown in Figure 1 20 B, the fluorescence intensity of BpPPESO 3 solution in water was dramatically increased u pon addition of the phospholipid (10CPC) due to the formation of a PPE lipid complex. Incubation of the PPE lipid solution with the enzyme PLC caused the fluorescence intensity to decrease. This decr ease in fluorescence intensity wa s attributed to the disr uption of PPE lipid complex, due to the hydrolysis of the phosphatidylcholine. The optimized assay provides a n easy, rapid and real time sensor for PLC wi th a detection limit as low as 1 nM.

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45 Figure 1 20. (A) Structures of polymer BpPPESO 3 10CPC and rea ction scheme for hydrolysis of 10CPC by PLC. (B) Proposed mechanism of PLC turn off assay. Reprinted with permission from Liu et al 73 DNA Sensing Along with RNA and proteins, DNA is one of the three major macromolecules that are essential for life. Most DNA molecules are double stranded helices, consistin g

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46 of two long polymers of the comp le mentary nucleotides. Many research groups have reported DNA sensors using various types of C PEs. Bazan et al used the FRET mechanism to detect a target DNA through triplex formation of DNA/PNA or DNA/DNA with cationic poly( fluorine co phenylene)s. 74 T his methodology wa s based on electrostatic interaction between cationically charged CPE and nega tively charged oligonucleotide. Figure 1 21. DNA sensing strat egy based on PPE DNA (top) and PPE DNA b e acon (bottom). Reprinted with permission from Lee et al 38 In a work reported by Kim and co workers, DNA probe sequences w ere successfully conjugated to PPE polymers using carbo d iimide chemistry. 38 The resulted single stranded DNA (ssDNA) coupled at the end of PPEs selectively hybri dized with HEX ( hexachlorofluorescein ) labeled target complementary DNA. As shown in Figure 1 2 1 the fluorescence energy for PPE wa s efficiently transferred to the target HEX DNA

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47 upon DNA/DNA hybridization. In addition, a special oligonucleotide probe wit h a quencher at the end wa s conjugated to the PPE polymers. The oligonucleotide can form a hairpin shape in buffer solution, leading to the fluorescence quenching of the PPE. Upon the addition of the complementary target DNA, a DNA double helix wa s formed and the quencher DABCYL ( 4 (4 dimethylamino)phenyl azo) benzoid acid ) wa s far away from the polymer chain, resultin g in the fluorescence recovery of PPE. Overview of This Dissertation The primary goal of the present study is to design and synthesize functi onal poly(phenylene ethynylene)s (PPEs) a nd study the structure property relationship. Their photophysical properties are studied by the spectroscopic analysis such as UV Vis absorption, fluorescence spectroscopy and fluorescence lifetime measurements. In addition, fluorescence correlation spectroscopy (FCS) and atomic force microscope (AFM) are used to study the aggregation behavior s T he PPEs in this dissertation have been successfully applied to develop the new fluorescence sensors, DSSCs and antibact er ial materials. In C hapter 2 a fluorescent oligo(phenylene ethynylene) was designed and synthesized through multiple step S onogashira reaction. This oligomer features an alkyl ammonium side group at one end and a trimethyl silyl protected acetylene group at the other end which is ready to act as a reaction site for the next Click R eaction Absorption and fluorescence of the oligomer were investigated and the results indicated that this oligomer was slightly aggregated in water The oligomer modified sili ca particles were able to show fluorescence in water under luminescence and produce singlet oxygen in the oxygen purged deuterated methanol solution

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48 In C hapter 3 a new family of cationic poly(phenylene ethynylene) polymers featuring guanidinium side gro ups was synthesized. The photophysical properties of the series of PPEs were investigated in methanol and aqueous solution by absorp tion, steady state fluorescence spectroscopy Both polymer s show ed slight aggregation in water. Fluorescence quenching exper iments with different quenchers such as AQS, K 4 Fe(CN) 6 PPi and Pi were conducted in methanol in order to test the molecular recognition capabilities. A fluorescent sensor for PPi in aqueous solution based on GU P1 /Triton complex was developed, which showe d a great selectivity over Pi. In C hapter 4 a new series of poly(arylene ethynylene ) (PAE) conjugated polyelectrolytes feat uring methylene carboxylate side chains have been prepared. The absorption and fluorescence properties of the P A Es were investigate d in methanol and water. The photophysical data suggest ed tha t this family of PAEs did not aggregate in aqueous solution. Stern Volmer fluorescence quenching studies were carried out using methyl viologen (MV 2+ ) as an electron acceptor in water. The linear shape of S tern V olmer plots and the low K sv values suggested that this set of PAEs d id not aggregate in water, which wa s also confirmed by FCS data. The tendency of aggregation in water wa s strongly suppressed, resulted in enhanced fluorescence quantum yi elds (~ 0.16). Through careful structure propert y relationship study, we conclude that the introduction of methylene carboxylate side groups is the key to reduce aggregation in water Some app lications based on this set of P A Es were d evelope d such as DSSC and mercury ion sensor. In C hapter 5 a new family of poly(phenylene ethynylene)s (PP Es) with methylene ammon ium side groups was synthesized The photophysical properties of

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49 the series of PPEs were investigated in methanol and aqueous solution by absorp ti on, and steady state fluorescence spectroscopy Both polymer P1 N and P2 N show ed very high fluorescence quantum yield s in water with characteristic molecular ly dissolved emission A fluorescent sensor for ATP was developed based on P1 N In the future, th is sensor can be applied as a potential fluorescence assay for phosphatase enzyme (ALP), which catalyzes the dephosphor yl ation of ATP in cells

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50 CHAPTER 2 SURFACE MODIFICATION OF SILICA PARTICLES USING A NOVEL WATER SOLUBLE OLIGO(PHENYLENE ETHYNYLENE) Bac kground Bacterial infection has become a global issue, and resistance to antibiotics makes the problem even worse. Antimicrobial agents capable of killing pathogenic microorganisms have gained interest in various areas such as medical devices, healthcare products, water purification systems, hospital, etc. 75 76 Cationic conjugated polymers and oligomers containing pendant quaternary ammonium groups have been among the most promising candidates as effect iv e antimicrobials and biocides because of the light switch properties, low cost and high killing efficiency. 17 19 21 T he interaction of cationic polymers or oligomers with bacteria usually involves three steps. First, the bacteria ar e attracted reversibly to the cationic chains driven by electrostatic and hydrophobic interactions Second, w hen irradiated with UV Vis light, the conjugate d polymers or oligomers absorb radiation and sensitize the formation of singlet oxygen ( 1 O 2 ). Last, t his reactive oxygen species can penetrate the cell membrane destroy membrane components and nuclei acid s and kill the bacteria 23 76 78 The killing efficiency i s well correlated with the singlet oxygen yield of poly(phenylene ethynyl ene )s (PPEs) and oligo(phenylene ethynylene)s (OPEs). 21 In general, end functional OPEs are proven to be more efficient to kill bacteria, due to the better solubil ity and higher singlet oxygen yields. 21 In a related work Schanze and co workers reported the preparation of silica pa rticles that contained a graft layer of a po ly(phenylene ethynylene) on the surfaces 17 The surfaces of silica particles were first functionalized with aryl iodide group s which served as graft point s under Sonogashira polymerization condition s However, the

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51 application of this surface modification stra t egy wa s limited by the low grafting yield, difficult synthesis of the silane reagent and inhomogeneity of the polymer chain length. In this work, a novel oligo(phenylene ethynylene) containing a trimethylsilyl acetylene end group was designed and s uccessf ull y synthesized After deprotection, t he oligomer c an be readily attached to the azide modified surfaces of silica particles using C lick R eaction Surface modification of 300 nm diameter silica particles was first accomplished by the reacti on of the sil ica surface s with trimethoxysilane bearing a chloride group, followed by the substitu tion reaction with sodium azide. The alkyl azide units were further utilized as the graf t points for the Click Reaction This grafting process was easily monitored by in frared spectroscopy (FTIR) and thermogravimetric analysis ( TGA). The surface modified silica beads fluoresced at 400 nm, correspond ing to the oligomers emission In oxygen saturated deuterated methanol solution, the functional silica particles can generate singlet oxygen This widely applicable me thod g i ve s access to silica based colloids with the important properties of oligo(phenylene ethynylene) for possible applications in antibacterial materials and fluorescence sensing. Results and Discussion Synthes is and S urface M odification Oligomer synthesis Th e novel oligomer ( OPEC1 ) i s func tionalized with a cationic a mmonium group at one end and a trimethylsilyl (TMS) acetylene at the other end (Figure 2 1). The cationic group impart s the oligomer wi th water sol ubil ity and the interaction with bacteria The acetylene will part icipat e in the Click R eaction after deprotection Compound 1 was prepared by the Sonogashira reaction of 1 iodo 4 bromobenzene with trimethylsilylacetylene followed by the TMS deprotectio n. Sonogashira reaction of

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52 compound 1 with 4 ( trimeth ylsilylacetylene ) iodobenzene resulted in compound 2 with 85% yield. Compound 3 was synthesized by the substitution reaction of 4 iodophenol and dimethylaminopropyl chloride. C ompound 4 was prepared by th e Sonogashira reaction of compound 3 and trimethylsilyl acetylene followed by a deprotection reaction The organic solvents soluble precursor 5 was synthesized by the Sonogashira reaction of compound 2 and compound 4 The reaction of compound 5 and methyl iodide in dichloromethane gave OPEC1 as white crystal s The final compound OPEC1 was characterized by 1 H NMR, 13 C NMR and high resolution mass spectrometry Figure 2 1. Synthesis of OPEC1

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53 Surface modification of silica particles Conventional methods of silica surface modification involve reaction of surface hydroxyl groups with commercially available silane coupling reagents such as 3 (trimethoxysilyl)propyl a mine. A similar approach was used to introduce the reactive a lkyl chloride s onto th e surface of silica particles ( 300 nm, Figure 2 2 ). Active points were introduced by the substitution of alkyl chlorides with sodium azide followed by the Click R eaction with alkynes. Then the Click R eaction of the deprotected 5 a nd azide functionalized silica particles was carried out in DMF with CuBr as catalyst to attach the fluorescent oligomer onto the silica surface. The final quarterized ammonium salt ( SiO 2 OPEC1 ) was obtained in dichloromethane with methyl iodide. Figre 2 2. Surface modification strategy (i) Toluene, reflux, 8 h; (ii) NaN 3 DMF, 70 o C, overnight; (iii) compound 5 DMF, Tetrabutylammonium fluoride CuBr, N,N,N ,N ,N pentamethyldiethylenetriamine, rt, 24 h; (iv) MeI, DCM Characterization of S urface M odified S ilica P articles Infrared s pectroscopy In order to obtain the infor mation of the graft process, infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) were applied As shown in Figure 2 3 the

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54 unmodified si lica particles ( SiO 2 OH ) exhibited a strong peak at 1110 cm 1 which was assigned to the Si O Si asymmetric stretch. In addition, a broad peak centered at 3400 cm 1 was due to OH stretch from both the silanol and adsorbed water. The FTIR spectrum of SiO 2 N 3 gave clear evidence for the presence of the azide groups The peak at 2100 cm 1 corresponded to the stretch of the azide group. In addition, multiple weak peaks around 2900 cm 1 indicated the presence of sp 3 C H bonds. After the Click R eaction the peak at 2100 cm 1 disappeared completely, confirming the success of the Click reaction with all the azide units reacted. In addition, the peaks around 1600 cm 1 confirmed the presence of aromatic compounds on the silica particles ( SiO 2 OPEC1 ) Figure 2 3 Comparison of infrared spectra of silica p articles: (A ) Unmodified silica particles ( SiO 2 OH ); (B) alkyl azide modified silica particles ( SiO 2 N 3 ); (C) OPEC1 grafted silica particles ( SiO 2 OPEC1 ).

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55 Thermogravimetric a nalysis Figure 2 4 Thermogravimetri c analysis of silica particles: unmodified silica particles SiO 2 OH ( ) ; a zide modified silica particles SiO 2 N 3 ( ) ; OPEC1 grafted silica particles SiO 2 OPEC1 ( ) To obtain the information of loading levels of the oligomer on the silica particles thermogravimetric analysi s (TGA) was used. Figure 2 4 show s the TGA analysi s of unmodified silica particles ( SiO 2 OH ), azide modified silica particles ( SiO 2 N 3 ) and OPE grafted silica particles ( SiO 2 OPEC1 ). The loss ~ 6.8% below 200 o C was due to the physisorbed wat er and residual organic solvent for all types of silica particl es The unmodified silica part icles exhibited a further 2% weight loss within 200 700 o C. This decrease ar o se from the loss of the strongly adsorbed water and the dehydration of silanol units. Both surface modified silica particles ( SiO 2 N 3 SiO 2 OPEC1 ) exhibited a

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56 greater weight loss with increase of temperature; this additional loss was associated with the presence of organic material. The thermal induced weigh t loss in the TGA increase d along the series SiO 2 OH < SiO 2 N 3 < SiO 2 O PEC1 indicating that the amount of organic materials increase d along the series. Calculations were carried out to estimate the functionality density from the TGA data. At 700 o C, a weight loss of 3% for the azide modified silica particles in the TGA curve was observed which was attri buted to the presence of spacer ~ propyl group The residual mass percentage was 88% and the surface grafting density of azide groups was calculated to be ~ 9.3 chains/nm 2 according to Equation 2 1 T he TGA curve of the OPEC1 grafted silica parti cles showed that the wei ght loss percentage correspond ed to the decomposition of OPEC1 chains was 8%, and the residue mass percent age was ~ 80% at 700 o C. The surface gra fting density of OPEC1 was calculated to be ~ 8.3 chains/nm 2 ( 2 1 ) wh ere is t he surface gra fting density W Org is the weight loss percentage of the organic component, W re is the residual weight percentage N A M Org is the molecular wei ght of the organic component, W SiO2 is the weight of silica particles ( ~ 3.69 x 10 14 g/sphere), and SA is the surface area of each silica particle ( ~ 3.42 x 10 5 nm 2 ). W SiO2 and SA were obtained from manufa cturer. Electron m icroscopy c haracterization Transmission electron microscopy (TEM) was used to determine the morphology and texture of the silica particl e surfaces. As shown in Figure 2 5A unmodified silica particles had clean, smooth and spherical surf aces After the attachment of alkyl azide

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57 functional group on the surfaces, the azide modified silica particles appeared about the same size and shape as the unmod ified silica particles (Figure 2 5B) In contrast the OPEC1 grafted silica particles ( SiO 2 O PEC1 ) s howed a rough irregular surface correspond ing to the presence of organic material. The organic compounds formed a thin layer outside the surface of silica parti cles and thus changed the shape of the particles. It is of note that the TEM images d id not show a significant change in the size of the silica particles after grafting of the oligomer. Figure 2 5 Transmission e lectron m icroscop y images of silica particles: ( A ) unmodified silica particles SiO 2 OH ; ( B ) azide modified silica particles SiO 2 N 3 ; ( C ) (D) OPEC1 grafted silica particles SiO 2 OPEC1

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58 F igure 2 6 Scanning e lectron m icroscop y images of silica particles: ( A ) unmodified silica particles SiO 2 OH ; ( B ) azide modified silica particles SiO 2 N 3 ; ( C ), (D) OPEC1 grafted silica particles Si O 2 O PEC1 In order to obtain more information about the shapes and features of the organic materials on the surfaces scanning electron microscop y (SEM) was used. As shown in Figure 2 6 A and B the unmodified and azide modified silica particles had smooth uniform surfaces. In contrast, the SEM image of OPEC1 grafted silica particles (Figure 2 6C and D) clearly showed the organic layer outside the surface The SEM images reveal ed the fact that the coverage wa s not uniform. Most of the area s were covered by a thin layer of organic material, but there were evidence of large aggregates at some places There are several reasons for these aggregates One possibility is that the

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59 oligomers form multiple layers coverage Another possibility is that some organic mater ial was chemically or physically adsorbed on the surfaces. Photophysical P roperties Absorption and f luorescence p roperties of OPEC1 Figure 2 7 Normalized absorption and emission spectra of OPEC1 in methanol (dash line) and water (solid line) The photo physical properties of OPEC1 were characterized in both water and methanol by absorption and fluorescence spectroscopy. Figure 2 7 shows the normalized absorption and fluorescence spectra of OPEC1 in methanol and water T he absorption maximum in methanol w a s at 335 nm and the fl uorescence maximum wa s ~ 400 nm In aqueous solution the maximum of the absorption spectrum wa s around 322 nm with a shoulder at 370 nm which may be assigned to the aggregate absorptio n. The emission peak in water wa s shifted by abo ut 30 nm to the long er wavelength and broadened due to the formation of aggregates.

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60 Fluorescence and singlet oxygen emission spectr um of SiO 2 O PEC1 The absorption and fluorescence of SiO 2 O PEC1 were also investigated in methanol However the attempts to obta in an absorption spectrum were un successful, because the silica particles were not transparent. Figure 2 8 A shows t he fluorescence spectra of SiO 2 O PEC1 and SiO 2 O H in methanol (10 mg/mL) The s urface modified silica particles SiO 2 O PEC1 showed a stron g fluorescence with a maximum ~ 4 0 0 nm Compared to the fluorescence spectrum of OPEC1 in methanol the fluorescence spectrum of SiO 2 O PEC1 was broader and weaker, likely due to the small amount of oligomers on the silica surfaces In contrast, unmodified silica particles SiO 2 O H d id not show any fluorescence but only a scattering peak at ~ 350 nm. Figure 2 8 (A) Fluorescence spectra of SiO 2 O H (dash line) and SiO 2 O PEC1 (solid line) in methanol (B) P hotograph y of SiO 2 O H and SiO 2 O PEC1 in methanol und er UV lamp irradiation [SiO 2 ] = 10 mg/mL. The presence of the organic material on the surfaces was confirmed by the strong fluorescence of SiO 2 O PEC1 in methanol. Figure 2 8B show s photography of unmodified and OPEC1 grafted silica particles suspensions i n methanol The OPEC1

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61 grafted silica particles solution emitted strong fluor escence, but no fluorescence was ob served from the unmodified silica particles. Figure 2 9 Singlet oxygen emission spectrum of SiO 2 O PEC1 in d euterated methanol In order to pr ove the ability of SiO 2 O PEC1 to generate singlet oxygen s inglet oxygen spectrum of OPEC1 grafted silica particles was measured in oxygen saturated d euterated metha nol. After purging oxygen for half an hour, the OPEC1 grafted silica particles SiO 2 O PEC1 w ere excited at 320 nm, and the emission signal of singlet oxygen appeared at ~ 1270 nm ( Figure 2 9 ). This data suggested that OPEC1 grafted silica particles SiO 2 O PEC1 can be explor ed as antibacterial material s However, the singlet oxygen signal was weak and noisy compared to the singlet oxygen spectra of oligo(phenylene ethynylene) s and poly( phenylene ethynylene) s soluti ons. 18 19 21 One reason for this could be that the concentration of the OPEC1 in the silica particle

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62 su rfaces is much lower. In addition, the formation of aggregates on the surface s of sili ca particles may impair the ability to generate the singlet oxygen. Fluorescence q uenching e xperiments Figure 2 10 Fluorescence spectra of OPEC1 and SiO 2 OPEC1 upon addition of different quenchers in water: (A) OPEC1 quenched by AQS; (B) OPEC1 quenched by K 4 Fe(CN) 6 ; (C) SiO 2 O PEC1 quenched by AQS; (D) SiO 2 OPEC1 quenched byK 4 Fe(CN) 6. [ OPEC1 [ SiO 2 OPEC1 ] = 3 mg/mL Quencher concentrations are from 0 to 3 The q uenching experiment s of this oligomer ( OPEC1 ) and the oligomer coated silica particles ( SiO 2 OPEC1 ) in water were investigated by AQS ( 9.10 a nthraquinone 2,6 disu lfonic ac id disodium salt ) and K 4 Fe(CN) 6 Figure 2 10 shows the fluorescence spectra of OPEC1 and Si O 2 OPEC1 upon addition of different quenchers in aqueous solution. In each case, the fluorescence wa s efficiently quenched. However, the fluorescence intensity of S i O 2 OPEC1 reached a saturation point whe re the further addition of quenchers induce d littl e change to the spectra This data suggests that

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63 oligomer s form multi layer aggregates on the surfaces of silica particles in aqueous solution. The quenchers can effi ciently quench the fluorescence of the outer layer of oligomer but can not reach the internal layer of fluorescent molecules. Figure 2 11. Stern Volmer plots of OPEC1 and Si OPEC1 upon addition of AQS and K 4 Fe(CN) 6 in aqueous solution. Figure 2 11 shows the Stern Volmer plots of OPEC1 and Si O 2 OPEC1 upon addition of AQS and K 4 Fe(CN) 6 The curves of OPEC1 upon addition of AQS and K 4 Fe(CN) 6 curve s upward, and the Stern Volmer constants are ca lculated as ~ 1. 9 x 10 6 M 1 for AQS and 1.1 x 10 6 M 1 for K 4 Fe(CN ) 6 respectively. In contrast, the Stern Volmer curves of Si O 2 OPEC1 reaches a pla te a u at the quencher concentration ~ 0. 5 with a larger Stern Volmer constants ( 5.8 x 10 6 M 1 for AQS and 5.9 x 10 6 M 1 for K 4 Fe(CN) 6 ) The formation of oligomer aggregat es on the surfaces of silica particles enables the inter chain exciton transfer pathway, resulting in a larger K sv value. It may be also due to higher charge density of the silica particles.

