water soluble p conjugated polymer for biosensor applications

WATER-SOLUBLE π-CONJUGATED POLYMER FOR BIOSENSOR APPLICATIONS ZHAN RUO YU NATIONAL UNIVERSITY OF SINGAPORE 2012 WATER-SOLUBLE π-CONJUGATED POLYMER FOR BIOSENSOR APPLICATIONS ZHAN RUO YU (B.S., FUDAN UNIV.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Zhan Ruo Yu 26 February 2013 Acknowledgements ACKNOWLEDGEMENTS First and foremost, I would like to express my deep and sincere gratitude to my supervisor, Associate Prof. Liu Bin, whose patience and kindness, as well as constructive suggestions, academic knowledge and experience, have been invaluable to me. I would like to take this opportunity to acknowledge Prof. Li Zhi and Dr. Xie Jianping, the members of my oral qualification examination committee, for their criticism and advice on the research topic, together with my thesis reviewers for their time, assistance and examination on this thesis. I am grateful to all group members, particularly Dr. Pu Kanyi, Dr. Shi Jianbing, Dr. Cai Liping and Dr. Liu Jie for their instructions on experiments and suggestions, Mr. Wang Guan for his help in the NMR experiments. I am also grateful to lab staff, Mr. Boey Kok Hong, Ms. Lee Chai Keng and Mr. Tan Evan Stephen for their kind support. I would love to thank my parents for their unlimited love and support during my stay abroad. The financial support from the National University of Singapore and Singapore Ministry of Education is gratefully acknowledged. i Table of contents TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS . ii SUMMARY . v LIST OF TABLES vii LIST OF SCHEMES . viii LIST OF FIGURES x LIST OF ABBREVIATIONS . xiii CHAPTER INTRODUCTION . 1.1 π-Conjugated polymers . 1.2 Sensors based on quenching . 1.3 Sensors based on Förster resonance energy transfer . 12 1.4 Sensors based on conformational change . 18 1.5 Heparin and assays for heparin . 22 1.6 Water-soluble nonionic conjugated polymers 26 1.7 Summary of CP-based optical sensors 29 1.8 Thesis outline 31 CHAPTER CATIONIC CONJUGATED POLYMER/HEPARIN INTERPOLYELECTROLYTE COMPLEX FOR HEPARIN QUANTIFICATION29 2.1 Introduction . 32 2.2 Experimental part 34 2.2.1 Materials 34 2.2.2 Instruments . 35 2.2.3 Synthesis 35 2.3 Results and discussion 36 2.3.1 Synthesis and characterization . 36 2.3.2 Effect of ionic strength and polymer concentration on PFBT-20% fluorescence . 38 2.3.3 Heparin titration . 39 ii Table of contents 2.3.4 Polysaccharide titration 41 2.3.5 Heparin quantification . 41 2.4 Conclusion 43 CHAPTER A CONJUGATED OLIGOELECTROLYTE/GRAPHENE OXIDE INTEGRATED ASSAY FOR LIGHT-UP VISUAL DETECTION OF HEPARIN45 3.1 Introduction . 45 3.2 Experimental part 47 3.2.1 Materials 47 3.2.2 Instruments . 47 3.2.3 Synthesis 47 3.2.4 Detection procedures . 50 3.3 Results and discussion 51 3.3.1 Synthesis and characterization . 51 3.3.2 Fluorescence quenching study . 53 3.3.3 Heparin detection . 54 3.3.4 Heparin quantification . 58 3.4 Conclusion 59 CHAPTER NAKED-EYE DETECTION AND QUANTIFICATION OF HEPARIN IN SERUM WITH A CATIONIC POLYTHIOPHENE . 61 4.1 Introduction . 61 4.2 Experimental part 62 4.2.1 Materials 62 4.2.2 Instruments . 62 4.2.3 Detection procedures . 62 4.3 Results and discussion 64 4.3.1 Chemical structure of P4Me-3TOEIM 64 4.3.2 Optical properties of P4Me-3TOEIM 65 4.3.3 Detection mechanisms . 65 4.3.4 Polysaccharide detection in water at room temperature 66 4.3.5 Polysaccharide detection in methanol/water at room temperature 68 4.3.6 Thermochromic property of P4Me-3TOEIM 69 iii Table of contents 4.3.6.1 Temperature dependent UV-Vis . 69 4.3.6.2 Temperature dependent LLS . 70 4.3.6.3 Temperature dependent CD 71 4.3.7 Heparin detection in fetal bovine serum medium 73 4.3.8 Heparin quantification . 75 4.4 Conclusion 77 CHAPTER TWO END FUNCTIONALIZED WATER-SOLUBLE NONIONIC CONJUGATED POLYMERS . 79 5.1 Introduction . 80 5.2 Experimental part 81 5.2.1 Materials 81 5.2.2 Instruments . 82 5.2.3 Synthesis 82 5.2.4 P1-B, streptavidin agarose resin binding . 91 5.2.5 P2-B, streptavidin agarose resin binding . 91 5.2.6 FRET experiment . 91 5.3 Results and discussion 92 5.3.1 Synthesis and characterization . 92 5.3.2 Optical properties . 96 5.3.3 Effect of surfactants on polymer optical properties . 97 5.3.4 Effect of ionic strength and nonspecific interactions on polymer fluorescence . 