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64 Summary In this chapter a novel water soluble oligo(phenylene eth ynylene) ( OPEC1 ) was synthesized with a quaternized am monium group at one end and a TMS protected acetylene at the other end Deprotected acetylene was used for grafting to silica particles by Click chemistry. The surface grafting process was monitored b y infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) The presence of the functional organic material on the surfaces of silica particles was confirmed and the surface morphology was investigated by SEM and TEM The absorption and fluorescen ce of OPEC1 were character ized in both methanol and water The modified silica particles Si O 2 OPEC1 exhibit ed a comparable fluorescence spectrum to that of OPEC1 and genera te d singlet oxygen in deuterated methanol Steady state fluorescence quenching exper iments of OPEC1 and Si O 2 OPEC1 were performed by AQS and K 4 Fe(CN) 6 in aqueous solution The data suggested that OPEC1 was aggregated on the surfaces of silica particles. The oligomer and functionalized silica particles are believed to be promising candidat es as biocid al materials. Experimental Materials Pd(PPh 3 ) 4 was purchased from Strem Chemical Company and used as received. 9.10 A nthraquinone 2,6 disu lfonic acid disodium salt (AQS) dimethylaminopropyl chloride 4 bromoiodobenzene and 4 iodophenol wer e purchased from Sigma Aldrich and used without further purification. Potassium carbonate, hydrochloric acid and potassium ferrous cyanide (K 4 Fe(CN) 6 ) were purchased from Fisher Scientific Company and used as received THF and DMF were purified by solvent dispensing system. Uniformly sized silica microspheres were purchased from Bangs Lab

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65 ( http://www.bangslabs.com ) as dry powder s All other chemicals w ere purch ased from Sigma Aldrich or Fisher Scientific compan ies a nd used without further purification An d for all experiments in water the solutions were prepared using water (pH = 6.5). Instrumentation NMR spectra were recorded on either Varian Gemini ( 300 MHz) or Inova 2 ( 500 MHz) spectrometer, and chemical shifts w ere reported in parts per million using CDCl 3 or DMSO d 6 as solvents. Infrared spectra were taken using KBr pellets on a Perkin Elmer Spectrum One FTIR spectrometer. Thermogravimetric analysis (TGA) data were obtained on a TA instruments Q5000 thermal ana lysis system. The samples were heated from room temperature to about 700 o C at a heating rate of 20 o C /min under a dry nitrogen atmosphere. UV V is spectra were collected on a Varian Cary 50 UV Vis spectrophotometer. Photoluminescence measurements were c arried out on a PTI f luorescence spectrophotometer. 1 cm square quartz cuvette was used for both absorption and emission measurements. Scanning e lectron m icroscope images were obtained on a Hitachi S 4000 FE SEM instrumen t. Transmi ssion e lectron m icroscope images were obtained on a Hitachi H 7000 TEM instrument. Singlet oxygen measuremen ts were carried out on a PTI f luorescence spectrophotometer equipped with a near IR detector. The sample solution was suspended in deuterated methanol and purged oxygen for half an hour before experiments. The sample was ex c ited at 330 nm. General Methods of Fluorescence Quenching OPEC1 oligomer solution (2 mL, 10 M) or a silica particles suspension ( SiO 2 OPEC1 2 mL, 3 mg/mL) was placed in a rectangular quartz cell and titrated with different quenchers (AQS, K 4 Fe(CN) 6 ) The fluorescence spectra were measured after

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66 addition of the quencher s Fluorescence peak inten sities were used for the construction of the Stern Volmer (SV) plots if not specifically mentioned. The ratio of initial fluorescence intensity to observed fluorescence intensity ( I 0 / I ) was plotted versus the quencher concentration. K sv values were obtaine d b y fitting of the lin ear region of S tern V olmer plots Synthe tic Procedures 4 ( Trimethylsilyl ethynyl)iodobenzene was synthesized following the literature procedure 79 4 Bromophenylacetylene (1) To a degassed mixture of 4 bromoiodobenzene ( 1 g 3.5 mmol) and trimethylsilylacetylene ( 0.45 mL 4.4 mmol) in 70 mL THF and 30 mL diisopropylamine, 1 5 mol% of Pd(PPh 3 ) 4 and 2 mol% of CuI w ere add ed T he reaction was allowed to run for 3 h at room temperature. T he r esulting mixture was then filtered and the filtrates were evaporated and dissolved in hexane The solution was then washed with saturated NH 4 Cl once and then deionized water twice. O rganic layers were combined, dried with anhydrous Na 2 SO 4 The final compound 1 was purified by f lash chromatograph y in hexane and obtained as a white powder (y ield: 80% ) 1 H NMR ( 3 00 MHz, CDCl 3 ) : 7.44 (d, 2 H), 7.33 (d, 2 H), 3.11 (s, 1 H). 13 C NMR ( 75 MHz, CDCl 3 ) : 133.55, 131.60, 123.15, 121.06, 82.59, 78.37 1 Bromo 4 ((4 trimethylsilylacetylenebenzyl)ethynyl)benzene (2). To a mixture of compound 1 ( 181 mg 1 mmol) and 4 trimethylsilylacetylene iodobenzene ( 40 mg 1 mmol) in degased 100 mL THF and 30 mL diisopropylamine, 1.5 mol% of Pd(PPh 3 ) 4 and 2 mol% of CuI w ere dispersed T he reaction was stirred for 8 h at room temperature. T he reaction mixture was then filtered and the filtrates were evaporated and dissolved in dichloromethane The solution was then washed with saturated NH 4 Cl

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67 once and then DI water twice. Organic layers were combined, dried with anhydrous Na 2 SO 4 Flash chromatograph y in dichloromethane gave the desired compound (y ield: 86% ) 1 H NMR ( 3 00 MHz, CDCl 3 ) : 7.50 (m, 6H), 7.40 (d, 2H), 0.28 (s, 9H). 13 C NMR (75 MHz, CDCl 3 ) : 133.20, 133.13, 131.86, 131.56, 123.35, 123.11, 122.93, 122.15, 104.73, 96.67, 90.38, 90.37, 0.16. 4 (3 (N,N dimethylamino)propoxy) iodobenzene (3) To a mixture of 4 iodophenol ( 2.20 g 10 mmol) and dimethylaminopropyl chloride ( 1.22 g 10 mmol) in 50 mL acetonitrile, 5 g K 2 CO 3 and 0.1 g KI were added The reaction was allowed to run for 6 h at 70 C. The resulting mixture was then filtrated and filtrates were evaporated and redissolve d in dichloromethane. C olumn chromatograph y in DCM / Me OH ( 9 : 1) gave the desired compound 3 (y ield: 75% ) 1 H NMR ( 3 00 MHz, CDCl 3 ) : 7.5 5(d, 2H), 6.67 (d, 2H), 3.96 (t, 2H), 2.62 (t, 2H), 2.24 (s, 6H), 1.90 (m.2H). 13 C NMR ( 75 MHz, CDCl 3 ) : 158.98, 130.23 117.04, 82.69, 66.43, 56.41, 45.70, 27.60. 1 Ethynl 4 (3 (N,N dimethylamino)propoxy)benzene (4). To a degased solution of compound 3 (2 g 4.96 mmol) in 50 mL THF and 30 mL diisopropylamine, Pd(PPh 3 ) 4 ( 30 mg ) and CuI ( 60 mg ) catalyst were added via spat ula Then trimethylsilylacetylene (0.75 mL 6 mmol) was added via syringe T he reaction was allowed to run overnight at room temperature. The reaction mixture was filtered and the filtrates were evaporated and redissolved in dichloromethane The solution was then washed with saturated NH 4 Cl once and then deionized water twice. Organic layers wer e combined, dried with anhydrous Na 2 SO 4 The organic solvents were removed under vacuum to yield a yellow powder, which was dissolved in DCM / MeOH ( 1:1). After dega sing for 30 min, potassium carbonate ( 4 g ) was added and the reaction was

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68 allowed to run for 3 h. After removing the solvents under vacuum, column chromatograph y in DCM / Me OH ( 9:1) gave the desired compound (y ield: 60% ) 1 H NMR ( 3 00 MHz, CDCl 3 ) : 7.42 (d, 2H), 6.86 (d, 2H), 4.00 (t, 2H), 3.00 (s, 1H), 2.68 (t, 2H), 2.30 (s, 6H), 1.98 (m, 2H). 13 C NMR ( 75 MHz, CDCl 3 ) : 159.48, 133.71, 114.61, 114.17, 83.88, 75.93, 66.32, 56.41, 45.52, 27.44. Compound 5. To a degased solution of compound 4 (301 mg 1 mmol) and compound 2 (500 mg 1.41 mmol) in 40 mL THF and 20 mL diisopropylamine, Pd(PPh 3 ) 4 ( 20 mg ) and CuI ( 30 mg ) were added The reaction was allowed to run overnight, followed by filtration. The filtrates were evaporated and the residue was disso lved in DCM. The solution was then washed with saturated NH 4 Cl once and then deionized water twice. The o rganic layers were combined and dried with anhydrous Na 2 SO 4 After removing the solvents under vacuum, column chromatography in DCM / MeOH (9:1) gave the des ired compound as a white powder (y ield: 78% ) 1 H NMR ( 5 00 MHz, CDCl 3 ) : 7.48 (d, 10H), 6.80 (d, 2H), 4.00 (t, 2H), 3.34 (t, 2H), 2.75 (s, 6H), 2.48 (m, 2H), 0.30 (s, 9H). 13 C NMR (1 25 MHz, CDCl 3 ): 159.49, 133.32, 132.14, 131.73, 131.60, 131.58, 123. 07, 123.35, 123.28, 115.18, 114.82, 104.82, 96.62, 91.82, 91.32, 90.91, 88.02, 66.47, 56.51, 45.65, 27.59, 0.13. HRMS ( APC I) m / z : [M+H] + calc d. f or C 32 H 34 NOSi, 476.2404 ; found, 476.2417. OPEC1. To a solution of compound 5 ( 200 mg ) in 15 mL DCM 1 mL MeI wa s added via syringe. After stirring overnight, the precipitate s were collected and dried under vacuum. Yield: 90% 1 H NMR ( 5 00 MHz, DMSO d 6 ) : 7.52 (m, 10H), 6.95 (d, 2H), 4.23 (t, 2H), 3.40 (t, 2H), 3.15 (s, 9H), 2.24 (m, 2H), 0.30 (s, 9H). 13 C NMR (1 25 MHz, DMSO d 6 ): 159.41, 138.76, 133.87, 132.67, 132.47, 132.37, 132.21, 123.82, 123.19,

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69 122.47, 118.04, 115.72, 115.00, 97.46, 92.40, 91.78, 91.37, 88.48, 65.67, 63.66, 53.03 23.21, 0.53. HRMS (ESI) m / z : [M I] + calcd. for C 33 H 36 NOSi, 490.2561 ; found, 490.2567. Surface Modification of Silica Particles Si O 2 N 3 Silica particles (200 mg) and trimethoxysilylpropylchloride ( 2 mL ) were mixed in 10 mL toluene and refluxed for 8 h. The surface modified silica particles were collected via centrifugation and washed with acetone several times. Then the particles were added to a mixture of sodium azide ( 2 g ) in 10 mL DMF. After stirring for overnight at 70 o C the mixture was poured into 20 mL deionized water. The azide modified silica particles were collected by centrifugation and washed by water 4 times and MeOH twice. The azide modified particles were dried under vacuum for 1 day. Si O 2 OPEC1. To a degass ed mixture of surface modified silica particles ( Si O 2 N 3 50 mg ) and compound 5 ( 50 mg 0.1 mmol ) in 10 mL DMF, TBAF solution ( 0.5 mL 1M in THF) was added via syringe. The reaction was allowed to run for 3h. Then 5 mg CuBr and 2 mL p entamethyldiethylenetriamine were added. The C lick R eaction wa s allowed to run for 24 h under argon atmosphere The silica particles were collected by centrifugation and washed by DMF 3 times, acetone 3 times and MeOH 3 times until no fluorescence in supernatant under UV light. The surface modified silica particles w ere dried under vacuum for 1 day. Then the particles were added to a solution of 1 mL MeI in 5 mL dichloromethane. After stirring for 12 h, the surface modified silica particles ( Si O 2 OPEC1 ) were collected by centrifugation and washed by acetone several ti mes. Then silica particles were dried under vacuum for 1 day.

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70 CHAPTER 3 CONJUGATED POLYELECTROLYT ES WITH GUANIDINIUM SIDE GROUPS : SYNTHESIS, PHOTOPHYSICS AND PYROPHOS PH ATE SENSING Background Conjugated polyelectrolytes (CPE s ) have attracted significant in terest during the past decades and become one of the versatile polymer materials in photovoltaic devices, organic solar cells and biochemical sensors due to their exceptional physical and photophysical properties, such as high fluorescence quantum yield, e lectrostatic interaction and extraordinary high sensitivity to the fluorescence quenchers. 6 7 14 51 52 60 80 In particular CPEs have been intensively explored as chemical and biological sensor s owing to their high sensitivit y and selectivity 43 54 61 81 The amplified quenching is attributed to the de localization and migration of the excitons along the polymer backbone, described as the molecular wire effect 51 T hree different mechanisms are proposed for the amplified quenching : ph oto induced electron transfer, F r ster resonance energy transfer and analyte induced aggregation. Specially, the an alyte induced aggregation mechanism provides the fluorescence sensors with unique high sensitivity and selectivity through the specific interaction between the polymer side group s and the analyte s 24 Pyrophosphate (PPi) anion plays an important role in numerous biological processes including ATP hydrolysis and DNA hybridization 82 The detection of PPi is investigated as a real time DNA sequencing method. 82 83 Of all the PPi receptors, guanidinium has attracted considerable interest recently. 84 86 T he guanidinium unit is n aturally present in the side g roup of the amino acid ( arginine ) and able to form strong ion interactions with oxoanions such as carboxylates, sulfates and phosphates. The guanidinium molecule has unique planar Y shaped co nfiguration and very high pKa ~

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71 13 which ensure s protonation over a wide pH range. 87 88 T he first example of a PPi sensor based on pyrene funtionalized mono guanidinium receptor was reported by Teramae s group. 89 It was found to self assemble to form a 2:1 ( host : guest ) complex with pyrophosphate. The PPi work ed as a spacer linking two host receptors. The binding constant was c alculated as 9.8 x 10 7 M 1 89 However, this sensor system has obvious limitations and only applicable in non aqueous solution such as methanol. Herein, we report a novel family of water soluble poly(phenylene ethynylene) s (PPEs) for PPi sensor, where guanidinium units are incorporated into the conjugated polymer system to impart both wate r solubility and molecular recognition properties. The photophysical properties of the series of PPEs were investigated in CH 3 OH and H 2 O solution by absorption, steady state fluorescence spectroscopy and fl uorescence lifetime measurement Similar to other PPEs, these polymers undergo spontaneous aggregation in aqueous solution, resulting in a broad fluorescence spectra and a low fluorescence quantum yield. 6 90 91 The a ddition of the non ionic surfactant (Triton X 100) into the weakly fluorescent aqueous solution of GU P1 increased fluorescence by forming polymer/s urfactant complex. The fluorescence of the polymer /surfactant complex in aqueous solution was effectively quenched by the addition of pyrophosphate (PPi) with high selectivity over Pi ; the quenching occurs because PPi binds to the guanidinium groups and in duces the aggregation of polymer/surfactant complex. In addition, it was found that diffusion time of the aggregated complex increased by 9 times in comparison with free PPE/surfactant complex. Therefore, a PPi sensor is developed utilizing the significant fluorescence spe ctra change and size change. Th is

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72 fluorescence sensor shows high selectivity for PPi over phosphate (Pi) in aqueous solution. R esults an d D iscussion Synthesis of P PEs with Guanidinium S id e G roups In this chapter we report t he synthesis of water soluble P PE s with novel guanidinium side chains as shown in Figure 3 1. GU P1 is a copolymer alternating with 1,4 ( phenylene ethynylene ) while GU P2 is the homopolymer. Both polymers have two guanidinium side chains for each repeat unit. The novel guanidinium side groups provide the good solubility in water and also molecular recognition capacity for the polymers. Figure 3 1. Structures of the PPEs with guanidinium side groups. Monomer synthesis Figure 3 2 shows the synthesis route for the monomer 5 Starting from the commercial available 1,4 bis(hydroxyethoxy)benzene, compound 1 and 2 were synthesized according the literature. 90 Azide substitution of compound 2 afforded compound 3 in a modest yiel d. Compound 4 was prepared by the reduction of PPh 3

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73 followed by the h ydroxylation. The reaction of amine groups and Boc 2 1 H pyrazole 1 carboxamidine provided the monomer 5 with 87% yield Figu re 3 2. Synthesis route for monomer 5 (i) CBr 4 PPh 3 MeCN 0 o C, 4 h; (ii) I 2 bis(trifluoroacetoxy)iodobenezene, CH 2 Cl 2 rt, 6 h; (iii) NaN 3 DMF, 40 o C, overnight; (iv) PPh 3 THF, H 2 O; (v) Boc 2 1 H pyrazole 1 carboxamidine DMF, rt, 24 h. Polymer synthesis and character ization The polymers were synthesized following the precursor route in which the monomers used a re unc harged and the polymerization l e a d s to a precursor conjugated polyme r that is uncharged. The ionic groups are unmasked in a subsequent reaction (e.g., base hydrolysis of the ester, or quaternization of the te rtiary amine by methyl iodide). T he synthesis route s for preparing the precursor organic soluble polymers the water soluble target p olymer s are shown in Figure 3 3 The precursor polymers were p repared by Sonogashira coupling of a stoichiometric amount of the monomer 5 with B OC protected guanidinium side chains and diethynylbenzene or trimethylsil y lacetylene

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74 comonomer ( GU P1 Boc or GU P2 Boc respectively) 62 The poly merization was carried out in organic solution in order to avoid elect rostatic repulsion of the ionic charged functional groups and to facilitate polymer characterization by gel permeation chromatography (GPC). Hydrolysis of the precursors was accomplished by treating the organic polymers with trifluo ro acetic acid (TFA) The water soluble P PEs were obtained as bright yellow solids in ~ 90 % yield after ly o ph i lization. Each polymer was characterized by 1 H NMR. Figure 3 3. Synthesis route for polymer GU P1 and GU P2 (i) Pd(PPh 3 ) 4 CuI, 1,4 di ethynylbe ze ne, THF, iPr 2 NH ; (ii) Pd(PPh 3 ) 4 CuI, TMS acetylene, THF, DBU; (iii) TFA, CHCl 3 Table 3 1. GPC data of GU P1 Boc and GU P2 Boc Polymer M n (kD) M w (kD) PDI GU P1 Boc 76 101 1.3 GU P2 Boc 42 79 1.9 The number and weight average molecular we ight ( M n and M w respectively) for the precursor polymers were characterized by GPC analysis against polystyrene

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75 standard s in THF As shown in Table 3 1, GU P1 Boc has a M n ~ 76 kD and PDI ~ 1.3, while GU P2 Boc has a M n ~ 42 kD and PDI ~ 1.9. Figure 3 4. 1 H NMR spectra of ( A ) monomer 5 ; ( B ) GU P1 Boc ; ( C ) GU P1 Figure 3 4 shows the representative 1 H NMR spectra of monomer 5 the precursor polymer GU P1 Boc and the water soluble polymer GU P1 Comparison between the spectra of monomer 5 and GU P1 Boc r eveal ed the appearance of the peaks at ~ 7.60 and 7.00 ppm which were assigned to be the proton s on the polymer backbone The resonance peaks appear ed to lose split pattern and become broader in the spectra of GU P1 Boc After hydrolysis, the 1 H NMR spectroscopy of GU P1 was accomplished in DMSO d 6 Due to the low solubility, the solvent peak and water residue peak show ed up strongly. No signals were observed in the range of 0.5 ~ 1.5 ppm and

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76 the peak at 8 8 ppm and 11.5 ppm di sappeared, indicating that the BO C groups were cl eaved with an exc ellent yield ( >95%). In addition, t he protons of guanidinium in DMSO d 6 showed up in the range of 7.0 8.0 ppm, close t o the peaks of aromatic protons. 92 Photophysical P roperties Absorption, fluorescence and fluorescence quantum yield The photophysical properties of th e P PEs were investigated by UV visible absorption fluorescence spectroscop y and fluorescence lifetim e measurement The absorption and fluorescence spectra are shown in Figure 3 5 Although GU P1 Boc and GU P2 Boc (Figure 3 5A ) show ed the similar pattern GU P2 Boc exhi bit ed a red shift in both absorption and emission spectra c om pared to GU P1 Boc This red shift wa s consistent with the differences between the homopolymer O p 61 and copolymer PPE CO 2 25 The solvent effects on water soluble polymers GU P1 and GU P2 were studied in CH 3 OH and H 2 O (Figure 3 5B and 3 5C ) M ethanol is typically consi dered to be a good solvent for P PEs because polymer aggregation is minimal. 6 In methanol, GU P1 exhibited an absorption maximum at 383 nm and a fluorescence maximum at 435 nm while GU P2 showed red shifted spectra with an absorption maximum at 415 nm and a fluorescence maximum at 473 nm. A bsorption spectr a are red shifted while fluorescence spectr a show a broad, red shifted excimer like band indicating that both GU P1 and GU P2 appeared to be aggregated i n aqueous solution Lacking of the bulky or highly charged side groups, GU P1 and GU P2 h ad a tendency to agg regate in aqueous solution driven by hydrophobic interactions of the polymer backbone leading to low fluorescence quantum yields (~ 2%), which are similar to other PPEs with linear side groups including PPE CO 2 and PPE SO 3 6 47 48 54

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77 Figure 3 5 Normalized UV Vis absorption and photoluminescence spectra of ( A ) GU P1 Boc (solid line) and GU P2 Boc (dash line) in CHCl 3 ; ( B ) GU P1 in MeOH (solid line) and H 2 O (dash line); ( C ) GU P2 in MeOH (solid line) and H 2 O (dash line). H 2 O has pH = 5. In summary, absorption and fluorescence studies suggest ed that both GU P1 and GU P2 exist ed as s lightly aggregated state s with low fluorescence quantum yield

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78 in water. In contrast, polymer GU P1 and GU P2 were more solvated in methanol with little evidence of the aggregat ion Tabl e 3 2 Photophysical data of GU P1 and GU P2 Polymer Solvent max ab (nm) max fl (nm) fl B GU P1 MeOH 383 435 0.16 H 2 O A 391 476 0.02 GU P2 MeOH 415 473 0.11 H 2 O A 415 500 0.02 A H 2 O pH = 5. B Quinine sulfate in 0.1 M H 2 SO 4 solut fl = 0.545). pH Effects on the absorption and fluorescence spectra Because GU P1 and GU P2 share the same solubilizing group (guanidinium), we focus on the pH effects on absorption and fluorescence spectra of GU P1 Figure 3 6. Abso rption (A) and fluorescence (B) spectra of GU P1 in H 2 O at different pH. Figure 3 6 shows the effects of pH on the absorption and emission spectra of GU P1 in aqueous solutions. At pH lower than 7, the absorption and fluorescence spectra remained the sam e, indicating that the polymers were well solvated in H 2 O.

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79 Guanidinium group had a pK a ~ 13, 87 88 and a s the pH increased the guanidinium side gr oup began to partially lose proton, leading to a decreased solubility and formation of aggregates. As shown in Figure 3 6A, the absorption shifted to the red and the intensity decreased from pH 7 to pH 11, signaling the formation of aggregates. The fluores cence of the polymers was more sensitive to the conformational changes induce d by the pH. As pH increased from 3 to 11, the fluorescence shifted to red and became broad and weak. The largest change in fluorescence spectra occurred between pH 7 and pH 9. P ar tial deprotonation of the side groups of the polymer result ed in a conformational change to big aggregates. 18 19 Fluorescence d ecay d ynamics The presence of aggregates in the P PEs causes the dynamic interaction between the excitons state in non aggregated chains and excitons localized on aggregate (trap) chains. In order to g et more information about the photophysical pro cess in the P PEs with guan idinium side chains, we measured the fluorescence de cay in both methanol and water using the time correlated single photon counting (TCSPC). T he detailed fitting parameters for solutions of GU P1 and GU P2 in pure methanol and in water at pH at 5.0 are sh own in Table 3 3 All the CPEs showed three exponential decay with components ranging from 0.15 ns to 2.12 ns. Here we provided a better phenomenological understanding of the decay kinetics including lifetim es ( i ) and relative amplitude contributions (RA%) as a function of emission wavelengths and solvents (Figure 3 7 ). The non expone ntial decay is due to the nature of inhomogeneous distribution of chromophores in the complex polymer system consisting of single chain dissolved chains and aggregated chains. In addition, conformational

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80 disorder of individual polymer chains gives arise to a distribution of conjugated segments with different chain lengths. Table 3 3 Fluorescence lifet im e of GU P1 and GU P2 in MeOH and w ater (pH = 5) MeOH H 2 O RA (%) GU P1 i (ns) 450 nm 500 nm 550 nm 600 nm i (ns) 450 nm 500 nm 550 nm 600 nm 1 = 0.15 73 72 75 58 1 = 0.17 88 82 76 75 2 = 0.46 27 27 23 36 2 = 0.57 12 17 22 22 3 = 1.26 0 1 2 6 3 = 1.98 0 1 3 3 2 0.99 0.99 1.00 1.02 2 1.09 0.99 1.01 0.99 GU P2 1 = 0.17 82 74 68 67 1 = 0.13 95 93 89 85 2 = 0.54 18 26 31 31 2 = 0.61 5 6 10 13 3 = 1.73 0 0 1 2 3 = 2.12 0 1 1 2 2 1.01 1.01 0.98 0.97 2 1.00 0.97 0.97 0.93 Global analysis of the emission decay of GU P1 in methanol yielded three decay components, with the fastest two components (0.15 ns and 0.46 ns) contributing to more than 94% of the overall amplitude (Figure 3 7A ). As th e detection wavelength increas ed from 450 nm to 600 nm, the fastest component had a decreasing contribution while t he second component (0.46 ns) slightly increased its contribution to the overall amplitude. Under acidic conditions in water (pH = 5.0), the fit of the fluorescence decay also featured three exponential decay s Similar results were obtained for GU P1 in H 2 O with two fast components (0.17 ns and 0.57 ns) contributing more than 97% to the overall amplitude (Figure 3 7B ). However, compared with Figure 3 7A the shorter

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81 lifetime component had a larger contribution while the longer lifetime components had relati vely smaller contributions to the overall amplitude, resulting a shorter mean lifetime in water. This can be attributed to the fact that P PE chains adopt ed a more favorable aggregation state in H 2 O, driven by the nature of hydrophobicity and those aggregat es act ed like energy traps. Table 3 2 show s that the fluorescence quantum yield of GU P1 in water is lower than that in methanol, a difference consistent with a larger fluorescence decay rate. Figure 3 7 Fluorescence lifetime of ( A ) GU P1 in MeOH, ( B ) GU P1 in H 2 O, ( C ) GU P2 in MeOH and ( D ) GU P2 in H 2 O. H 2 O has pH = 5 at various emission wavelengths. The fluorescence decay of the GU P2 solution in methanol featured a similar three component decay with = 0.17 n s, 0.54 ns and 1.73 ns (Figure 3 7C ). In the

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82 aqueous solution, the decay of GU P2 was domina t ed by a short lived component ( = 0.13 ns, > 85%), indicating that P PE polymers ha d a stronger tendency to exist as aggregate states in water (Figure 3 7D ). There are two issues to consider with respect to the wavelength dependence of the amplitudes. First, this wavelength dependence may be due to multiple emissive segments, such as the aggregated chains in water. Second, the longer lifetime components ha ve a larger contribution at a longer wavelength. It may be explained by the fact that the emission at longer wavelength has a slower decay rate, where the contribution from the aggregated polymer chains and longer chain segments is larger. Overall, the em ission decay kinetics reveal ed the fact that the fluorescence decay behavior of P PEs wa s compl icated The changes in the fluorescence decay dynamics indicate d that energy wa s transferred to and quenched by aggregated states. At short wavelength the decay w a s rapid, likely due to energy transfer from single chains to aggregate traps. At longer wavelength the lifetime wa s longer, reflecting the lower radiative decay rate for the aggregated chains. 48 The shorter fluorescence lifetime in polar solvent was consistent with the lower fluorescence quantum yield due to the fact that polymer wa s aggregated. Steady State Fluorescence Quenchi ng of GU P1 and GU P2 in Methanol The guanidinium unit was reported to self assemble to form a 2:1 complex with biological relevant pyrophospha te (P 2 O 7 4 PPi) in MeOH. 87 89 In order to investigate the binding efficiency of PPi, fluorescence qu enching experiments of GU P1 and GU P2 in methanol wer e performed using PPi AQS, K 4 Fe(CN) 6 and Pi as quencher s Figure 3 8 shows the fluorescence of GU P1 in methanol upon addition of different quencher solutions. All quenchers quenched the fluorescence o f GU P1 in methanol efficiently.

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83 AQS and K 4 Fe(CN) 6 are known efficient energy transfer quencher s and the fluorescence of the polymer was quenched without any shape change as seen in Figure 3 8A and B. Strong fluorescence quench ing w ere observed for PPi a nd Pi accompanied by a slight broadening of the emission spectra and a loss of vibronic structure at higher quencher concentrations. This can be attributed to the fact that PPi and Pi can induce the aggregation of the polymer in MeOH. Figure 3 8. Fluo rescence spectra of GU P1 in MeOH upo n the addition of different quenchers. (A) AQS ; (B) K 4 Fe(CN) 6 ; (C) PPi; (D) Pi. [ GU P1 ] = 2 Figure 3 9 show s Stern Volmer quenching plots of GU P1 ( 2 AQS, K 4 Fe(CN) 6 PPi, and Pi. In each case the SV plots were curved upward and consequently we characterized each plot by a K sv value calculated from the linear

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84 portion of the c urves at low quencher concentration along with the [Q 90 ] value, which is defined as the quencher concentration that affords 90% fluorescence quenching. Fluorescence quenching data are summarized in Table 3 4 Figure 3 9 Stern Volmer Plot s of GU P1 ( 2 M) with various concentration of the quenchers in MeOH. AQS ( ) ; K 4 Fe(CN) 6 ( ) ; PPi ( ) ; Pi ( ) Table 3 4 Stern Volmer constant and [Q 90 ] GU P1 in MeOH with different quenchers. Polymer Quencher K sv (M 1 ) [Q 90 ] ( ) GU P1 PPi 3.6 x 10 6 0.8 K 4 F e(CN) 6 6.8 x 10 5 3.0 AQS 6.1 x 10 5 4.4 Pi 5.3 x 10 5 6.0 Among all the quenchers, PPi exhibited the strongest quenching with the highest K sv value of 3.6 x 10 6 M 1 and the lowest [Q 90 ] value of 0.8 AQS and K 4 Fe(CN) 6

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85 known as widely used electro n deficient quencher molecules had S tern Volmer coefficients of 6.8 x 10 5 M 1 and 6.1 x 10 5 M 1 respectively. In addition, the GU P1 showed a good selectivity for PPi over Pi; Pi had much weaker ability to quench the fluorescence with a K sv value of 5.3 x 10 5 M 1 and a [Q 90 ] value of 6.0 T he significant fluorescence quenching by PPi was presumably a consequence of aggregation induced by the complexation of two guanidinium side groups of the polymer GU P1 with PPi. Although AQS had two sulfate groups, there was no evidence that AQS can cause the aggregation under the experimental condition s likely due to the low binding affinity to gua nidinium and less basic nature. Figure 3 10. Fluorescence spectra of GU P2 in MeOH upon addition of different quenchers. (A) AQS; (B) K 4 Fe(CN) 6 ; (C) PPi ; (D) Pi. [ GU P 2 ] = 2

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86 Figure 3 11 Stern Volmer Plot s of GU P 2 different quenchers in MeOH. AQS ( ) ; K 4 Fe(CN) 6 ( ) ; PPi ( ) ; Pi ( ) Table 3 5 Stern Volmer constan t and [Q 90 ] GU P 2 in MeOH with different quenchers. Polymer Quencher K sv (M 1 ) [Q 90 ] ( ) GU P 2 PPi 4 .6 x 10 6 1.2 K 4 Fe(CN) 6 1 5 x 10 6 1.1 AQS 8 0 x 10 6 0.4 Pi 1 .3 x 10 6 4.5 Fluorescence quenching of GU P2 by different qu enchers were conducted in MeOH and shown in Figure 3 10. Figure 3 11 shows the Stern Volmer plots of GU P2 in MeOH, where the Stern Volmer constants were calculated from the linear range at low concentration and the [Q 90 ] values were obtained. Compared with GU P1 all the

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87 quenchers ha d a higher K sv value due to the fact that GU P2 had a higher charge density. AQS and K 4 Fe(CN) 6 exhibited K sv values, 8 x 10 6 M 1 and 1.5 x 10 6 M 1 respectively. In MeOH, GU P2 also had a good selectivity for PPi over Pi, as seen in Table 3 5. The Stern Volmer constant for PPi wa s 4.6 x 10 6 M 1 3 times higher than that for P i ~ 1.3 x 10 6 M 1 However, the [Q 90 ] of PPi for GU P2 wa s 1.2 which wa s larger than the [ Q 90 ] for GU P1 The differences can be explained by the possib ility for formation of an intra chain complex, where two neighbor guanidinium units bind with only one PPi molecule. In general, it was shown that PPi can quench t he fluorescence of polymers much more efficient ly than Pi. I n part the enhanced quenching efficiency ar o se because the associati on constant for the guanidinium / PPi complex wa s larger compared with the other anions. In addition, another effect contributed t o the significantly enhanced fluorescence quenching: analyte induced aggregation of the polymer chains As the concentration increase d PPi act ed like a bridge, inducing polymer aggregation. In particular, analyte induced polymer aggregation turn ed on path ways for three dimensional exciton migration and significantly increase d the que nching ability of the quencher. 93 95 Application of GU P1 to PPi Sensing Intera ction of GU P1 with PPi in water g ave unexpected results, the fluorescence intensity of GU P1 increased first and decreased afterward. This was likely due to fact that polymer alone wa s aggregated in aqueous solution Previous work showed that addit ion of surfactant to CPEs cause d in the fluorescence recovery 96 A similar result was observed that the addition of non ionic surfactant Triton X 100 to a solution of GU P1 in water gave a substantial increase in fluorescence

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88 intensity. The significantly enhanced fluorescence intensity and blue shif ted spectrum of polymer chains by surfactant molecules. A titration was carried out to quantify the effects of Triton X 100 concentration on the polymer fl uorescence. As s hown in Figure 3 12 A upon the addition of Triton X 100 from 0 to 200 M the fluorescence intensity of GU P1 increased by about 15 fold at 435 nm. The significant change of the fluorescenc e intensity suggest ed that surfactant molecules inhibit ed the aggre gation of the poly mer chains as reported 97 98 The improved properties can be used in PPi sensor development i n aqueous solution. Figure 3 12 ( A ) Fluorescence spectra and emission change of GU P1 (3 ) in H 2 O (pH = 6.5) upon the addition of Triton X ; ( B ) fluorescence spectra of GU P1 /trito n complex and emission change upon the addition of PPi from 0 to 30 [ GU P1 ] = 3 [Triton X 100] = 200 Insets are the Stern Volmer pl ots. As an effort to develop a PPi sensor, a fluorescence quenching experiment of the mixture of GU P1 /Triton ([ GU P1 ton X was carried out upon addition of various concentration of PPi. As shown in Figure 3 12B addition of PPi induced significant fluorescence quenching via analyte induced aggregation mechanism, with a modest Stern Volmer value of 1.7 x 10 5 M 1 and a [Q 90 ]

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89 ~ 22 As a comparison, the effect of Pi was studied by addition of Pi to the GU P1 /T riton X 100 mixture. V ery little or no quenching was observed, indicating that GU P1 /Triton mixture had a high selectivity of PPi over Pi (Figure 3 13). Although Pi can neutralize the polymer charges, clearly it is una ble to induce the polymer aggregation Figure 3 13 F luorescence spectra of GU P1 /triton complex and emission change upon the addition of Pi from GU P1 Inset is the Stern Volmer plots. Table 3 6 Diffusion time and hydrodynamic radius calculation. Number GU P1 Triton X PPi (s) R H (nm) 1 2 0 0 1.46 x10 4 3.14 2 2 200 0 2.98 x 10 4 6.41 3 2 200 50 2.57 x 10 3 55.3 In order to de termine the size change in our P PE system, fluorescence correlation spectroscopy (FCS) was applied to measur e the diffusion behavior of

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90 aqueous GU P1 GU P1 / Triton complex and GU P1 / Triton /PPi complex. Dif fusion time and subsequently calculated hydrodynamic radius are summarized in Figure 3 14 and Table 3 6 Figure 3 14 Normalized correlation curves for GU P1 (black), GU P1 /Triton X 100 (Red) and GU P1 /Triton X 100/ PPi (blue) in aqueous solutions ( pH = 6.5 ) The solid lines are single specific fitting curves. Black: GU P1 GU P1 (2 GU P1 The results indicate d that GU P1 /Triton complex showed a slightly longer diffusio n time (29.8 ms) compared to GU P1 alone (14.6 ms). The increase of the diffusion time wa s probably attributed from the hydrophobic inter action between surfactants and polymer chain s that le d to the formation of PPE/surfactant complex. After PPi wa s introd uced, a significant further increase o f the diffusion time (55.3 ms)

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91 wa s observed, indicating the formation of very large aggregates. The size change of the polymer contribute d to its fluorescence quenching, which wa s consistent with the idea that P PEs wer e cross linked together by PPi to form inter chain aggregates. Figure 3 15 Proposed PPi sensing mechanism Therefore, a PPi sensing strategy is established based on the interaction between the natural substrate PPi, surfactant and a fluorescent water soluble conjugated polyelectrolyte as illustrated in Figure 3 15 The fluorescence intensity of the P PE in water wa s increased significantly by the addition of Triton X 100, due to the formation of a CPE surfactant complex. Pyrophosphate induced aggregation cause d substantial fluorescence quenching, which in turn allow ed sensing of dissolved PPi in aqueous solution. S ummary We have successfully prepared and characterized a new series of conjugated polyelectrolyt es based on the poly(phenylene ethynylene) ba ckbone featuring

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92 Hydrolysis of the precursor polymers followed by dialysis against pure water affords water soluble cationic P PEs. Investigations of the p hotophysical propert ies of the P PEs led to the conclusion that both GU P1 and GU P2 we re aggregated in water, with lower fluorescence qu antum yield, red shifted broad fluorescence spectra and shorter fluorescence lifetime. Steady state fluorescence quenching of GU P1 and GU P2 with a series of quenchers reveal ed the fact that two guanidinium side units can form a complex with PPi, which in turn induce d the aggregation of polymer chains and dramatically decrease d the fluorescence intensity. A fluorescence tu rn off sensor for pyrophosphate wa s developed by taking advantage of the int eraction between water soluble P PE, surfactant and PPi. In aqueous solution, the spectroscopic properties of the polymer/surfactant we re sensitive to the concentration of PPi with high selectivity o ver Pi due to analyte induced aggregation mechanism. The change in the polymer aggregation state wa s readily examined by fluorescence correlation spect roscopy, which directly provide d the size of polymer chains. We are currently developing biological assay s for enzymes with PPi as substrates using this system and we believe the design principles can be applied to other anion species of interest. Experimental Materials Pd(PPh 3 ) 4 was purchased from Strem Chemical Company and used as received. Triton X 100 s o dium pyrophosphate, sodium phosphate, Boc 2 1 H pyrazole 1 carboxamidine 9 10 anthraquinone 2,6 disu lfonic acid disodium salt (AQS), and 1,8 d iazabicyclo[5.4.0]undec 7 ene (DBU) were purchased from Sigma Aldrich and used

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93 without further purification. Sodium azide, trifluoroacetic acid, hydrochloric acid and potassium ferrous cyanide (K 4 Fe(CN) 6 ) were purchased from Fisher Scientific Company and used as received THF and DMF were purified by solvent dispensing system. All chemicals were from commercial sources unless specially mentioned. S tock solutions (1.0 mM polymer repeat unit ) of all P P Es were prepared in H 2 O (pH = 5) and were stored at 4 o C. Instruments and General M ethods NMR spectra were record ed using a Gemini 300 NMR operating at 300 MHz for 1 H NMR and at 75 MHz for 13 C NMR for small organic compounds 1 H NMR spectra of the polymers were measured in Inova2 500 NMR operating at 500 MHz for 1 H NMR Gel permeation chromatogra phy (GPC) analysis w as carried out on a system comprised of a Shimadzu L C 6D pump, Agilent mixed D column and a Shimadzu SPD 20A photodioide array (PDA) detector, with THF as eluent at 1 mL/min flow rate. The system was calibrated against linear polystyrene standards in THF. UV Vis absorption spectra were measured on a Shimadz u UV 1800 spectrophotometer. Luminescence spectra were measured on a PTI (Photon Technology International) fluorescence spectrometer. Fluorescence lifetimes were determined by time correlated single photon counting on a FluoTime 100 spectrometer ( Pico Quan t ) equipped with 370 nm diode laser as excitation source. Fluorescence quantum yields were reported relative to known standards. The optical density of solutions at the excitation wavelength was <0.1 and corrections were applied for differences in the refr active index of standard and sample solutions. Fluorescence correlation spectroscopy (FCS) measurements were taken on a homemade setup using a 405 nm diode laser as the excitation light. F luorescein ( 30 nM )

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94 in 10 mM phosphate buffer (pH = 8) was used as th e calibration standard for the system. Synthetic P rocedures 1,4 Bis(2 bromoethoxy)benzene ( 1 ) and 2,5 d iiodo 1,4 bis(2 bromoethoxy) benzene ( 2 ) were synthesized according to literature procedures 90 2,5 Diiodo 1,4 bis(2 azidoethoxy)be nzene (3). To a solution of compound 2 (3.0 g, 5.2 mmol ) in 50 mL of dry DMF, sodium azide (6.77 g, 0.1 mol) was charged. The mix ture was allowed to stir at 40 o C overnight. Upon the completion of reaction, the mixture was poured into 100 mL of cold water The white pre cipitate was collected by fil tration and dried under vacuum (y ield: 2 5 g, 8 2%). 1 H NMR ( 300 MHz, CDCl 3 ): 3.64 (t, 4H), 4.10 (t, 4H), 7.22 (s, 2H). 13 C NMR ( 75 MHz, CDCl 3 ): 50.49, 69.24, 86.32, 123.36 153.23. 2,5 Diiodo 1,4 bis(2 aminoethoxy)benzene (4). To a solution of c ompound 3 (2.5 g, 10 mmol) in 180 mL of THF and water mixture ( v/v, 2/1) triph e nylph osp h ine (8.18g, 31.2 mmol) was charged. The mixture was heated to reflux for 2 h. The solvent was removed under vacuum and the residue was taken by 20 mL acetone. C oncentrated HCl ( 5 mL ) was added slowly to the solution, whereupon a white precipitate was f ormed The precipitate was collected by vacuum filtration and re dissolved in 30 mL water and basified with 20 mL of 1N NaOH solution The white solid was collected by vacuum filtration and dried under vacuum (y ield: 1.2 g, 52%). 1 H NMR ( 300 MHz, DMSO d 6 ): 1.50 (s, 4H), 2.84 (t, 4H), 3.92 (t, 4H), 7.35 (s, 2H). 13 C NMR ( 75 MHz, DMSO d 6 ): 40.74, 41.72, 87.90, 123.41, 153.13. Compound 5. To a stirred solution of compound 4 (1 g, 2.23 mmol) in DMF (20 Boc 2 1 H pyrazole 1 carboxamidine (1. 52 g, 4.90 mmol). The

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95 resulting solution was stirred at room temperature for 24 h. The white precipitates were collected by vacuum filtration and washed thor oughly by large amount of water (y ield: 1.8 g, 87%). 1 H NMR ( 500 MHz, CDCl 3 ): 1.49 (s,18H), 1.51 (s, 18H), 3.88 (t, 4H), 4.06 (t, 4H), 7.19 (s, 2H), 8.81 (m, 2H), 11.48(s, 2H). 13 C NMR ( 125 MHz, CDCl 3 ): 28.32, 28.55, 40.33, 69.07 79.60, 83.37, 86.57, 123.55, 153.01, 153.17, 156.67, 163.74. HRMS (ESI) m / z : [M+Na] + calcd. for C 32 H 50 I 2 N 6 O 10 Na, 955.1570 ; found, 955.1572 Synthesis of GU P1 Boc. To a degassed solution of c ompound 5 (93.3 mg, 0.1 mmol) and 1,4 diethynylbenzen e (12.6 mg, 0.1 mmol) in 25 mL THF/iPr 2 NH ( v/v 4/1), tetrakis(triphenylphosphine)palladium(0) (10 mg, 8.7 ol) and CuI (10 mg, 52 m ol) were added. The solution was stirred at 60 C for 48 h. The solvent was removed under vacuum. The residue was re dissolved in 20 mL CHCl 3 and then passed through a short alumina column to remove all the catalyst. The result ing solution was concentrated to 3 mL and then poured into a large volume of hexane. The polymer p recipitate s were collected as yellow fine powder and the polymer was further purified by multiple precipitation in hexane (y ield: 70% ) 1 H NMR ( 500 MHz, CDCl 3 ): 1. 3 0 (s, 18H), 1. 45 (s, 18H), 3.90 (br, t, 4H), 4. 20 (br, t, 4H), 7. 0 1 (s, 2H), 7.5 5 (s, 4H), 8.90 (br, 2H), 11.52 (s, 2H). GPC (THF, Polystyrene standards): M n = 76.2 kD, M w = 101 kD, PDI = 1.33. Synthesis of GU P2 Boc. To a solution of c ompound 5 ( 142 mg, 0.15mmol) in 15 mL THF, 0.8 mL DBU and 0.2 mL deionized water were added. After degassing for 30 mins, CuI (10 mg, 52 ol) and Pd(PPh 3 ) 4 (10 mg, 8.7 ol) were added under the protection of a rgon. Then 22 L trimethylsilylacetylene was added to the so lution by syringe. The solution was stirred at room temperature for 3 days before directly passing through a short alumina column. The resulting solution was concentrated and poured

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96 into a large volume of hexane. The polymer precipitated as a fine powder a nd then further purified by several steps of dissolution in CHCl 3 followed by precipi tation fro m hexane ( y ield: 50% ) 1 H NMR ( 500 MHz, CDCl 3 ): 1. 2 5 ( s, br 18H), 1. 45 (s, 18H), 3.9 0 (br, 4H), 4. 2 0 (br, 4H), 7. 0 0 (s, 2H), 8.80 (br, 2H), 11. 4 5 (br, 2H). GPC (THF, Polystyrene standards): M n = 41.5 kD, M w = 79.4 kD, PDI = 1.91. Hydrolysis GU P1 Boc (80 mg, 0.1 mmol) or GU P2 Boc (65 mg, 0.1 mmol ) was dissolved in 20 mL CHCl 3 and cooled down in an ice/water bath. T rifluoroacetic acid (TFA 20 mL ) was added to the polymer solution drop wise. Upon the completion of the addition, the reaction mixture was allowed to warm to room temperature and stirred for another 24 h. The excess TFA and the solvent were removed under vacuum and the residue was taken by 3 mL ethanol followed with addition of 3 mL hydrochloric acid. Then the polymer solution was poured into a large volume of acetone and precipitated as a yellow powder. The polymer was further purified by several steps of dissolution in water followed with precipitation from acetone. The polymer was dissolved in 20 mL water and filtered through a cellulose membrane (pore size: 0.22 um). Final purification of the polymer was accomplished by dialysis of aqueous solution of the polymer against water ( p H = 5) using Fisher Brand cellulose membrane (12 kD molecular weight cut off) for 3 days. Light yellow solid was obtained in a yield of 80~90 % after ly o ph i lizat ion GU P1 : 1 H NMR ( 500 MHz, DMSO d 6 ): 3.6 5 ( br 4H), 4.2 0 ( br 4H), 7. 0 0 8.0 0 ( br 16 H). GU P2 : 1 H NMR ( 500 MHz, DMSO d 6 ): 3.6 0 ( br 4H), 4.2 5 ( br 4H), 7 .2 0 7. 7 0 ( br 10 H) 7.75 8.1 0 (br, 2H).

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97 CHAPTER 4 VARIABLE BAND GAP POLY(ARYLENE ETHYN YLENE)S FEATURING METHYLENE CARBOXYLATE SIDE CHAINS Background Over the past decades, significant efforts have been devoted to synthesize new conjugated polyelectrolytes (CPEs) and explore their application s in photovoltaic devices, solar cells, 49 and che mical and biosensors 24 In particular, P A E type CPEs have attracted more and more attention, owing to their extraordinary properties such as high fluorescence quantum yield, water solubility, and high sensitivity to fluorescence quenchers due to excito n migration. 52 99 100 In addition the amplified fluores cence quenching effects are enhanced in aggregate d states when exciton inter cha in migration is accessible. 54 91 101 However, the application s of poly(arylene ethynylene)s ( PAEs ) is sometimes limited by low quantum yield, bad solubility and unexpected sensing behavior caused by the self assembly into aggregates in aqueous solutions. 52 94 As mentioned earlier in C hapter 3, methanol is typically considered as a goo d solvent for conjugated polyelectrolytes whi ch preserve their good optical properties such as the high fluorescence quantum yields, sharp structured fluorescence spectra, and low S tokes shift from absorption maxim um to emissio n maxim um In aqueous soluti on, PA Es exhibit a red shift in UV Vis spectra with a pronounced shoulder and a like It wa s found that t he difference s in photophysical properties we solv 6 7 For this reason, sizable efforts have been made to reduc e aggregation tendencies by addi ng surfac tants, attach ing bulky ionic side groups or twist ing the polymer backbone. 48 64 96

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98 The traditional PPE type CPEs always had an oxygen on the linker directly attached to the polymer backbone and in a related work reported by Schanze and coworkers, three OPEs without oxygen linkers exhibited extraordinary photophysic al properties in aqueous solution. 102 The remarkable photophysical properties were attributed to the introduction of methylene car boxylate side groups, resulting in unaggregated OPEs in aqueous solution. Therefore, a new series of poly(arylene eth y nylene) s (PAE s ) conjugated polyelectrolytes featuring methylene carboxylate side chains have been prepared. The series cons ist of four mem bers of polymers which share the same anionic side groups : methylene carboxylate ( CH 2 CO 2 Na).The repeat unit of the poly(arylene ethynylene) backbone com prises of a bis( methylene carboxylate )phenylene ethynylene unit alternating with a second arylene ethy nylene moiety and four different aryl s were used, Ar = 1,4 phenyl ( P1 ) 2,5 thienyl ( P2 ) 2,5 (3,4 ethylenedioxy)thienyl ( P3 ) and 1,4 (2,3,5,6 tetrafluoro)phenyl ( P4 ) (Figure 4 1) The p hotophysical properties of the PAEs were studied in both methanol and water by absorption, fluorescence spectroscopy and fluorescence lifetime measurements Stern Volmer fluorescence quenching studies were carried out using methyl viologen (MV 2+ ) as an electron acceptor in aqueous solution. The photophysical data suggested t hat the aggregation tendency was significantly suppressed, resulting in non aggregated PAEs in aqueous solution. Some applications based on this set of PAE s were developed. Fluorescence quenching and fluorescence correlation spectroscopy were applied to s tudy the interaction between polymer P1 and different metal ions. As an effort to develop new dye sensitized solar cell P2 H ( acid form polymer) was deposited on the surface s of

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99 ZnO single crystal. AFM images indicate d that most of the polymers exist ed as a single chain with a height around 3 nm. Finally, a fluorescen t sensor based on P4 / r hodamine system was developed to detect mercury ( II) io n in water with a high selectivity over other metal ions. Results and Discussion Synthesis of PAEs with M ethylene C arboxylate S ide C hains In this study, we report new water soluble PAEs with novel methylene carboxylate side chains as shown in Figure 4 1. Each polymer contains a bis( methylene carboxylate) phenylene ethynylene unit and different arylene ethynylene units have been introduced into the polymer backbone to tune the band gap. Four different arylene units were used, Ar = 1,4 phenyl, 2,5 thienyl, 2,5 (3,4 thylenedioxy)thienyl and 1,4 (2,3,5,6 tetrafluoro)phenyl. Figure 4 1. S tructures of poly(arylene ethynylene)s with methylene carboxylate side groups.

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100 Monomer synthesis The monomer, didodecyl (2,5 diiodo 1,4 phenylene)diacetate ( C1 ) was synthesized as shown in Figure 4 2. In this route, the monomer was prepared from com mercial ly available dich loro p xylene in 6 steps with the overall yield of 14%. Figure 4 2. Synthesis of monomer C1 (i) Ac 2 O, pyridine, overnight; (ii) I 2 NaIO 4 AcOH, Ac 2 O, rt, 6 hrs; (iii) K 2 CO 3 CH 2 Cl 2 CH 3 OH, rt, ov ernight; (iv) PCC, NaIO 4 ,rt, 6 hrs; (v) C 12 H 25 OH, H 3 PO 4 150 o C, 6 hrs ; (vi) TMSA, Pd(PPh 3 ) 2 Cl 2 CuI, rt, overnight; (vii) tetrabutylammonium floride (TBAF), CHCl 3 rt, 1h. T (1,4 phenylene)diethanol ( 1 ) was prepared following the literature proced ure. 103 (1,4 phenylene) diethanol ( 1 ) with excess of acetic anhydride afforded compound 2 in quantitat ive yield. Iodination of compound 2 with iodine and sodium periodinate in acetic acid and acetic anhydride mixture gave compound 3 in 80% yield. Subsequent deprotection of the ester 3 with K 2 CO 3 afforded compound 4 in 85% yield. Diacid compound 5 was prepared by oxidation of compound 4 wit h pyridinium chlorochromate (PCC) and periodic acid. Then F isher esterification of

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101 compound 5 with dodecyl alcohol in the presence of a catalytic amount of H 3 PO 4 gave the desired monomer C1 which was easily isolated as white solid s i n 85% yield. The purit ies of intermediates and the monomer w ere proven by 1 H NMR, 13 C NMR spectroscopy and mass spectrometry. Compound 6 was synthesized according to the procedures described in literatures. 104 105 Sonogashira reaction with trimethylsilyl acetylene (TMSA) and subsequent deprotection provide d compound 8 with a good yield ~ 85%. Polymer synthesis and characteriz ation I n order to prevent electrostatic repulsion of ionic charged groups in aqueous media and facilitate gel permeation chromatography (GPC) analysis to measure accurate molecular weight the polymerization was carried out in organic solvents using a cursor route As shown in Figure 4 3, a set of anionic PAE based conjugated polyelectrolytes with variable band gap were synthesized using palladium catalyzed Sonogashira coupling chemistry, which consist of four polymers that share the same anionic side groups, methylene carboxylate ( CH 2 CO 2 Na). The tuning of the band gap was accomplished by copolymerizing monomer C1 with different trimethylsilyl (TMS) protected diacetylene arylene compounds. After the reaction was stirred at 60 o C for 24 hours, compoun d 6 (1 mg) and compound 8 (1 mg) were added as end cappers. After additional 24 hours, t he organic precursors were isolated as solids and purified by multiple precipitations in methanol. Each polymer was characterized by 1 H NMR spectroscopy. The GPC analys is was carried out in THF against polystyrene standards and the n umber average molecular weight and polydispersity data for P1 E P4 E are listed in Table 4 1.

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102 The hydrolysis of the precursor polymers was easily accomplished by treating the ester precurso rs with trifluoroacetic acid followed by NaOH in THF / H 2 O mixture. The resulting polymers were precipitated in acetone containing 5 ~ 20% MeOH and collected by centrifugation. Further purification was carried by out by multiple p recipitation in acetone foll ow ed by the dialysis using 12 kD molecular weight cut off (MWCO) dialysis membranes for 3 days. The water soluble PAEs ( P1 P4 ) were obtained as solids in 90 ~ 100% yields after ly o ph i lization. Figure 4 3. Synthesis of PA Es through precursor route. (i) Pd(PPh 3 ) 4 CuI, TBAT, compound 6 compound 8 THF, i Pr 2 NH, 60 o C, 48 h; (ii) TFA, CHCl 3 5 h ; NaOH, THF, H 2 O, 60 o C, 2 days. Table 4 1. GPC analys i s for precursor polymers ( Pn E ) Structures Names Ar M n (kD) PDI P1 E 19 1.8 P2 E 42 1.9 P3 E 19 1.8 P4 E 65 2.6

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103 Figure 4 4 shows the representative 1 H NMR spectra of monomer C1 the precursor polymer P1 E and the water soluble polymer P1 1 H NMR spectra analysis reveal ed that there wa s only one differen ce between the spectra of monomer C1 and P1 E : the resonance peak at = 7.6 0 ppm, which wa s assigned to all aromatic protons of the polymer backbone. In addition t he resonance peaks become broader and weaker in the spectra of P1 E Figure 4 4. 1 H NMR spectra of ( A ) monomer C1 ; ( B ) P1 E ; ( C ) P1 After ester hydrolysis, the 1 H NMR spectr um of P1 was measure d in CD 3 OD T he solvent peak and water residue peak show ed up strongly because of the low solubility of P1 No signals were observed in the range of 0.5 ~ 1.5 ppm and the peak at 4.3 ppm

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104 disappeared, indicating that the dodecyl groups were cleaved with an excellent yield ( > 95%). Photophysical P roperties Absorption and fluorescence spectroscopy The photophysical properti es of precursor polymers were studied in CHCl 3 by UV Vis absorption and fluorescence spectroscopy. Fig ure 4 5 shows a systematically red shift in UV Vis absorption and fluorescence spectra of dodecyl ester precursor polymers in the order of P1 E < P4 E < P2 E < P3 E in CHCl 3 Across the entire series, the absorpt ion maximum shift ed from 375 nm ( P1 E ) to 43 4 nm ( P3 E ), whereas the fluorescence maximum shift ed from 418 nm ( P1 E ) to 484 nm ( P3 E ). The origin of the red shifts is likely due to the differen ce in HOMO LUMO levels of the polymers. Specially, the shifts of P2 and P3 polymers aros e from the elect ron donor character of thi e n yl an d 3,4 ethylenedioxy 2,5 thienyl units. It is of note that incorporation of tetrafluorophenyl into the polymer backbone only induces a small red shift in absorption and fluorescence spectra compared with phenyl, indicating that tetrafluorophenyl is not a good pi electron acceptor unit. Figure 4 5. Normalized a bsorption (A) and f luorescence (B) spectra of P1 E ( ), P2 E ( ), P3 E ( ), P4 E ( ) in CHCl 3

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105 In our previous studies, it was found that the optical properti es of these water soluble PAEs we re strongly dependent on solvents. 6 48 Here the photophysical properties of PAEs with methylene carboxylate side chains were investigated by UV Vis and fluorescence spectroscopy in MeOH and H 2 O. Normalized absorption and fluoresc ence spectra of PAEs in MeOH and H 2 O are shown in Figure 4 6. Figure 4 6. Normalized absorption and emission spectra of PAEs containing methylene carboxylate side chains in MeOH (solid line) and H 2 O (d ot line). (A) P1 ; (B) P2 ; (C) P3 ; (D) P4 MeOH conta ins 10 mM NaOH; H 2 O at pH = 8.0. In general, the absorption and fluorescence spectra of PAEs i n aqueous solution we re broader and red shifted compared with the spectra in MeOH. The absorption and fluorescence spectra of P1 and P2 ha d very small red shifts (< 6 nm) and the fluorescence spectra retain ed the sharp structure, indicating that P1 and P2 d id not

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106 aggregate in H 2 O. In contrast, the absorption spectra of P3 and P 4 sho w ed bigger red shifts (9 nm and 16 nm, respectively) and the structured fluo rescence bands we re replaced by a combination of the molecular ly dissolved polymer emission and a like fluorescence band at higher wavelengths. This photophysical data impli ed that P3 and P 4 polymer s in H 2 O we re partially aggregated. Table 4 2 Photophysical data of PAEs ( P1 P4 ). Polymers MeOH A H 2 O B max abs /nm max em /nm FL C max abs /nm max em /nm FL P1 368 409 0.19 374 411 0.16 P2 388 448 0.26 394 454 0.17 P3 427 475 0.14 453 475 0.03 P4 373 421 0.26 382 415 0.16 A MeOH contains 10 mM NaOH ; B H 2 O has pH = 8.0; C Quinine sulfate in 0. 1 M H 2 SO 4 was used as a FL = 0.454. The fluorescence quantum yields of the series of PAEs ( P1 P4 ) were measured in MeOH and H 2 O and summarized in Table 4 2. In general all polymers ( P1 P4 ) we re fluorescent in MeOH with a fluorescence quantum yield ~ 0. 20. In aqueous solution polymers ( P1 P2 and P4 ) exhibit ed a comparable high quantum yield (~ 0. 16) The hi gh fluorescence quantum yields we re likely due to the re lieved aggregation by attaching the novel methylene carboxylate side chains. However, the emis sion quantum yield of the polymer ( P3 ) drop ped t o 0.0 3. It is likely due to the fact that P3 has o xygen atoms on linkers directly attached to the polymer backbone, which may promote aggregation and quench the excited states. Regarding to the oxygen effects see more discussion s in the last chapter ( C hapter 6).

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107 pH Effects on the absorption and fluorescence In order to probe the pH effects on photop h ysical properties, the absorption and fluorescence spectra of P1 and P2 in water were measured as a function of pH (Figure 4 7) The pH of the polymer solution was adjusted by addition of dilute HCl solution or NaOH solu tion according to a pH meter. Figure 4 7. Absorption (A) and emission spectra (B) of P1 in aqueous solutions as a function of pH. [ P1 ] P2 in aqueous solutions as a function of pH. [ P2 ] In general, the absorption and emission of both polymers show ed a strong de p e nd ence on pH. At pH 11 the absorption of P1 show ed a maxim um at 374 n m and the emission show ed a structure d emission with a maximum at 411 nm. As the pH decrease d the absorption spectra of P1 exhibit ed a bathochromic shift with a shoulder

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108 band at 410 nm and the fluor escence spectra intensities decreas ed The largest chang e in the fluorescence spectra wa s visible at pH = 4, in line with the p K a values of phenylacetic acid ~ 4.31. 106 At this point, 67% of the carboxylate groups on the polymer chains we re protonated. As a result of carboxylate protonation the interaction with solvent molecules and the repulsion s between side chains we re decreased lead ing to the aggregation or precipitation of polymer chains A similar change in absorption and fluorescence spectra was observed for P2 as the pH decreased from 11 to 2. It was found that the p rotonation of the carboxylate side g roups caused the aggregation or prec ipitation, leading to a decreas e in absorption spectra and fluorescence intensities The biggest change in fluor escence spectra happened when pH decreased f ro m pH = 5 to pH = 4 At the same time (pH = 4), t he absorption spectra began to show the shoulder Fluorescence lifetime measurement In order t o gain more information about photophysical properties of the precursor polymer s Pn E fluor escence lifetime s w ere measured in CHCl 3 In general, all four precursor polymers exhibited bi exponential decay s with the same short lifetime component ( ~ 0.3 ns) A s the detection wavelength increased the longer lifetime species had more contribution t o the overall amplitudes. The non exponential decay can be explained by the polymer polydispersity and different emissiv e segments with different chain length s As seen in Table 4 3, the global analysis of the emission of P1 E yielded two decay components ( 1 = 0.30 ns, 2 = 0.76 ns). As the detection wavelength increased from 420 nm to 500 nm, the contribution of the first component decreased while the second component increased contribution to the overall emission lifetime This may be explained by the fa ct that the emission at the longer wavelength had a slower

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109 decay rate because the contributions from the longer polymer chains and lon ger chain segments were larger. Similar fluorescence lifetimes were observed for P2 E and P3 E Despite the similar featu res, P4 E had a much longer lifetime component ( ~ 1.36 ns), which typical i n the donor acceptor type CPEs. 48 Table 4 3. Fluorescence lifetime ( i ns) and relative amplitudes (RA %) for precursor polymer Pn E in CHCl 3 RA (%) A P1 E (ns) B 420 nm 450 nm 470 nm 500 nm P2 E (ns) 450 nm 470 nm 500 nm 520 nm 1 = 0.30 100 97 94 88 1 = 0.34 91 90 83 74 2 = 0.76 0 3 6 12 2 = 0.73 9 10 17 26 2 1.03 1.00 0.97 1.01 2 0.99 0.99 1.01 1.03 P3 E (ns) 470 nm 500 nm 550 nm 600 nm P4 E (ns) 420 nm 450 nm 500 nm 550 nm 1 = 0.38 86 88 82 74 1 = 0.36 98 93 76 57 2 = 0.71 14 12 18 26 2 = 1.36 2 7 23 36 2 1.03 1.02 1.05 1.01 2 1. 08 1.01 1.01 1.00 A D ata were process ed by global fitting algorithm. B Typical limits of error on i are less than 3%. In order to understand the relationship between the optical properties, chemical structures and aggregation behavior, the fluorescence decay of the PAEs with methylene carboxylate side chains were measured in MeOH and H 2 O using the time correlated s ignal photon counting (TCSPC). T o keep the carboxylate side chains un protonated, MeOH solutions contained 10 mM NaOH and aqueous solutions we re adjusted to pH = 8.0. As seen in Table 4 4, all the PAEs showed relatively complicated multi exponential lifetimes, which were attributed to the existence of aggregation, inhomogeneity of polymer chains length and emissive segments with different length s

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110 In general, the fluorescence decays of PAEs in MeOH featured two exponential decays. The lifetimes of PAEs in MeOH we re similar to the precursor polymer in CHCl 3 and t he fluorescence decay of PAEs in H 2 O was more complicated For all polymers in MeOH, t he global analysis of the fluorescence decay prov id ed two exponential decays as shown in Figure 4 8 The first component had shorter lifetime and the second component had longer lifetime. As the detection wavelength increased, the contribution from the fi rst component gradually decreased, while the second one increased its contribution to the overall amplitude. This resulted in larger lifetimes at longer wavelengths. For polymers ( P 1 and P2 ) i n aqueous solution, global analysis of the fluorescence decays g ave bi exponential decays with two components similar to those in MeOH. The shorter lifetime component decreased its contribution as the detection wavelength increased. The similar fl u orescence decay in aqueous solution suggested that those polymers ( P1 an d P2 ) were as well solvated as in MeOH, indicating that those polymers were not aggregated. Although the fluorescence decay of P4 in aqueous solution exhibited three decay components, the first two components had similar lifetimes and similar behaviors to those in MeOH. T he third component ( 3 = 8.72 ns) had a n essenti al role at wavelength 550 nm (~ 25% contribution), whereas the broad excimer band showed up. P4 exhibited a s light aggregation behavior but the fluorescence quantum yield was much higher (~16%) suggesting that only a small amoun t of P4 was aggregated. In the previous studies, it was found that some PPEs aggregated in H 2 O and the aggregates acted like a n energy trap and quencher resulting in a shorter lifetime in water than in MeOH. 48 A similar result was observed for the fluorescence decay of P3

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111 First, the global analysis of the fluorescence decay of P3 in aqueous solution yielded three exponential decays with a much longer lifetime component ( 3 = 2.20 ns). This longer time component had ~10% contribution at 600 nm, where the excimer like band was observed, indicating that the aggregates had a longer lifetime. In addition, the shortest lifetime had a dominant role in the overall amplitude, re sulting in a much shorter mean lifetime in aqueous solution. Figure 4 8. Fluorescence lifetime at different detection wavelengths: (A) P1 in MeOH; (B) P1 in H 2 O; (C) P2 in MeOH; (D) P2 in H 2 O; (E) P3 in MeOH; (F) P3 in H 2 O; (G) P4 in MeOH; (H) P4 in H 2 O. MeOH contains 10 mM NaOH. H 2 O has pH = 8.0.

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112 Table 4 4. Fluorescence lifetime ( i ns) and relative amplitudes (RA, %) for Pn in basic MeOH and H 2 O ( pH = 8.0). RA (%) A MeOH H 2 O P1 (ns) B 420 nm 450 nm 470 nm 500 nm (ns) 420 nm 450 nm 470 nm 500 nm 1 = 0.35 68 74 73 67 1 = 0.40 42 56 60 53 2 = 0.63 32 26 27 33 2 = 0.68 5 8 42 40 47 2 1.05 1.00 1.03 1.07 2 1.03 1.01 1.00 1.02 P2 (ns) 450 nm 470 nm 500 nm 520 nm (ns) 450 nm 470 nm 500 nm 520 nm 1 = 0.42 77 71 71 66 1 = 0.40 67 64 64 61 2 = 0.72 23 29 29 34 2 = 0.66 23 26 26 29 2 1.01 0.99 1.00 1.01 2 1.0 8 1.00 1.00 1.10 P3 (ns) 470 nm 500 nm 550 nm 600 nm (ns) 470 nm 500 nm 550 nm 600 nm 1 = 0.22 42 39 37 35 1 = 0.22 73 70 67 54 2 = 0.53 58 61 63 65 2 = 0.52 27 30 31 36 3 = 2.20 0 0 2 10 2 1.02 1.04 1.05 1.01 2 1.04 0.98 0.98 1.03 P4 (ns) 420 nm 450 nm 500 nm 550 nm (ns) 420 nm 450 nm 500 nm 550 nm 1 = 0.49 94 92 89 82 1 = 0.52 88 87 72 48 2 = 1.09 6 8 10 16 2 = 1.49 12 12 19 27 3 = 8.72 0 1 9 25 2 1.00 1.00 1.00 0.96 2 1.04 1.04 1.01 1.08 A Data were proc ess ed by global fitting algorithm. B Typical limits of error on i are less than 3%.

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113 In conclusion the introduction of the methylene carboxylate side groups successfully suppressed the aggregation of PAEs in aqueous solution, since polymers ( P1 P2 and P 4 ) showed similar decay behavior in aqueous solution to that in methanol solution. The oxygen on the linker to the polymer backbone of P3 may induce some aggregation and thus have a much shorter lifetime in aqueous solution. Fluorescence correlation spectr oscopy The photophysical data suggested that P1 and P2 did not aggregate while P3 and P4 were partial ly aggregated. In order to gain more information regarding the solution properties of PAEs fluorescence correlation spectroscopy was applied to detect the diffusion time of each polymer and calculate the hydrodynamic radius. The experiments were carried out in aqueous solutions (pH = 8.0) with of each polymer using fluorescein (30 nM) as standard. The hydrodynamic radius of the PAEs in aqueous solution wa s estimated from the FCS diffusion time according to the equations listed below. The diffusion time and hydrodynamic radius for the polymers a re summarized in Figure 4 8 and Table 4 5. ( 4 1 ) ( 4 2 ) ( 4 3 ) w here D 0 is the diffusion coefficient of the standard, 0 is the diffusi on time of the standard, W r is the focus volume of the fluorescence microscope, d is the s a m ple diffusion time, D is the diffusion coefficient of samples, T is the temperature, K is the Boltzmann constant, is the viscosity of water and R H is the hydrod ynamic radius.

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114 Table 4 5. Diffusion time and hydrodynamic radius of PAEs in aqueous solution (pH = 8.0) Polymer Concentration Pn E M n (kD) ( x 10 5 s) R H (nm) P1 2 19 6.17 1.49 P2 2 48 6.72 1.63 P3 2 19 4.77 1.15 P4 2 65 7.55 1.83 Figure 4 9 Normalized correlation curves for PAEs in aqueous solutions. Balck: P1 ; Red: P2 ; Blue: P3 ; Pink: P4 pH = 8.0. [ PAE single specific fitting curves. In general the diffusion times of all PAEs were in the range of 10 5 s and the diffusion curve in Figure 4 9 w as quite smooth and similar to each other. Polymer P4 ga v e the larg est diffusion time (7.55 x 10 5 s) and radius (1.83 nm) because P4 had the

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115 highest molecular weight. P3 showed the smallest particle size (radius ~ 1.15 nm). The diffusion time for P1 and P2 were 6.17 x 10 5 s and 6.72 x 10 5 s, respectively Typically, larger molecular weight polymers give lon ger diffusion time s However, the diffusion time and the radius of P3 w ere smaller than P1 despite similar molec ular weight. This can be explained by the polymer structure differences : t he polymer backbone of P1 wa s more rigid and linear while the polymer backbone of P3 ha d some flexibility f o r rotat ion because of the more flexible 2,5 (3,4 e thylenedioxy)thienyl uni t. Overall, the relatively small diffusion time and hydrodynamic radius suggested that all PAEs did not form large aggregat es in aqueous solution However, there was also possibility that some of them ( P3 and P4 ) formed some loose aggregates, which can n ot be detected by FCS techniques. Steady state fluorescence quenching with methyl viologen (MV 2+ ) In the previous studies, it was found that unaggregated PPEs usually had smaller K sv values (~ 10 5 M 1 ) compared with the aggregated PPEs in aqueous solution inc luding PPE CO 2 and PPE SO 3 The aggregated polymers had a much larger Stern Volmer constant (10 6 M 1 ~ 10 7 M 1 ), when quenched by a quencher like methyl viologen (MV 2+ ) 48 54 91 101 Steady state fluorescence quenching of PAEs were conducted in aqueous solution with methyl viologen. MV 2+ is known as an efficient quencher via the photo induce d electron transfer mechanism. The detailed fluorescence spectra of PAEs (2 n the addition of MV 2+ were shown in Figure 4 10 The fluorescence of all PAEs was efficiently quenched by MV 2+ in aqueous solution Figure 4 1 1 shows t he Stern Volmer plots of PAEs upon addition of MV 2+ In each case the S tern V olmer plot showed a linear dependence on the quencher concentrations Consequently we

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116 characterized the plots using K sv values calculated at low quencher concentrations according to Stern Volmer equation ( Equation 4 4). (4 4) w here I 0 is the emission intensity in the absence of the quencher, and I is the emission intensity in the presence of the quencher, [Q] is the quencher concentration, and the K sv is the effective association constant for the complex formed between the polymer and the quencher. Figure 4 10 Fluorescence spectra of PAEs upon the addition of MV 2+ quencher. ( A ) P1 ; ( B ) P2 ; ( C ) P3 ; ( D ) P4 The K sv values for P1 P2 P3 and P4 we re 3.9 x 10 5 M 1 2.5 x 10 5 M 1 3.4 x 10 5 M 1 and 1.9 x 10 5 M 1 respectively. The K sv values for all PAEs were much smaller at

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117 the range of 10 5 M 1 suggesting that no strong aggregation in aqueous solution was observed However, in the quenching experiments of P3 and P4 the excimer band s at long er wavelengths were efficiently quenched within the first titration of MV 2+ indicating that only a small amount of polymers were aggregated and th ey were quenche d by MV 2+ with a higher efficiency. Figure 4 1 1 Stern Volmer plots of PAEs upon the addition of MV 2+ quencher. P1 ( ) ; P2 ( ) ; P3 ( ) ; P4 ( ) Application of P1 to M etal I on S ensing in A queous S olution The interaction of P1 with different metal ions was studied in aqueous solution. This experiment was carried out in water (pH = 8.0) with a serie s of 6 different metal ions : Na + Zn 2+ Hg 2+ Cu 2+ Fe 2+ and Fe 3+ From the study of pH dependence, pure polymer P1 was not aggregated at pH = 8 The f luorescence quenching experiment with

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118 Na + was used as a control to reflect the influences of photo bleach ing and ion strength. Figure 4 1 2 show s the fluorescence spectra of P1 upon addition of various concentrations of different metal i ons. As shown in Figure 4 1 2 the addition of 25 Na + and Zn 2+ to P1 solution failed to induce any fluorescence quenching, while 25 of Cu 2+ Fe 2+ and Hg 2+ can induce modest quenching. In contrast, the strongest quenching of polymer fluorescence w as induced by 7.5 of Fe 3+ with a loss of the vibronic structure in fluorescence spectra. Figure 4 1 2 Fluorescence spectra of P1 in H 2 O (pH = 8.0) u pon addition of different metal ions. ( A ) Na + ; ( B ) Cu 2+ ; ( C ) Fe 2+ ; ( D ) Fe 3+ ; ( E ) Hg 2+ ; ( F ) Zn 2+ [ P1 ] = 2 ; Fe 3+ concentration ranged from 0 7.5 ; other metal ions concentrations ranged from 0 25

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119 Figure 4 1 3 (A) Stern Volmer plots (A) of P1 with different metal ions in aqueous s olution (pH = 8.0) (B) C omparison of K sv values for different metal ions. The Stern Volmer (SV) plots for all the 6 metal io ns a re shown in Figure 4 1 3 A In the experiment concentration range, a linear curve was observed for all metals except Cu 2+ and Fe 3 + which showed an upward curvature above 1 concentration.

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120 Stern Volmer quenching constants ( K sv ) for all the me t al ions were extrapolated by fitting the linear regions of their SV plots and were compared in the bar graph shown in Figure 4 1 3 B. For all the metal ions, K sv values were observed in the range of 10 4 M 1 ~ 10 5 M 1 and t he largest K sv value was obtained for Fe 3+ ( 2.5 x 10 5 M 1 ) Figure 4 1 4 Diffusion time of P1 in the presence of different metal ions in H 2 O (pH = 8.0) obtained by the fluorescenc e correlation spectroscopy using the fl uorescein standard. [ P1 ] = 2 In order to study the aggregation behavior of the polymer in the presence of different metal ions, fluorescence correlation spectroscopy was applied to detect the diffusion time and the data w ere shown in Figure 4 1 4 For metal ions such as Zn 2+ Na + and Hg 2+ the diffusion times of P1 / metal ions were ~ 5.5 x 10 5 s, close to the diffusion

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121 time of pure P1 in aqueous solution. The a ddition of 25 Cu 2+ Fe 2+ and Fe 3+ were able to induce significant aggregation, and the diffusion time s were increased mor e than 3 fold. In particular, Fe 3+ can induce the aggregation very efficiently, due to the fact that Fe 3+ i s trivalent and ha s more charge density T he formation of polymer aggrega t es enable s the interchain exciton transfer resulting in a much more effici ent quenching. In conclusion the fluorescence of polymer P1 was most sensitive to Fe 3+ which can be explained by the fact that Fe 3+ can significantly induce the polymer aggregation and also quench the exited state of the polymer. The total quenching effe ct was a combination of the metal ion induced aggregation and electron transfer mechanism. T his system can be explored for fluorescence sens ing of Fe 3+ with high sensitivity and selectivity. Application of P2 in Dy e S ensitized Solar Cells In a previous st udy, our group collaborated with Dr. Parkinson s group on investigation of interfacial morphology and photoelectrochemistry of CPEs on metal oxide semiconductor. It was found that the magnitude of sensitized photocurrent was related to the surface coverage and the degree of aggregation of CPEs. 16 Therefore, we decided to continue our collaboration with Dr. Alexander Nepomnyashchii in Dr. Parkinson s group using p olymer P2 since P2 was not aggregated in solution and may stay unaggregated in film. Conjugated polyelectrolytes (CPEs) have been extensively studied as sensitizers in dye sensitized solar cells (DSSC s ) owing to their high absorption, tunable band gap g ood charge transport properties and low cost 7 49 107 108 The b inding to oxide semiconductor surface s can be also controlled for CPEs through the degree of

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122 carboxylate substitution on the polymer backbone The morphology of the CPEs at the interface where charge separation occurs plays a pivotal role in the solar electrical power conversion and wavelength response of the photocurrents 109 110 For instance, inefficient exciton transport ma y occur for aggregated polymer whose photo generated excitons must hop several chains to reach the semiconductor interface. 111 112 Aggregation may also decrease the efficiency of light harvesting, charge injection and charge collection depending on the structure and physical properties of the aggregates 113 114 In contrast, non aggregated CPE chains are expected to inj ect electrons more efficiently. As described in the previous section polymer P2 did not aggregate in solution Therefore, we hypothesized that the unaggregated polymer in solution may be a promising candidate to stay un aggregated as film on the surfaces of semiconductors. The acid form of the polymer ( P 2 H ) was prepared by adding HCl solution (1 N, 3 mL) to 10 mg of P2 in H 2 O solution (3 mL) and collected by centrifugation (Figure 4 15 ) After the deposition of the acid form P2 H on the surface s of ZnO s i ngle crystals, a tomic f orce m icroscopy ( AFM ) and photoelectrochemical measurements were carried out to investigate the aggregation behavior. Figure 4 15 Synthesis of P2 H

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123 The photophysical properties of P2 and P2 H were initially investigated and compared in Figure 4 1 6 T he absor ption and fluorescence spectra in different solvents showed no substantial so lvent effect In particular, the high fluorescen ce quantum yield s ( 16% in water 26% in MeOH and 3 0% in DMF respecti vely ) indicat ed polymer P2 and P2 H d id not aggregate in solution and may ha ve the potential to form films without aggregation. Figure 4 1 6 Normalized a bsorption (A) and fluorescence spectra (B) o f P2 in MeOH ( ), P2 in H 2 O ( ) and P2 H in DMF ( ). MeO H contains 10 mM NaOH. H 2 O has pH = 8. It is known that o btaining topographic information of the de posited PP Es polymer chains within a mesporous oxide film is challenging. Therefore, in this work a tomically flat n type z inc oxide (0001) single crystal sur faces were used as a substrate to allow detailed structure determination with a tomic f orce m icroscopy (AFM) and to correlate photocurren t response with the sensitizer concentration in the s olution In addition, z inc oxide is a semiconductor material with a wide band gap of 3.2 3.4 e V 115 which is suitable for studying sensitization by organic dyes. 116 According to the absorption spectrum, P 2 H h a d an a bsorption maximum at around 390 nm or 3.0 eV which wa s

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124 below the ZnO band gap. The driving force of 0.2 0.4 V was big enough to enable the photoex cited electron injec tion from P2 H into ZnO. Figure 4 17 A shows c lear atomically flat te rraces of around 250 nm across obtained for polished and annealed ZnO crystals. A d ipping method submerging the ZnO in to various concentrations of P 2 H in DMF solutions for 5 min s was u sed for the surface coverage studies. This process was found to be the most reliable and reproducible for the formation of uniform coverage of the carboxylate polymer on the single crystal surfaces Figure 4 1 7 Non contact tapping mode AFM images of P 2 H deposited on Zn O (0001) surface from DMF solutions of different concentrations: ( A ) 0, (B) 6 and (D) 60 g/m L (C) C ross section analysis for the red line in (B). DMF solution was the solvent used for deposition with a dipping time of 5 minutes. Data o btained by Dr. Nepomnyashchii.

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125 Polymer deposition at a low concentration of P 2 H led to sub monolayer coverage of the pa rticles in AFM images (Figure 4 1 7 B ). The cross section analysis for the red line in Figure 4 1 7 B reveal ed that most of the particles were around 2 to 3 nm in height, which seem ed to be a single polymer chain (Figure 4 17 C) It is of note that t he particle heights a re use d to estimate their size s since the AFM microscope has the highest resolution on height. Exposure to solution with a h igher concentration result ed in an increase of surface coverage without a sign of aggregation (Figure 4 1 7 D) To verify our premise that the bl o bs in the AFM images corresponded to individual polymer chains, all the particles in Figure 4 1 7 B were measur ed and a histogram showing the distribution of heights was compared with the distribution of calcu lated radius in Figure 4 1 8 For each polymer chain, the radius of the solid particle was calculated from the molecular weight obtained from GPC a nalysis usin g the equations listed below and the hemispherical model. Therefore, the GPC curve can be transferred into a distribution curve of calculated radius. The distribution of calcul ated radius of polymer chains was in close agreement with the distribution of he ights. T he only difference wa s that the calcula ted radius wa s ~ 1 nm bigger which can be attributed to the fact that particles in solid state we re flatter than hemispherical model. The good correlation between the real particles heights and the calculated radius supported our premise and suggested that this novel polymer did not aggregate on surface.

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126 w here, M ester is the molecular weight of the ester polymer M 1 is the molecular weight of each repeat unit of acid polymer M 2 is the molecular weight of each repeat unit of ester polymer M acid is the molecular weight of the acid polymer N A is Avogadro number, m is the mass of each acid polymer chain the density is assumed to be 1.1 g/cm 3 ac cording to density of P3HT solid 117 T he hemispherical model is chosen a nd r is the radius of each polymer particle. Figur e 4 1 8 ( A ) Distribution of the particles with different heights obtained from Figure 4 1 7 B D ata obtained by Dr. Nepomnyashchii ( B ) Distribution of the polymer chains over calculated radius.

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127 Figure 4 19 A shows the i ncident photon to current efficiency (I PCE) spectra for adsorbed P 2 H on the surfaces of ZnO single crystals as a function of polymer concentration, using iodi de/iodine couple as mediator Eff icient electron injection from polymer to ZnO crystals wa s observed The sensitized photocurrent from P 2 H started at 480 nm and reache d a maximum value at ~ 41 0 nm close to adsorption maxima in film and DMF solution The increases of the photocurrent with the coverage showed an adsorption isotherm like behavior where a s aturation point wa s reached at ~ 30 g/m L in Figure 4 19 B Figure 4 1 9 (A ) IPCE spectra for a ZnO electrode dipped into various concentration of P 2 H in DMF solution (B) IPCE values as a function of the dipping solution conce ntration for curves shown in A. Data obtained by Dr. Nepomnya shchii. The isotherm behavior was similar to the isotherms obtained for monomeric ruthenium dyes monomeric and h aggregated cyanine dyes on the TiO 2 single crystal surface s ( anatase and rutile ) 118 multilayers in the AFM images suggest ed that the individual chains were covalently bond ed to the oxide surface and not aggregat ed into multilayers Otherwise decreases in the IPCE values can be observ ed for other polymeric sensitizers with increasing coverage due to the formation of thick polymer layers where photoexcited carriers or

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128 excitons created near the polymer surface cannot reach the interface to be separated by e lectron injection into the semiconductor conduction band. 113 114 The maximum IPCE for P 2 H on th e surfaces of ZnO wa s ~ 0.04%. There are several explanations for the low IPCE values. One is that the dye coverage is sub monolayer coverage The other is that ZnO crystals absorb light starting from ~ 4 0 0 nm overlap ping with the absorption maximum of the polymer. In addition, the relative low band gap between the polymer and the ZnO leads to lower electron injection efficiency. In conclusion the synthetic strategy to produce a sensitizing polymer that was unlikely to aggregate was successful The absence of aggregations in solution and at surfaces was verified by photophysical measurements, AFM imaging, and photocurrent spectroscopy. Non aggregat ing polymer ic sensitizers may be useful in designing more efficient solar cell s as it allows for more control of surface polyelectrolyte binding Application of P 4 in M e r cury (II) I on S ensing Mercury ion (Hg 2+ ) is a toxic heavy metal ion, available in many natural sources such as coal and gold mining, fossil fuel, solid waste. 119 Owing to its notorious biological membrane disruption, accumulation and long residence in nervous systems, mercury pollution cause s se rious nervous diseases such as A crodynia, M i n a mata and Hunter R ussel syndrome. 120 121 Therefore, great efforts have been devoted to the design and develop ment of an ea sy, rapid and effective sensor to detect and quantitatively measure the mercury ion sensors in aqueous solution. Among all the sensors, the fluorescence sensors based on conjugated polyelectrolytes ( CPEs) seem to be the ideal candidate for both sensing and bio imaging of the metal io ns in various samples, due to their s high sensitivity, fast analysis, non destructive sample preparation and the highly effective quenching effects known as amplified quenching effects 52 122 125 Recently, Ma and

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129 coworkers reported th at rhodamine B thiolactone ( S Rho ) can form a c omplex with Hg 2+ 123 The r hodamine Hg 2+ complex ( S Rho Hg 2+ ) exhibited an absorption maxima at ~ 560 nm, which was overlap ping w ith the fluorescence spectra of the polymer P4 (Figure 4 2 1 ) Also the positive charged S Rho Hg 2+ ion complex will be attracted to the negative charged P4 by the e lectrostatic interaction. Here we report a novel and highly sensitive Hg 2+ sensor bas ed on P4 and rhodamine B thiolactone, by taking advantage of the F r ster energy transfer ( FRET ) between the polymer and rhodamine B thiolactone Hg 2+ complex. Figure 4 20 Structures of P4 and S Rho Figure 4 2 1 Normalized fl uorescence spectrum of P4 (solid line) and absorption spectrum of S Rho Hg 2+ (dashed line)

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130 As discussed before, the conjugated polymer P4 was synthesized through a precursor route using a Sonogashira reaction, in which both monomers were soluble in orga nic solvent Subsequent deprotection of the ester groups afforded the water soluble highly emissive polymer P4 Figure 4 2 2 (A) Fluorescence spectr a of P4 ( 1 ) and P4 (1 )/ S Rho (1 ) upon the addition of Hg 2+ (300 nM) in H 2 O /DMSO (99/1, v/v ) (B ) F luorescence spectra of P4 (1 )/ S Rho (1 ) upon the addition of v arious concentration of Hg 2+ in H 2 O /DMSO (99/1, v/v ). Excitation at 373 nm Figure 4 2 2 A shows the fluorescence spectra of P4 ( 1 ) P4 (1 )/ S Rho (1 ) mixture and P4 ( 1 )/Hg 2+ ( 300 nM) mixture in H 2 O /DMSO (99/1, v/v ). The addition of S Rho ( 1 ) to the P4 ( 1 ) did not affect the fluorescence spectra indicating that t he interaction between S Rho and P4 was negligible w ithout Hg 2+ ion I n addition the fluorescence spectrum of P4 remained the same upon the addition of Hg 2+ (300 nM) In contrast the addition of Hg 2+ ion into the P4 (1 )/ S Rho (1 ) mixture caused significant quenching to the fluorescence spectra indicating the combination of S Rho and Hg 2+ played the import ant role. M ore than 95% of the emission intensity at 525 nm was quenched upon the addition of 300 nM Hg 2+ Formation of the positive charged S Rho /Hg 2+ ion complex through a ring opening process 123 resulted in efficient

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131 fluorescence quenching of P4 via the F r ster energy transfer mechanism. The Stern Volmer plots of P4 (1 )/ S Rho (1 ) upon the addition of different metal ions in H 2 O /DMSO (99/1, v/v pH 8) w ere shown in Figure 4 2 3 A. The S tern Volmer plot for Hg 2+ curved upward at higher concentration and exhibited a linear profile at the low concentration (<50 nM). The K sv value was derived from the linear range to be ~ 2.3 x 10 7 M 1 which wa s the highest value among all reported Hg 2+ sensor s based on CPE. In addition, the selectivity of this sensor is remarkably high for Hg 2+ The fluorescence intensity change of P4 (1 )/ S Rho (1 ) upon addition of different metal ions (300 nM) was shown in Figure 4 2 3 B. Metal ions such as Ca 2+ Cu 2+ Fe 2+ Fe 3+ and Zn 2+ failed to induce significant change, while Hg 2+ quenched most of the fluorescence intensity. Figure 4 2 3 (A) St ern Volmer plots of P4 (1 )/ S Rho (1 ) upon the addition of different metal ions in H 2 O /DMSO (99/1, v/v) ; (B) fluorescence intensity changes of P4 (1 )/ S Rho (1 ) upon the addition of different metal ions (300 nM) in H 2 O /DMSO (99/1, v/v) pH = 8. Excitation at 373 nm, fluorescence intensity was monitored at 525 nm. The proposed sensing mechanism was displayed in Figure 4 2 4 The rhodamine B thiolactone undergoes a ring opening process and forms a complex with Hg 2+ The

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132 positive charges on the comp lex played an essential role in binding to the carboxylate groups of P4 The overlapped spectra between donor fluorescence and acceptor absorption provided the fundamental pathway to the F r ster ener gy transfer effects. Overall, a novel fluorescence turn off sensor for Hg 2+ ion has been developed based on P4 and S Rho The fluorescence of P4 / S Rho was selectively quenched by Hg 2+ with an outstanding efficiency. The quenching mechanism was proposed and rationaliz ed as F r ster energy transfer mechanism. In a ddition, the fluorescence sensor provided an easy and fast way to detect Hg 2+ in aqueous solution. Figure 4 2 4 Proposed sensing mechanism for Hg 2 + Summary In this chapter a new family of anionic poly(arylene ethynylene)s (PAEs) featuring methylene ca rboxylate side groups have been prepared. The polymerization was carried out in organic solvents by Sonogashira coupling reaction according to the precursor route. Subsequent base promoted ester hydrolysis and purification provided the water soluble conj ugated polyelectrolytes (CPEs). The repeat unit of the PAEs

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133 backbone consist ed of a phenylene ethynylene unit alternating with a second arylene ethylene moiet y and four different arylenes were used, Ar = 1,4 phenyl, 2,5 thienyl, 2,5 (3,4 ethylenedioxy)thie nyl and 1,4 (2,3,5,6 tetrafluoro)phenyl. The different arylene units induced variation in the HOMO LUMO band gap across the series of polymers, resulting in a n absorption maxim um ranging from 360 nm to 450 nm and a fluorescence maxim um ranging from 409 nm to 475 nm. The photophysical properties such as absorption, fluorescence and fluorescence lifetime were investigated in MeOH and H 2 O. All polymers except P3 showed comparable optical prope rties in aqueous solution to th ose in MeOH. Through the careful stru cture propert y relationship analysis, the weak fluorescence and l ow fluorescence quantum yield w ere attributed to the oxygen attached to the polymer backbone, which induced aggre gation and quenching. The synthetic route for the alternative polymer without oxygen linkage was much more challenging, but as a result, the photophysical properties were improved drastically. Most of the polymers had a fluorescence quantum yield ~ 16% in aqueous solutions. The s teady state fluorescence quenching of the PAEs were co nducted by MV 2+ in aqueous solution. The fluorescence of the PAEs was quenched efficiently with K sv value s ~ 10 5 M 1 indicating that most of the polymers chains were existed as single chains. And the small diffusion times measured by fluorescence correlat ion spectroscopy supported the same conclusion that polymers did not aggregate in water Beyond the basic photophysical studies, several applications based on the PAEs were successfully developed. First, the interaction of P1 with the metal ions were inve stigated and the results suggested that P1 was remarkable sensitive to Fe 3+ and a p romising Fe 3+ sensor could be built. The AFM images of the acid polymer P2 H on the

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134 surface of ZnO single crystal were obtained. By comparison of the particles heights in th e AFM images and the calculated radius from GPC data, it was found that the b lo bs in the AFM images represent ed the single chains which formed a globular structure. Further photoelectrochemical experiments of the P2 H were carried out for the application of Dye Sensitized Solar Cell (DSSC). Finally, a novel highly sensitive fluorescenc e sensor for mercury (II) ion w as designed and develope d bas ed on P4 / S Rho complex with a high selectivity This sensor takes advantages of the F r ster energy transfer mecha nism and the overlap between the fluorescence of P4 and absorption of S Rho Hg 2+ complex ions. The fluorescence of the P4 was quenched by the S Rho Hg 2+ metal ions with a K sv value ~ 2.5 x 10 7 M 1 Experimental Materials Pd(PPh 3 ) 4 and Pd(PPh 3 ) 2 Cl 2 were pu rchased from Strem Chemical Company and used as received. 1,4 B is(chloromethyl)benzene, sodium perchlorate, 1 dodecanol, diisopropylamine, tetrabutylammonium hexafluorophosphate (TBAPF 6 ) and copper iodide were purchased from Sigma Aldrich Chemical Company. Acetic acid, acetic anhydride, sulfuric acid, potassium carbonate ferric chloride, mercury (II) chloride, ferrous chloride, zinc chloride and sodium chloride were purchased from Fisher Scientific Company and used as received Tetrabutylammonium difluorot riphenylsilicate (TBAT) was purchase d from TCI America Company. THF and DMF were purified by solvent dispensing system. All other chemicals and solvents were purchased from Sigma Aldrich, Fisher Scientific and used as received. S tock solutions (1.0 mM) of all P A Es were prepared in deionized H 2 O (pH = 9) and have been stored at 4 o C. For all experiments with the P A Es in aqueous solution, the pH was adjusted to 8.0 by addition

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1 35 of a dilute solution of sodium hydroxide. An d for all experiments with the P A Es in MeOH, the m ethanol solution contains 10 mM NaOH to keep the solution basic. Instrumentation NMR spectra were record ed using a Gemini 300 NMR operating at 300 MHz for 1 H NMR and at 75 MHz for 13 C NMR for small organic compounds 1 H NMR spectra of the poly mers were measured in Inova2 500 NMR operating at 500 MHz for 1 H NMR Gel permeation chromatogra phy (GPC) analysis w as carried out on a system comprised of a Shimadzu LC 6D pump, Agilent mixed D column and a Shimadzu SPD 20A photodioide array (PDA) detecto r, with THF as eluent at 1 mL/min flow rate. The system was calibrated against linear polystyrene standards in THF. UV absorption spectra were measured on a Shimadzu UV 1800 spectrophotometer. Luminescence spectra were measured on a PTI (Photon Technology International) fluorescence spectrometer. Fluorescence lifetimes were determined by time correlated single photon counting on a FluoTime 100 spectrometer ( Pico Quant ) equipped with 370 nm diode laser as excitation source. Detection filters were used with a width ~ 10 nm. A 1 cm square quartz cuvette was used for solution spectra, and emission was collected at 90 o relative to excitation beam. Fluorescence quantum yields are reported relative to known standards quinine sulfate in 0. 1 M H 2 SO 4 solution The op tical density of solutions at the excitation wavelength was < 0.1, and corrections were applied for differences in the refractive index of standard and sample solutions. FCS measurements were taken on a homemade setup using a 405 nm diode laser (Coherent, CUBE) as the excitation light. Fluorescein (30 nM in 10 mM phosphate buffer, pH = 8) was used as the calibration for the system. The concentrations of ol igomer and polymer samples were 2 uM for all experiments.

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136 General M ethods for S urface and P hotoelectro chemical C haracterization Sample preparation : ZnO (0001) single crystals were obtained from MTI Corporation (Richmond, CA). Atomically flat surfaces without substantial pits present were achieved by polishing with 30 and 50 nm diameter silica (Buehler, Lak e Bluff, IL) and further annealing at 1000 o C for 12 hours. The initial solution wi th CPEs was filtered using 200 M syringe filter ( Nalgene, Rochester, NY) prior the photoelectrochemical and AFM measurements ZnO (0001) single crystal electrodes were dipp ed inside the solutions and covered with CPEs Then the crystals were attached with plastic screws to specially built cell to assure presence of electrical contact. Atomic f orce m icroscopy (AFM) i nvestigations : AFM measurement has been carried out using no n contact AC mode of AFM and Asylum Research (Santa Barbara, CA) microscope. Olympus silicon rectangular probes with a 42 N/m force constant, tip radius of 9+/ 2 nm and resonant frequency of approximately 300 kHz were used to probe the crystal surface. Cro ss section analysis was applied to determine the size (height) of the particles. Exactly the same crystals as used for the photoelectrochemical measurements were applied to achieve direct correlation between photoelectrochemical and surface properties. Dif ferent areas of ZnO (0001) single crystals were analyzed to obtain reproducible results. Ph otoelectrochemical measurements : Photoelectrochemical measurements were carried out in aqueous solutions containing 0.1 M KNO 3 and 20 mM KI. Three electrode setup wi th Zn O (0001) as a working electrode, platinum wire as a counter electrode and Ag/AgCl as a reference electrode was used in all experiments. Princeton Instruments (Princeton, NJ) 174A potentiostat was applied in IPCE measurements. IPCE spectra

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137 were collect ed using a Stanford Research Systems (Sunnyvale, CA) model SR830 DSP lock in preamplifier. The collected signal from preamplifier was transmitted to a SRS model SR830 DSP lock in amplifier. 100 W Oriel tungsten lamp (Newport Corp., Santa Clara, CA) (cut of f filter 350 nm) was used as a source of excitation. The light was passed through a computer controlled monochromator (2 nm step interval) and further chopped at 14 Hz to provide a modulated photocurrent signal (model SR540 chopper, Stanford Research Instr uments). The collected photocurrent signal was then corrected for photon flux by collecting lamp power spectrum with thermopile detector (Pomona Electronics, Way Everett, WA). Incident power through monochromator for certain wavelength was collected using Thorlabs C Series photodiode (Newton, NJ). I V curves were collected for the same crystals using 120 mW purple laser and IVIUM potentiostats (Eindhoven, The Netherlands). Synthe tic Procedure Compound 1 103 6 104 105 1,4 bis(trimethylsilyl)ethynyl)tetrafluor obenzene, 126 and 5,7 bis((trimethylsilyl)ethynyl) 2,3 dihyd rothieno[3,4 b][1,4]dioxine 47 w ere prepared according to the literature procedure s The rhodamine B thiolactone was synthesized from commercial available rhodamin e B according to the literature. 123 1,4 bis(2 acetoxy ethyl)benzene (2). To a mixture of acetic anhydride (50 mL), pyridine (200 mL) and 4 dimethylamino pyridine ( 20 mg ), compound 1 (10 g, 60 mmol) was added and the mixture was allowed to stir at room temperature for overnight. The solvents and excess of acetic anhydride were removed under vacuum. The residue was purified by silica c hromatography to give 2 as a white solid ( yield: 15 g, 100%). 1 H NMR (300 MHz, CDCl 3 ): 7.15 (s, 4H), 4.26 (t, 4H), 2.91 (t, 4H), 2.03 (s, 6H). 13 C NMR (75

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138 MHz, CDCl 3 ): 171.19, 136.28, 129.24, 65.12, 34.89, 21.18. HR MS (ESI) m/z : [M +Na] + calcd. for C 1 4 H 18 O 4 Na 273.1097; found, 273.1283. 2,5 Diiodo 1,4 bis(2 acetoxy ethyl)benzene (3). Sodi um periodate (7.3 g, 34 mmol, 40% excess) and iodine (26.2 g, 104 mmol, 40% excess) were stirred into a mixture of glacial acetic acid (129 mL) and acetic anhydride (64.5 mL) at 0 o C. Concentrated sulfuric acid (43 mL, 860 mmol) was then added slowly to t he stirring suspension. Compound 2 (21.5 g, 86 mmol) was added to this solution and stirred continuously for 6 h at room temperature. The reaction mixture was then poured into an ice water mixture containing previously dissolved Na 2 SO 3 The precipitate was collected and recrystallized in ethanol to give compound 3 as a white solid ( yield: 34.5 g, 80%). 1 H NMR (300 MHz, CDCl 3 ): 7.67 (s, 2H), 4.24 (t, 4H), 3.00 (t, 4H), 2.06 (s, 6H). 13 C NMR (75 MHz, CDCl 3 ) : 171.13, 141.26, 140.57, 100.43, 63.34, 38.89, 2 1.16. HR MS (ESI) m/z : [M ] + calcd. for C 14 H 18 I 2 O 4 503.9289; found, 502.9225. (2,5 Diiodo 1,4 phenylene)dienthanol ( 4 ). To the solution of 3 (8.4g, 16.7 mmol) in dichloromethane (50 mL) and methanol (200 mL) was added potassium carbonate (25 g, 181 mmo l) The mixture was stirred at room temperature overnight. The solvent was removed under vacuum. Water (300 mL) was add ed and the suspension was vigorous ly stirred at room temperature for 2 h. The solid was collected by vacuum filtration to give 4 as a whi te solid ( yield: 5.9 g, 85%). 1 H NMR (300 MHz, Acetone d 6 ): 7.71 (s, 2H), 3.86 (m, 4H), 2.94 (t, 4H), 1.38 (t, 2H). 13 C NMR (75 MHz, Acetone d 6 ): 142.26, 140.50, 100.25, 61.15, 42.84. HR MS ESI m/z : [M+Na] + calcd. for C 10 H 12 I 2 O 2 Na, 440.8819, found 440.8 827.

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139 (2,5 Diiodo 1,4 phenylene)diacetic acid ( 5). To acetonitrile (80 mL) p eriodic acid (4.8 g, 21 mmol) was added and the mixture was stirred at room temperature for 15 min. The mixture was cooled down to 0 o C, and co mpound 4 (2.0 g, 4.8 mmol) was then added, followed by the addition of freshly prepared pyridinium chlorochromate (44 mg, 2 mol%) in acetonitrile (10 mL). The reaction mixture was stirred at room temperature for 6 h. After removal of most solvent under vacuum, the residue was poured in to water (100 mL). The precipitate was collected and recrystallized in toluene to afford 5 as a white solid ( yield: 1.7 g, 80%). 1 H NMR (300 MHz, DMSO d 6 ): 12.50 ( s, 2H), 7.80 (s, 2H), 3.68 ( s 4 H). 13 C NMR (75 MHz, DMSO d 6 ): 171.98, 141.33, 139.99, 10 2.18, 45.12. HR MS ESI m/z : [M +Na] + calcd. for C 10 H 8 I 2 O 4 Na, 468.8404, found, 468.8404. (2,5 diiodo 1,4 phenylene)diacetate ( C 1). A mixture of compound 5 (9.0 g, 20 mmol), dodecyl alcohol (80.0 g, 465 mmol) and 85% phosphoric acid (0.5 mL) was placed in a flask equipped with a Dean Sta rk trap. After reaction at 150 o C for 6 h, the solvent was removed by vacuum distillation. The residue was recryst allized in isopropanol to give C 1 as a white solid ( yield: 13.4 g, 85%). 1 H NMR (300 MHz, CDCl 3 ): 7.73 (s, 2H), 4.12 (t, 4H), 3.71 (s, 4H), 1.63 (m, 4H), 1.26 (m, 36H), 0.88 (t, 6H). 13 C NMR (75 MHz, CDCl 3 ): 170.09, 140.84, 138.85, 100.81, 65.64, 45.31, 32.14, 29.88, 29.81, 29.59, 29.43, 28.75, 26.12, 22.93, 14.36. HR MS ES I m/z : [M + Na] + calcd. for C 34 H 56 I 2 O 4 Na, 805.2160 ; found, 805.2160 Compound 7 : Compound 6 (6.4 g, 20 mmol) was dissolved in a mixture of THF (50 mL) and isopropylamine (150 mL), and combined with Pd(PPh 3 ) 2 Cl 2 (28 mg, 0.4 mmol) and CuI (15 mg, 0.8 mmol). After bubbling A r for 40 mi ns trimethylsilylacetylene

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140 (4.8 g, 50 mmol ) was added, and stirred overnight at room temperature. The solvent was removed and the residue was extracted with dichloromethane/water. The organic layer was washed with saturated ammonium chloride, water and br ine, and then dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the crude product was purified by silica chromatography (hexane/dichloro methane, 2/1) to give compound 7 as light yellow solid ( yield: 5.5 g, 95%). 1 H NMR (300 MHz CDCl 3 ) : 7.43 (d, 2H ), 7.21 (d, 2H), 3.50 (s, 2H), 1.43 (s, 9H), 0.24 (s, 9H). 13 C NMR (75 MHz, CDCl 3 ) : 170.45, 135.45, 132.28, 129.33, 42.87, 28.23, 0.24. HR MS ESI m / z : [M+ Na] + calcd. for C 17 H 24 O 2 SiNa, 311.1438; found, 311.1448. Compound 8 : To the solution of compound 7 (2.9 g, 10 mmol) in chloroform (50 mL) was added tetra n butylammonium floride (TBAF, 12 mL, 12 mmol, 1M in THF). After reaction over 1 h, the reaction mixture was passed through a silica column and gave 8 as light yellow solid ( yield: 2.0 g, 9 5%). 1 H NMR (300 MHz, CDCl 3 ) : 7.46 (d, 2H), 7.24 (d, 2H), 3.52 (s, 2H), 3.06 (s, 1H), 1.43 (s, 9H). 13 C NMR (75 MHz, CDCl 3 ) : 170.55, 135.71, 132.38, 129.40, 83.69, 81.29, 42.77, 28.22. HR MS ESI m / z : [M+Na] + calcd. for C 14 H 16 O 2 Na, 239.1042, found, 239.1043. General polymerization pr ocedure for PPEs. Monomer C 1 (0,1 mmol) and 0.1 mmol of the co monomers (1,4 bis(trimethylsilyl)ethynyl)benzene for P1 2,5 bis((trimethylsilil) ethynyl)thiophene for P2 5,7 bis((trimethylsilyl)ethynyl) 2,3 dihydrothieno[3,4 b][1,4]dioxine for P3 1,4 bi s(trimethylsilyl)ethynyl)tetrafluorobenzene for P4 respectively ) were dissolved in a mixture of THF (20 mL) and diisopropylamine (5 mL). The solution was degassed for 1 hour, followed by the addition of tetrabutylammonium difluorotriphenylsilicate ( TBAT, 108 mg, 0.2 mmol). The reaction

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141 was allowed to stir at room temperature for 3 hours. The n Pd(PPh 3 ) 4 (15 mg) and CuI (10 mg) w ere added under the protection of argon. The reaction was stirred at 70 o C for 24 hours, followed by the addition of the Endcap 6 (1 mg). After 6 hours, the Endcap 8 (1mg) was ad ded After another 1 8 hours, the solvent was removed under vacuum and the residue was dissolved in CHCl 3 The resulting solution was passed through a short column of alumina to remove all catalyst and concent rated to 2 mL. Then the polymer was precipitated in a large amount of methanol (100 mL) and collected by centrifugation. The polymer was further purified by multiple cycles of dissolving in CHCl 3 and precipitation in methanol. Hydrolysis. T he precursor Pn E (~ 40 mg) was dissolved in chloroform ( 15 mL) and treated with excess of TFA ( 15 mL) over 5 hours at ambient temperature. The solvents were completely removed under vacuum. The residue was dissolved in THF (20 mL) and then a solution of sodium hydroxide (10 equivalents to ester group) in methanol/water (3 mL, 2/1, v/v) was added. The mixture w as stirred at 50 o C overnight. The solvents THF and methanol were removed under vacuum, and water (10 mL) was added to the residue, followed by heating at 50 o C for 1 day The mixture was concentrated to about 2 mL, precipitated in acetone (25 mL), and centrifuged. The precipitate was dissolved again in water (pH 9, 2 mL). Multiple precipitation was repeated 3 ~ 5 times in acetone (containing 5 ~ 20% methanol). An y insoluble in water was removed by centrifuge before precipitation operation. After filtration on a membrane filter with a 0. 22 m pore size the polymers were subjected to dialysis using Fisher Brand dialysis membrane (molecular weight cutoff 8 k D ) again st water (pH 8) over 3

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142 days. Light yellow to brown solid s w ere obtained in a yield of 80 ~ 90% after ly o ph i lization. Acid polymer P2 H. To a solution of P2 (10 mg) in 3 mL water (pH 8), 3 mL of dilute HCl solution (3 N) was added. The precipitate was colle cted by centrifugation and dried under vacuum for 3 days. The acid polymer was used without any further purification. P 1 E 1 H NMR ( 500 MHz, CDCl 3 ): 7.50 (br, s, 6H), 4.14 (br, m, 4H), 3.85 (br, s, 4H), 1.68 (br, m, 4H), 1.25 (br, m, 36H), 0.85 (br, t, 6 H). GPC (THF, polystyrene standard): M n = 19 kD M w = 34 kD PDI = 1.8 P 2 E 1 H NMR ( 500 MHz, CDCl 3 ): 7.50 (br, s, 2H), 7.18 (br, s, 2H), 4.15 (br, t, 4H), 3.83 (br, s, 4H), 1.65 (br, m, 4H), 1.27 (br, m, 36H), 0.88 (br, t, 6H). GPC (THF, polystyrene st andard): M n = 42 kD M w = 80 kD PDI = 1.9 P 3 E 1 H NMR ( 500 MHz, CDCl 3 ): 7.50 (br, s, 2H), 4.38 (br, s, 4H), 4.1 6 (br, t, 4H), 3.84 (br s, 4H), 1.65 (br, m, 4H), 1.2 5 (br, m, 36H), 0. 90 (br, 6H). GPC (THF, polystyrene standard): M n = 19 kD M w = 34 kD PDI = 1.8 P 4 E 1 H NMR ( 500 MHz, CDCl 3 ): 7.65 (br, s, 2H), 4.18 (br, m, 6H), 3.90 (br, m, 2H), 1.66 (br, m, 4H), 1.28 (br, m, 36H), 0.83 (br, t, 6H). GPC (THF, polystyrene standard): M n = 65 kD M w = 169 kD PDI = 2.6 P 1 1 H NMR ( 500 MHz, CD 3 OD ): 7 .55 (br, 6H), 3.78 (br, 4H). P 2 1 H NMR ( 500 MHz, CD 3 OD ): 7.50 (br, 2H), 7.20 (br, 2H) 3.75 (br, 4H). P 3 1 H NMR ( 500 MHz, CD 3 OD ): 7.53 (br, 2H), 4.38 (br, 4H), 3.78 (br, 4H). P 4 1 H NMR ( 500 MHz, CD 3 OD ): 7.60 (br, 2H), 4.10 (br, 2H), 3.85 (br, 2H).

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143 CHAPTER 5 HIGHLY FLUORESCENT CONJUGATED POLYELECTROLYTES FEATURING METHYLENE AMMONIUM SIDE GROUPS Backgrou nd Over past decades conjugated polyelectrolytes (CPEs) have attrac t ed considerable attention as chemical and biosensors for the detection and anal ysis of a variety of molecules of environmental and biological interest including small molecules, ions and biological targets. 7 52 Among all the sensors, fluorescent sensors based on cationic CPEs are of particular interest. First, most biologically important species are negatively charged su ch as DNA, protein and G ram negative bacteria. Second, the amplified quenching effects of CPEs allow developing the sensors with extremely high sensitivity and rapid detection. However, despite the advantages of CPEs, their solution processi ng is sometimes limited by bad solubility low quantum yield and unexpected sensing behavior induc ed by a strong tendency to self aggregate in aqueous solution. 64 127 128 In C hapter 4, it was shown that PPEs with me thyl ene carboxylate side groups had much less tendency to aggregate in aqueous solution. Through the structure property studies, we found that the enhanced fluorescence quantum y ield was correlated with the absence of oxygen on the linker to phenylene rings. In this chapter, a novel family of water soluble poly(phenylene ethynylene s ) (PPEs) with cationic methylene ammoniu m side groups were synthesized by Sonogashira reaction. The backbone of this PPE series shares a same bis(methylene trimethylammonium) phenylene ethynylene unit alternating with a second aryl e ne ethynylene unit (phenylene for P1 N and thiophene for P2 N respectively). The photophysical properties of the series of PPEs were investigated in methanol and aqueous solution by absorption,

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144 fluorescence spectroscopy and fluorescence lifetime measurement The photophysical data suggested that both polymers existed as single polymer chains in aqueous solution with remarkab ly high fluorescence quantum yields (0.16 for P2 N and 0.30 for P1 N respectively) Steady state fluorescence quenching of this series of PPEs were performed by AQS and K 4 Fe (CN) 6 in aqueous solution A fluorescence sensor for ATP has been successfully devel oped based on the highly fluorescent cationic PPE ( P1 N ). The fluorescence of P1 N in MES buffer solution was efficiently quenched by ATP with a high selectivity over ADP, AMP, PPi and Pi This sensor can be applied to build a fluorescence assay for alkali ne phosphatase (ALP), which catalyzes the dephosphorylating of ATP in cells. 129 Results and Discussion Synthesis of PPEs with Cationic Methylene Ammonium Side Groups Figure 5 1. Structures of P1 N and P2 N In this chapter, a series of water soluble PPEs with cationic methylene ammonium side groups were synthesized through Sonogashira reaction. The backbone of this serie s of PPEs consists of a bis(methylene ammo nium )phenylene ethynylene unit alternating with a second arylene ethynylene unit. For P1 N the second aryl ene unit wa s phenyl ene while the arylene wa s thiophene for P2 N (Figure 5 1).

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145 Monomer synthesis Figure 5 2 shows the synthetic route for the monome r N1 Compound 1 was prepared by the substitution reaction of the commercial available dichloro p xylene and potassium acetate. I odination reaction of compound 1 in a mixture of acetic acid and acetic anhydride resulted in compound 2 in 80% yield. Compound 3 was synthesized by the base assisted hydrolysis with quantitative yield. Compound 4 was obtained by the reaction of PBr 3 and compound 3 Monomer N1 was synthesized by refluxing trimethylamine with compound 4 for 24 hours. Figure 5 2. Synthetic route for the monomer N1 (i) KOAc, DMF, 60 o C, overnight; (ii) I 2 NaIO 4 AcOH, (Ac) 2 O, H 2 SO 4 60 o C, overnight; (iii) K 2 CO 3 CHCl 3 MeOH, rt overnight; (iv) PBr 3 THF, rt, 24 h; (v) NMe 3 CHCl 3 reflux, 24 h. Polymer synthesis and characterization The polymers ( P1 N and P2 N ) were synthesized in a direct route where the monomers used were soluble in DMF/H 2 O mixture. The polymerization directly le d to water soluble conjugated polyelectrolyte s

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146 Figure 5 3 shows the synthesis r oute for water soluble polymer s P1 N and P2 N The polymers were prepared by Sonogashira coupling of a stoichiometric amount of the monomer N1 and die t hynylben zen e or diethynylthiophene ( P1 N or P2 N respectively). The polymers were purified by multiple p recipitations in acetone, followed by the dialysis against deionized water using Fisher brand dialysis membrane (12 kD Molecular Weight Cut off) for three days. The final polymers were obtained after ly o ph i lization as brown yellow solids in ~ 70% yield. Ea ch polymer was characterized by 1 H NMR. Figure 5 3. Synthesis route for P1 N and P2 N (i) Pd(PPh 3 ) 4 CuI, DMF, H 2 O, 70 o C, 2d. Figure 5 4 shows the 1 H NMR spectra of monomer N1 and the water soluble polymer s P1 N and P2 N The 1 H NMR spectra of P1 N and P2 N were obtained in D 2 O at 50 o C after H 2 O signal suppression In Figure 5 4B, the peaks from 7.4 ppm to 8.2 ppm were the protons of the aromatic rings. In Figure 5 4C, the peak ~ 8.2 ppm was from the protons of the ben zene ring and the peak ~ 7.6 ppm was from the protons of the thiophene. The peaks ~ 5.0 ppm in both Figure 5 4B and C were assigned to the benzyl protons.

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147 Figure 5 4. 1 H NMR spectra (500 MHz) of (A) monomer N1 ; (B) P1 N ; (C) P2 N in D 2 O. Polymer NMR spe ctra were obtained in D 2 O at 50 o C after water signal suppression by Dr. Ion. Fluorescence correlation spectroscopy Obtaining accurate molecular weights of CPEs by GPC in aqueous solution was challenging because of the need of special instruments. Therefo re, fluorescence correlation spectroscopy was applied to detect the diffusion time of each polymer and calculate the hydrodynamic radius. The experiments were carried out in aqueous solution s (pH = 6 5 ) with of P1 N and P2 N using fluorescein (30 nM) as standard. The diffusion times and calculated hydrodynamic radius are shown and compared with polymer P1 and P2 ( C hapter 4) in Table 5 1. It is of note that the diffusion time of the polymer sample is related to the focus volume of the FCS instrument and the focus

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148 volume may vary slightly with each experiment The diffusion time of P1 N was smaller (10.02 x 10 5 s) compared with the diffusion time of P2 N ( 12.54 x 10 5 s). The hydrodynamic radius of P1 N was 1.86 nm and the hydrodynamic radius of P2 N was 2.32 nm. Considering that P1 N and P1 had the same polymer backbone, the molecular weight of P1 N was calculated according to the e quation s below If we used the spherical model, Equation 5 1 was chosen; if we consider our polymer to be a rod like particl e we should use Equation 5 2 See the detail s of FCS calculation in Appendix C. (5 1 ) (5 2 ) w here M is the molecular weight of the ionic polymer, R is the hydrodynamic radius of the polymer ( spherical model ) and L is the length of the polymer sample (rod model) P2 and P2 N had the same polymer backbone and same conformation in aqueous solution, thus the molecular weight of P2 N was estimated based on the molecular weight of P2 Then molecular weight of P1 N was estimated to be 13 ~ 20 kD and the molecular weight of P2 N was 38 ~ 7 7 kD. Table 5 1 Diffusion time and hydrodynamic radius of P P Es in aqueous solution. Polymer ( x 10 5 s) D (x 10 10 m 2 s 1 ) R H (nm) L (nm) M n (kD) P1 N A 10.02 1.30 1.86 14.9 20 C 13 D P 1 B 6.17 1.62 1.49 11.9 19 ( P1 E ) P 2 N A 12.54 1.04 2.32 18.6 77 C 38 D P 2 B 6.72 1.48 1.63 13.0 48 ( P2 E ) A P1 N and P2 N solution had pH = 6.5. B P1 and P2 soluti on had pH = 8.0. C Molecular weight calculated by spherical model. D Molecular weight calculated by rigid rod model.

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149 Photophysical Properties Absorptio n, fluorescence and quantum yield The p hotophysical properties of the P PEs were investigated by UV Vis a bsorption fluorescence spectroscopy and fluorescence lifetime measurements in MeOH and H 2 O. Figure 5 5 shows the normalized absorption and fluorescence spectra of P1 N and P2 N in both methanol and water. In general both polymers wer e well solvated in Me OH with st ructured emission and very small S tok e s shift between absorption maximum and fluorescence maximum. Unlike traditional PPEs, solvents effects we re much suppressed for both P1 N and P2 N In methanol, P1 N exhibit ed an absorption maximum at 385 nm and a fluorescence maximum at 415 nm. The spectra of P1 N in aqueous solution were very similar to those in methanol, indicating that P1 N wa s molecular ly dissolved in H 2 O. A similar result wa s observed for polymer P2 N. The spectra of P2 N in methanol and water we re almost the same with an absorption maximum around 425 nm and a fluorescence maximum around 464 nm. Figure 5 5. Normalized absorption and fluorescence spectra of P1 N (A) and P2 N (B) in MeOH (solid line) and H 2 O (dash line). H 2 O at pH = 6.5.

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150 Table 5 2. Photophysical data of P1 N and P2 N Polymer Solvent max abs (nm) max fl (nm) F B P1 N MeOH 386 415 0.30 H 2 O A 385 414 0.30 P2 N MeOH 425 464 0.17 H 2 O A 424 462 0.14 A H 2 O at pH = 6.5. B Qu i nine sulfate in 0.1 M H 2 SO 4 solution as a standard ( F = 0.545). The absence of aggregation wa s confirmed by the remarkably high fluorescence quantum yield in aqueous solution Table 5 2 summarizes the photophysical properties of P1 N and P2 N in both methanol and water. The fluorescence quantum yield of P1 N wa s ~ 0.30 in aqueous solution, the same as that in methan ol. The fluorescence of quantum yield of P2 N in methanol wa s ~ 0.17, due to the presence of thiophene unit which increased the rate of intersystem crossing The quantum yield of P2 N in aqueous solution wa s still quite high ~ 0.14. In conclusion, t he shap e and structure of the fluorescence spectr a combined with the high fluorescence quantum yield s support ed our premise that the positively charged methylene trimethylammonium side groups can significantly suppress the aggregation of PPEs in aqueous solution. Fluorescence lifetime measurement In the previous studies, it was found that the presence of aggregates in CPEs cause d the dynamic interaction between the excitons state in non aggregated chains and excitons located on the aggregated chains. 48 The aggregates in those c ases act ed as an energy trap and quencher, resulting in a much shorter lifetime in aqueous solution. In order to support the idea that the aggregated chains were abs ent, the fluorescence

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151 decay of P1 N and P2 N in both methanol and water were measured using the time correlated single photon counting (TCSPC). Table 5 2 and Figure 5 6 show the average fluorescence lifetime of P1 N and P2 N in MeOH and H 2 O (pH = 6.5) at each detection wavelength with ~ 10 nm band width Figure 5 6. Fluorescence lifetime at di fferent wavelengths: (A) P1 N in MeOH; (B) P1 N in H 2 O; (A) P2 N in MeOH; (B) P2 N in H 2 O. In general the fluorescence decays of P1 N and P2 N in both MeOH and H 2 O featured bi exponential decays The global analysis of the fluorescence decay of P1 N in Me OH yielded two components ( 1 = 0. 38 ns and 2 = 0. 65 ns). The first component had a dominant contribution (>78%) over all the detection wavelengths As the detection wavelength increased, the contribution of the second component increased, resulting in

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152 a slightly longer lifetime at longer wavelength. In aqueous solution, the global analysis of the fluorescence decay of P1 N gave a similar result with two decay components. The average lifetime of P1 N in aqueous solution was the same as that in methanol, indicating that P1 N was mole cularly dissolved in aqueous solution. The fluorescence decay of P2 N in both MeOH and H 2 O were processed by global fitting algorithm. Two decay components were obtained for the fluorescence of P2 N in both MeOH and H 2 O. It was found that the fluorescence of P2 N in MeOH was not dependent on the detection wavelength with the first component ( 1 = 0. 17 ns) contributing ~ 25% and the second component contributing ~ 75%. Similar results were observed for the fluorescence decay of P2 N in aqueous solution. Com pared with P1 N the longer lifetime component had a dominant role in the overall amplitude T he mean fluorescence lifetime of both P1 N and P2 N were similar ~ 0.42 ns in both MeOH and H 2 O. Table 5 3. F luorescence lifetime of P1 N and P2 N RA (%) A MeO H H 2 O B P1 N (ns) 420 nm 450 nm 470 nm 500 nm (ns) 420 nm 450 nm 470 nm 500 nm 1 = 0. 38 94 89 86 78 1 = 0.4 2 99 98 96 88 2 = 0.6 5 6 11 14 22 2 = 1.17 1 2 4 12 2 0.96 1.0 2 1.0 0 1.0 2 2 1.0 5 1.0 3 0 97 0 1.0 1 P2 N (ns) 450 nm 470 nm 500 nm 520 nm (ns) 4 50 nm 470 nm 500 nm 520 nm 1 = 0. 17 29 19 22 25 1 = 0. 18 20 21 25 25 2 = 0. 50 71 81 78 74 2 = 0. 47 80 79 75 75 2 1.0 0 1 01 1.0 3 0 99 2 0.99 1.0 6 1.00 1. 02 A D ata were process ed by global fitting algorithm. Typical limits of error on i are les s than 3%. B H 2 O at pH = 6.5.

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153 Steady S tate Fluorescence Q ue nching Experiments As mentioned in previous chapters, PPEs are attractive targets for study of the amplified quenching effects. In order to investigate the amplified quenching effects, steady s t ate fluorescence quenching experiments of P1 N and P2 N were performed by AQS ( 9.10 anthraquinone 2,6 disu lfonic acid disodium salt ) and K 4 Fe(CN) 6 in aqueous solution. Figure 5 7. Fluorescence spectra of PPEs in H 2 O upon addition of quenchers. (A) P1 N by AQS; (B) P1 N by K 4 Fe(CN) 6 ; (C) P2 N by AQS; (D) P2 N by K 4 Fe(CN) 6 [ Polymer ] = 2 H 2 O at pH = 6.5. Figure 5 7 shows the fluorescence spectra of the polymers upon additions of different quenchers. Addition of AQS ( 2 M) in the aqueous solution of P1 N resulted in significant quenching (>95%) with a loss of the vibronic structure in the fluorescence

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154 spectra. Compared with AQS, K 4 Fe(CN) 6 exhibited a stronger quenching ability that addition of K 4 Fe(CN) 6 (0.7 M) quenched most of the fluorescence of P1 N Similar results were obtained for polymer P2 N Figure 5 8. Stern Volmer plots of P1 N and P2 N with various concentrations of the quenchers in H 2 O. P1 N by AQS ( ); P1 N by K 4 Fe(CN) 6 ( ); P2 N by AQS ( ); P2 N by K 4 Fe(CN) 6 [ Polymer ] = 2 M ; H 2 O at pH = 6.5. Table 5 4. Stern Volmer constant and [Q 90 ] for 2 polymer in H 2 O with AQS and K 4 Fe(CN) 6 Polymer Quencher K sv (M 1 ) [Q 90 ] ( M ) P1 N AQS 5.9 x 10 5 1.2 K 4 Fe(CN) 6 8.2 x 10 5 0.6 P2 N AQS 6.4 x 10 5 1.3 K 4 Fe(CN) 6 1.1 x 10 6 0.6 Figure 5 8 sh ows the Stern Volmer plots of PPEs ( P1 N and P2 N ) with various concentrations of the quenchers. In general, the Stern Volmer plot of each case curved

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155 upward and quencher K 4 Fe(CN) 6 had a better efficiency to quench the fluorescence of both P1 N and P2 N S tern Volmer constants were calculated at the linear range when the quencher concentration was low. For P1 N quencher AQS had a smaller K sv value ~ 5.9 x 10 5 M 1 while K 4 Fe(CN) 6 exhibited a bigger K sv value ~ 8.2 x 10 5 M 1 and a bigger [Q 90 ] value ~ 0.6 M The K sv values for P2 N were slightly larger compared with those for P1 N (6.4 x 10 5 M 1 for AQS and 1.1 x 10 6 M 1 for K 4 Fe(CN) 6 respectively). This can be explained by the different conformation s of P1 N and P2 N in aqueous solution: P1 N is more lik e rigid rod while P2 N was able to form helix structure because of the thiophene unit. Table 5 4 summarizes the Stern Volmer constants and the [Q 90 ] values, which is defined as the quencher concentration when 90% of the fluorescence is quenched. The [Q 90 ] values of AQS were 1.2 M for P1 N and 1.3 M for P2 N respectively. The [Q 90 ] values of K 4 Fe(CN) 6 were smaller compared with AQS (0.6 M for both P1 N and P2 N ). Application to Adenosine Triphosphate S ensing Adenosine triphosphate (ATP) is a multifunctional nucleoside availabl e in cells. It is often referred to as molecular unit of currency, because ATP transports chemical energy within cells for metabolism. One ATP molecule contains three phosphate groups (Figure 5 9 ) Under the catalysis of the alkaline phosphatase (ALP), ATP can lose one 5 phosphate group to form ADP (adenosine diphosphate) or two 5 phosphate groups to form AMP (adenosine monophosphate). 130 Herein, we report that the conjugated polymer P1 N can be utilized as probes for sensitive and selective fluorescence sensor for ATP due to formation of conjugated polymer/phosphate substrates complex by electrostatic interaction

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156 Figure 5 9. Dephosphorylation of adenosine triphosphate (ATP) by alkaline phosphatase (ALP). Figure 5 10. Fluorescence spectra of P1 N (2 ) in MES buffer (10 mM, pH = 6.5) upon addition of ATP (A), ADP (B), AMP (C) ,PPi (D) and Pi (E).

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157 Figure 5 11. (A) Fluorescence spectra of P1 N (2 ) in MES buffer (10 mM, pH = 6.5) upon addition of 10 of different quenchers. (B) Stern Volmer plot s of P1 N (2 ) in MES buffer (10 mM, pH = 6.5) upon addition of different quenchers. Figure 5 10 shows the fluorescence spectra of P1 N in MES (2 (N mopholine) ethane sulfonic acid) buffer (10 mM, pH 6.5) upon addition of different phosphate substrates i ncluding ATP, ADP, AMP, PPi and Pi. The addition of 25 M of ATP can induce about 90% quenching of the fluorescence intensity of P1 N In contrast, the addition of 800 M of other quenchers (ADP, AMP, PPi and Pi) reduced the fluorescence intensity by less than 40%. A more distinct comparison of the fluorescence spectra of P1 N with 10 M of different q uenchers is shown in Figure 5 11 A. In the MES buffer solution, 10 M of Pi and AMP barely induced any change to the fluorescence spectra compared to the pure polymer solution. After the introduction of 10 M of ADP, the fluorescence was quenched by ~ 10%. In contrast, more than 80% of the fluorescence of P1 N was quenched by 10 M of ATP, indicating that P1 N was highly selective for ATP over the other phosphat e substrates. As a comparison, 10 M of PPi quenched ~ 40% fluorescence of P1 N Figure 5 1 1 B shows the Stern Volmer plots of P1 N in MES buffer solution upon addition of different quenchers. While ADP, AMP, PPi and Pi failed to induce substantial quenchin g ATP quenched the fluorescence very

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158 efficiently with a K sv value ~ 3.8 x 10 5 M 1 There are several advantages of this sensor compared with the ATP sensor in literatures. 131 132 First, the polymer itself was highly fluorescent with a quantum yield ~0.30 in aqueous solution. The sensitivity of the fluorescent sensor based on P1 N is higher than zinc complex base d chemo sensors. Second, this sensor had a better selectivity for ATP over PPi compared to the poor ability to differentiate them by sensors in literatures 24 129 Addition of 10 M ATP quenched more than 90% of the fluorescence intensity at 415 nm, while the same amount of PP i quenched less than 30%. In addition, the direct detection of ATP using this sensor is fast and rapid, since the polymer P1 N is well solvated in aqueous sol ution. In conclusion, a sensitive and selective fluorescence sensor for ATP has been successfully developed based on a highly fluorescent cationic PPE ( P1 N ). The fluorescence of P1 N in MES buffer solution was efficiently quenched by ATP. This sensor can be applied to build a fluorescence assay for alkaline phosphatase (ALP) in the future study. S ummary In this chapter, a new series of conjugated polyelectroly tes based on the poly(phenylene ethynylene) backbone featuring methylene ammonium side groups ha ve been synthesized and characterized These polyme rs were prepared using a direct which polymerization directly le d to cationic water soluble PPE s ( P1 N and P2 N ) Investigations of the photophysical properties of the P PEs proved that both P1 N and P2 N exist ed as single chains in water, with high fluorescence quantum yield s comparable fluorescence spectra and fluorescence lifetime to those in methanol

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159 Steady state fluorescence quenching of P1 N and P2 N with AQS and K 4 Fe(CN) 6 reveal ed the am plified quenching effects. A fluorescence turn off sensor for ATP is developed by taking advantage o f the interaction between water soluble P1 N and the phosphate substrates driven by the electrostatic interaction. In MES buffer solution, the fluorescen ce of the polym er P1 N wa s sensitive to the concentration of ATP with high selectivity over ADP, AMP PPi and Pi We are currently developing biological assays for enzymes such as ALP using this system and we believe the design principles can be applied to other anion species of interest. Experimental Materials Pd(PPh 3 ) 4 was purchased from Strem Chemical Company and used as received. S odium phosphate (Pi) 2 (N mo r pholine)ethane sulfonic acid (MES), trimethylamine, 9.10 anthraquinone 2,6 disu lfonic acid di sodium salt (AQS), sodium adenosine triphosphate (ATP), sodium adenosine diphosphate (ADP), sodium adenosine monophosphate (AMP) and sodium pyrophosphate (PPi) were purchased from Sigma Aldrich and used without further purification. Potassium acetate sul fur ic acid, acetic acid, acetic anhydride and potassium ferrous cyanide (K 4 Fe(CN) 6 ) were purchased from Fisher Scientific Company and used as received THF and DMF were purified by solvent dispensing system. All other chemicals were purchased from commerc ial sources unless specially mentioned. S tock solutions ( 0.5 mM) of all P P Es were prepared in H 2 O and have been stored at 4 o C. For all photophysical experiments, the P PEs solutions were prepared in deionized H 2 O with pH adjusted to 6.5. MES buffer (10

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160 mM pH = 6.5) was prepared by dissolving MES into deionized water and the pH was adjusted by adding dilute HCl according to the pH meter. Instruments and General M ethods NMR spectra were record ed using a Gemini 300 NMR operating at 300 MHz for 1 H NMR and at 75 MHz for 13 C NMR for small organic compounds 1 H NMR spectra of the polymers were measured in Inova2 500 NMR operating at 500 MHz at 50 o C after water signal suppression The polymer NMR spectra were measured by Dr. Ion. UV absorption spectra were measu red on a Shimadzu UV 1800 spectrophotometer. Luminescence spectra were measured on a PTI (Photon Technology International) fluorescence spectrometer. Fluorescence lifetimes were determined by time correlated single photon counting on a FluoTime 100 spectro meter ( Pico Quant ) equipped with 370 nm diode laser as excitation source. Fluorescence quantum yields are reported relative to known standards. The optical density of solutions at the excitation wavelength was <0.1 and corrections were applied for differen ces in the refractive index of standard and sample solutions. FCS measurements were taken on a homemade setup using a 405 nm diode laser (Coherent, CUBE) as the excitation light. Fluorescein (30 nM in 10 mM phosphate buffer, pH = 8) was used as the calibra tion for the system. Synthetic P rocedures 1,4 Phenylenebis(methylene) diacetate (1) To a solution of dichloro p xylene (3.9 g, 22 mmol) in 100 mL DMF, potassium acetate (21.2 g, 0.22 mol) was add ed. T he reaction was stirred at 60 o C for overnight. After c ooling to room temperature, the mixture was poured into cold water. The white precipitate was collected by filtration an d washed several times with water ( y ield : 3.5 g, 73 % ) 1 H NMR ( 300 MHz, CDCl 3 ): 7.30

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161 (s, 4H), 5.0 (s, 4H), 2.05 (s, 6H). 1 3 C NMR ( 75 MHz, CDCl 3 ): 170.85, 136.09, 120.53, 65.97, 21.08. (2,5 Diiodo 1,4 phenylene)bis(methylene) diacetate (2) Sodium periodate (1.68 g) and diiodine (6.2 g, 40% excess) were stirred into a mixture of glacial acetic acid (15 mL) and acetic anhydride (8 mL) a t 5 o C for 30 mins Then c oncentrated sulfuric acid (2 mL) was then added slowly to the stirring suspension. Compound 1 (2 g, 9 mmol) was added to this solution and stirred continuously for 24 h at 60 o C. The reaction was then poured into an ice water mi xture saturated with Na 2 SO 3 All precipitate was collected by filtration and washed with cold ethanol. Recrystallization of the product from ethanol re sulted in pure white solid ( y ield: 3.3 g, 77% ). 1 H NMR ( 300 MHz, CDCl 3 ): 7.80 (s, 2H), 5.05 (s, 4H), 2.17 (s, 6H). 1 3 C NMR ( 75 MHz, CDCl 3 ): 170.56, 140.21, 139.59, 97.78, 68.99, 21.13. (2,5 Diiodo 1,4 phenylene)dimethanol (3) To a solution of compound 2 (1 g, 2.1 mmol) in a mixture of CHCl 3 (100 mL) and MeOH (100 mL), pota ssium carbonate (10 g) was added. The reaction was stirred at room temperature for 24 h. The solvents were then removed under vacuum and 300 mL water was added to the residue. The resulting mixture was stirred for 2 h. The insoluble white solid was collect ed and dried under vacuum ( y ield: 0.75 g, 80%). 1 H NMR ( 300 MHz, DMSO d 6 ): 7.00 (s, 2H), 5.55 ( t, 2H), 4.36 (d, 4H). 1 3 C NMR ( 75 MHz, DMSO d 6 ): 144.10, 136.97, 96.53, 66.39. 1,4 Bis(bromomethyl) 2,5 diiodobenzene (4) To a solution of compound 3 (0.88 g, 2.5 mmol) in THF (50 mL), PBr 3 (0.24 mL) was added slowly at 0 o C The re action was stirred at room temperature for 24 h. The organic solvent was removed under vacuum and the residue was dissolved in CHCl 3 (100 mL) and washed with water

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162 for 3 times. After drying over anhydrous Na 2 SO 4 the organic solvent was removed to yield co mpound 4 as a white solid ( y ield: 0.72 g, 60%). 1 H NMR ( 300 MHz, CDCl 3 ): 7.86 (s, 2H), 4.45 (s, 4H). 1 3 C NMR ( 75 MHz, CDCl 3 ): 141.90, 140.80, 99.70, 36.80. Monomer N1 To a solution of compound 4 (0.47 g, 0.96 mmol) in a mixture of ethanol (30 mL) and acetone (30 mL), trimethylamine in water solution (25%, 30 mL) was added. The reaction was reflux ed for 24 h. Then the solvents were removed under vacuum and the residue was dried ( y ield: 0.55 g, 93%). 1 H NMR ( 500 MHz, D 2 O ): 8.20 (s, 2H), 4.75 (s, 4H), 3.20 (s, 18H). 1 3 C NMR ( 125 MHz, D 2 O ): 145.59, 135.14, 103.56, 70.78, 53 .50. General procedure for polymerization. Monomer N 1 ( 63.4 mg, 0 1 mmol) and 0.1 mmol of the corresponding co monomers (1,4 di ethynyl)benzene for P1 N 2,5 di ethynyl)thiophene for P2 N ) were dissolved in a mixture of DMF ( 15 mL) H 2 O (5 mL) and diisopropy lamine (5 mL). The solution was degassed for 1 hour, followed by the addition of Pd(PPh 3 ) 4 (15 mg) and CuI (10 mg) under argon atmosphere The reaction was stirred at 70 o C for 48 hours Then the resulting mixture was concentrated to 2 mL and poured into ac etone (50 mL). The yellow fiber precipitate was collected by centrifugation and dissolved into H 2 O (2 mL). The polymer was further purified by multiple precipitation in acetone, followed by the dialysis against Millipore water using Fisher Brand dialysis m embrane (12 kD molecular weight cut off) for 3 days. Brown yellow solids were obtained after ly o phlization P1 N : 1 H NMR ( 500 MHz, D 2 O ): 7.5 0 ~ 8.25 (br, 6H), 5.00 (s, 4 H) 3.32 (br, 18H). I ntegration of the peak ~ 5.00 pm was slightly less than 4 due to the suppression of the water signal nearby.

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163 P2 N : 1 H NMR ( 500 MHz, D 2 O ): 8.2 0 (br, 2H), 7.6 5 (br, 2H), 5.00 ( br, 4H), 3.33 (br, 18H) I ntegration of the peak ~ 5.00 pm was slightly less than 4 due to the suppression of the water signal nearby.

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164 CHAPTER 6 CONCLUSION In conclusion, the design and synthesis of functional poly(phenylene ethynylene)s (PPEs) have been present ed and discussed Their photophy sical properties and aggregation behaviors in different solven ts have been investigated as a guide to sensor application. Especially, the discovery of the methylene carboxylate or methylene ammonium side groups to suppress aggregation of PP Es provides the polymer chemists with new approaches to develop and synthesize novel non aggregated conjugated polyelectrolytes. By taking advantage the analyte induced aggregation mechanism, several fluorescent sensors have been built. Traditional PPE typ es CPEs As discussed in C hapter 1, PPEs with linear side groups always aggregated in aqueous solution, driven by the hydrophobic interaction and stacking. In order to improve the photophysical properties for sensor application, usually surfactant was needed. For example, surfactant (Triton X 100) drastically improved the fluorescence property of GU P1 which made the PPi sensor possible. Many e fforts have been made to avoid the aggregation. Swager and coworkers incorporated Iptycenes as building blocks into the PPE backbone. I ptycenes can provide steric blocking, which can inhibit strong interactions between polymer chain s which have a strong te ndency to form non emissive exci mer complexes. 52 128 133 Hecht and coworkers discovered that the introduction of the oligo(ethylene glycol) as side groups can significantly induce the steric hindrance between polymers, resulting in non aggregated PPEs. 62 However, the bulky oligo(ethylene glycol) side groups wrap the polymer backbone and block other molecule from getting close. 39 63 64 Our group (Schanz e group) also has devoted a lot of

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165 efforts to get non aggregated PPEs by changing different solubilizing groups and attaching bulky polyionic side groups 24 48 There is one problem that has not been answered or even asked: What caused the PPEs to aggregate in aqueous solution? Before this dissertation, almost all of the PPEs in literatures share a common feature. T hey have an oxygen atom on the solubilizing group directly attached to the polymer backbone. The reason for this is simple: the monomers of PPEs were synthesized from 1,4 diiodo 2,5 dihydroxylbenzen e It is easy to put different solubilizing groups to the PPE type CPEs by a substitution reaction. In 2013, Feng and Schanze discovered that a set of OPEs without that oxygen linkage exhibited remarkable fluorescence quant um yield in aqueous solution (> 0.80). 134 Following his discovery, this dissertation will explore the methylene carboxylate and methylene ammonium side groups to achieve non aggregated PPEs. Non oxygen PPE type CPEs In thi s dissertation, two sets of PPE type CPEs were synthesized and characterized in C hapter 4 and 5 Their photophysical properties and aggregation behavior in aqueous solution were carefully investigated. It was found that the introduction of both methylene c arboxylate ( CH 2 CO 2 Na) and methylene ammonium ( CH 2 NMe 3 Br) significantly suppressed the aggregation of PPEs in aqueous solution. Most of the PPEs had comparable fluorescence properties in H 2 O compared with those in MeOH. The fluorescence quantum yields o f most PPEs were ~ 0.16, much bigger than the traditional PPEs. These differences between the traditional PPEs and non oxygen PPEs were attributed to the aggregation behavior in aqueous solution. In a collaboration work with Dr. Feng, the TEM images of PPE s with oxygen linker showed

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166 large aggregates while non oxygen PPEs stayed unaggregated as small particles. Until now, we could not answer what causes the differences with complete confidence but we have some hypothesis that may provide a way to explain. In an unpublished simulation experiment by Wink e l in our group, it was found that OPE (20 repeat units) with oxygen had higher HOMO energy (~ 0.3 eV higher) than the non oxygen OPE. In the traditional PPEs, the benzene with oxygen linker may behave like a d onor, and the other benzene without oxygen is lower in energy. This donor acceptor like effect may promote in ter chain aggregation and stabilize the aggregates. However, in C hapter 4, we designed two donor acceptor polymers ( P3 and P4 ) and both polymers di d not form large aggregates Another possibility is that the oxygen in traditional PPEs interacts with water molecule in aqueous solution resulting in a very fast energy relaxation pathway. This idea can also explain the low quantum yield of P3 in aqueous solution, because P3 had an oxygen linker directly attached to the backbone. In addition, t he photo induced electron transfer between the polymer backbone and the oxygen lone pairs may also exist. Non aggregated PPEs Taken together, the basic principle t o achieve non aggregated PPE type CPEs is to prevent the inter chain interaction and suppress stacki ng. Based on our discovery, it is a good idea to change the oxygen linker to methylene linker, which results in non aggregated PPEs in aqueous solution. If you want to get even better photophysical properties, you can combine the advantage s of polyionic side groups and methylene linker. The non aggregated PPEs can be favorably used in sensor application based on analyte induced aggregation mechanism. High performance devices including DSSCs can be obtained using non aggregated PPEs.

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167 APPENDIX A NMR SPECTRA Figure A 1. 1 H NMR spectrum (500 MHz, CDCl 3 ) of compound 5 ( C hapter 2). Figure A 2. 1 H NMR spectrum (500 MHz, DMSO d 6 ) of OPEC1 ( C hapter 2).

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168 Fig ure A 3. 1 H NMR spectrum (500 MHz, CDCl 3 ) of GU P1 Boc ( C hapter 3). Figure A 4. 1 H NMR spectrum (500 MHz, DMSO d 6 ) of GU P1 ( C hapter 3).

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169 Figure A 5. 1 H NMR spectrum (500 MHz, CDCl 3 ) of GU P2 Boc ( C hapter 3). Figure A 6. 1 H NMR spectrum (500 MHz, DMSO d 6 ) of GU P2 ( C hapter 3).

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170 Figure A 7. 1 H NMR spectrum (500 MHz, CDCl 3 ) of P1 E ( C hapter 4). Figure A 8. 1 H NMR spectrum (500 MHz, CD 3 OD) of P1 ( C hapter 4).

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171 Figure A 9. 1 H NMR spectrum (500 MHz, CDCl 3 ) of P2 E ( C hapter 4). Figure A 10 1 H NMR spect rum (500 MHz, CD 3 OD) of P2 ( C hapter 4).

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172 Figure A 11. 1 H NMR spectrum (500 MHz, CDCl 3 ) of P3 E ( C hapter 4). Figure A 1 2 1 H NMR spectrum (500 MHz, CD 3 OD) of P 3 ( C hapter 4).

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173 Figure A 13. 1 H NMR spectrum (500 MHz, CDCl 3 ) of P4 E ( C hapter 4). Figure A 14. 1 H NMR spectrum (500 MHz, CD 3 OD) of P4 ( C hapter 4).

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174 Figure A 15. 1 H NMR spectrum (500 MHz, D 2 O 50 o C ) of P1 N ( C hapter 5). Figure A 16. 1 H NMR spectrum (500 MHz, D 2 O 50 o C ) of P2 N ( C hapter 5).

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175 APPENDIX B MASS S PECTRA Figure B 1. Mass spect rum of Compound 5 ( C hapter 2). Figure B 2. Mass spectrum of OPEC1 ( C hapter 2).

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176 APPENDIX C FCS CALCULATION In principle, f luctuations in the fluorescence signal are quantified by tem poral autocorrelation of the recorded emission signal s collected within the confocal volume. The normalized autocorrelation function, defined as 72 ( C 1 ) is used to characterize the temporal fluctuations In Equation C 1 describes the fluctuation of the fluorescence signal as deviations from the temporal average of the signal at time t A three dimensional fitting model represen ting a single component system is written as: ( C 2 ) w here is the longitudinal radius and is the transversal or waist radius of the conf ocal volume ; and the structure parameter, equates to N is the average number of fluorescent molecules in the confocal volume ; is the average time of fluorescent molecules diffusing in the detection volume, which i s characteri stic for a specific molecule. The relationship of to the molecular diffusion coefficient D (m 2 s 1 ) is given by: ( C 3 ) The waist radius is obtained from its convers ion equation : ( C 4 ) where is the diffusion coefficient of the standard calibration dye.

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177 The translational diffusion coefficient D of a mol ecule is related to its size by the Stokes Einstein equation : ( C 5 ) where k ; T is the temperature ; is the viscosity of the solvent ; and R is th e hydrodynamic radius. Equation C 5 can be used to estimate the size of diffusing particles by assuming the particles has a spherical shape with radius R which is related to the molecular weight MW of the molecule with a specific gravity by ( C 6 ) where V is the volume of molecule. Thus we have ( C 7 ) These equations show that the radius R and diffusio n coefficient D are weakly dependent on the molecular weight. By combining E quation C 5 and C 7 we have: ( C 8 ) This relationship is useful for estimation of the mole cular weight of a spherical particle from its diffusion coefficient. For most of our polymers discussed in this presentation, they are not spherical but more like rigid rod. Therefore, the hydrodynamic radius calculated using the spherical model usually ga ve us a smaller number than the actual size. We can use some simple consideration to calculate the frictional confident for rod like particles. 135 Su ppose a rod like particle has a length 2a and radius b. The volume is given by the formula: (C 9)

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178 W here V rod is the volue of the rod like particle. The axial ratio P is defined as: (C 10) For a rod li ke particle, the frictional confident can be found to be 136 137 (C 11) W here f is the frictional confident for the rod like particle, f 0 is the frictional confident for a spherical particle and defined as: (C 12) W here is the viscosity of the solvent, R 0 is the radius that has a volume equal to the volume of rod with axial ratio P Therefore, the relationship between a and R 0 is: (C 13) Combined with Equation C 10, one can find that: (C 14) If we combine Equations C 5, C 11, C 12 and C 14, we can find that: (C 15) If we define a factor C = ln2 P 0.3, the value of C will be 4 < C < 5, when 40 < P < 100. In the PPEs system, the radius b does not change as molecular weight increases. Therefore, t he relationsh ip between the molecular weight of the particles and the length will be linear, if we assume the radius b does not change. We can find: (C 16)

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179 LIST OF REFERENCES (1) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem Rev 2000 100 2537. (2) Gunes, S.; Neugebauer, H.; Sariciftci, N. S. Chem Rev 2007 107 1324. (3) Kraft, A.; Grimsdale, A. C.; Holmes, A. B. Angew Chem Int Edit 1998 37 402. (4) Tor si, L.; Dodabalapur, A.; Rothberg, L. J.; Fung, A. W. P.; Katz, H. E. Science 1996 272 1462. (5) Montali, A.; Smith, P.; Weder, C. Synthetic Met 1998 97 123. (6) Tan, C. Y.; Pinto, M. R.; Schanze, K. S. Chem Commun 2002 446. (7) Jiang, H.; Taraneka r, P.; Reynolds, J. R.; Schanze, K. S. Angew Chem Int Edit 2009 48 4300. (8) Shi, S. Q.; Wudl, F. Macromolecules 1990 23 2119. (9) Chen, L. H.; McBranch, D. W.; Wang, H. L.; Helgeson, R.; Wudl, F.; Whitten, D. G. P Natl Acad Sci USA 1999 96 12287. (10) Bolink, H. J.; Brine, H.; Coronado, E.; Sessolo, M. Acs Appl Mater Inter 2010 2 2694. (11) Seo, J. H.; Gutacker, A.; Walker, B.; Cho, S. N.; Garcia, A.; Yang, R. Q.; Nguyen, T. Q.; Heeger, A. J.; Bazan, G. C. J Am Chem Soc 2009 131 18220. (12) Seo, J. H.; Namdas, E. B.; Gutacker, A.; Heeger, A. J.; Bazan, G. C. Adv Funct Mater 2011 21 3667. (13) Sirringhaus, H. Adv Mater 2005 17 2411. (14) Jiang, H.; Zhao, X. Y.; Shelton, A. H.; Lee, S. H.; Reynolds, J. R.; Schanze, K. S. Acs Appl Mater In ter 2009 1 381. (15) Fang, Z.; Schanze, K. S. Abstr Pap Am Chem S 2011 241 (16) Sambur, J. B.; Averill, C. M.; Bradley, C.; Schuttlefield, J.; Lee, S. H.; Reynods, J. R.; Schanze, K. S.; Parkinson, B. A. Langmuir 2011 27 11906. (17) Ogawa, K.; Chemburu, S.; Lopez, G. P.; Whitten, D. G.; Schanze, K. S. Langmuir 2007 23 4541. (18) Chemburu, S.; Ji, E.; Casana, Y.; Wu, Y.; Buranda, T.; Schanze, K. S.; Lopez, G. P.; Whitten, D. G. J Phys Chem B 2008 112 14492.

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187 BIOGRAPHICAL SKETCH Xuzhi Zhu was born in Wuhu, Anhui Province, China in 1988. Xuzhi Zhu started his undergraduate studies at Fudan University (Sha nghai, China) in 2005. Four years later, he received his bachelor s degree of science in Macromolecular Science and Engineering. During the same year, Xuzhi went to United States to continue his graduate studies in Department of Chemistry at University of Florida, where he joined Dr. Schanze s group. In th e past four years, he focused his research on the topic of Conjugated Polyelectrolytes under the supervision of Dr. Kirk S. Schanze. Xuzhi received his Ph. D. from University of Florida in the summer of 20 13. In 2013, Xuzhi will go back to China to work in chemistry industry and in education industry