99 5.3.5 Biotinylated polymer streptavidin binding on surface . 101 5.3.6 Biotinylated polymer streptavidin binding in solution 103 5.4 Conclusion 105 CHAPTER CONCLUSION AND RECOMMENDATION . 107 REFERENCES 114 LIST OF PUBLICATIONS . 120 iv Summary SUMMARY The demand for the detection of chemical and biological substances in fields including clinical diagnosis, environmental monitoring, forensic analysis and antiterrorism promotes the fast growing of powerful analytical technologies. In this regard, water-soluble conjugated polymers (CPs) with electron delocalized backbones and highly polar side chains have emerged as a versatile building block for the construction of biosensors. Despite the fact that various CP based sensors have already been successfully developed, continuous effects are still needed to further extend CP applications to those lacking novel sensing methods and to develop new CP materials with improved performances. In this thesis, three CP type fluorescent/colorimetric sensors based on different mechanisms (Förster resonance energy transfer, quenching and conformational change) are developed for the detection of heparin. Heparin is a drug commonly used in surgery and long term care to prevent blood coagulation. Close monitoring of heparin levels is of great importance to avoid possible serious complications induced by heparin overdose. In general, these CP-based sensors have common advantageous features such as simple formulation, quick response, high selectivity, feasible for visual detection and reasonable quantification ranges, and they may find applications in research work requiring quick detection and quantification of purified heparin samples or heparin in biological media. Current CP based sensors mostly use conjugated polyelectrolytes (CPEs) as sensory materials. Nonspecific interactions between CPEs and interfering substances are inevitable and may adversely affect the sensors’ selectivity. In this thesis, two functionalized water-soluble nonionic polymers (NCPs) are developed. The special monomer units render these NCPs with desirable features including soluble in water, optically stable under high ionic strength and minimal nonspecific interactions with interfering proteins. In addition, through conjugation between functional groups and biotin molecules, these NCPs are endowed with streptavidin recognition capability. These v Summary functionalized NCPs may serve as templates for the development of new NCP probes by incorporation of the same monomer units and conjugation with other biorecognition elements. vi List of tables LIST OF TABLES Table 1.1 A brief summary of CP-based optical sensors. Table 4.1 Comparison of fluorescent/colorimetric detection/quantification. assays for Hep vii Chapter B 480 Intensity (a.u.) 400 Cy5-SA, @ 649 nm P2-B, 0.5 @ 434 nm P2-NH2, 0.5  320 240 160 80 500 550 600 650 700 750 800 Wavelength (nm) Figure 5.5 (A) PL spectra of P1-B and P1-NH2 solutions after incubation with Cy5-SA in 1× PBS, pH = 7.4. After dilution with × PBS, pH = 7.4, P1-B concentrations are 0, 0.50, 1.0, 1.5 and 2.0 µM, P1-NH2 concentration is 2.0 µM, and [Cy5-SA] = 3.3 × 10-8 M. (B) PL spectra of P2-B and P2-NH2 solutions after incubation with Cy5-SA in 1× PBS, pH = 7.4. After dilution with × PBS, pH = 7.4, P2-B concentration is and 0.50 µM, P1-NH2 concentration is 0.50 µM, and [Cy5-SA] = 3.3 × 10-8 M. Excitation wavelengths for P1 type and P2 type are 438 and 434 nm, respectively. 5.4 Conclusion We have successfully synthesized two neutral end functionalized conjugated polymers. P2 with a thick OEG shell has excellent water solubility and high quantum yield (0.45). In addition, the introduction of OEG side chains also renders both polymers with good optical stability in high slat solutions (up to M) and minimal nonspecific interactions with biomolecules. Biotinylated polymers show reasonable binding abilities in solid surfaces, and can function as energy donors to detect the biotin-streptavidin recognition event in homogenous solutions. These desirable optical properties (high quantum yield, optically stable) as well as specific surface and solution recognition capabilities make the materials attractive in biological applications. In terms of material design, OEG chains were introduced to the fluorene unit to achieve the water solubility of the final polymers. The monomer units will enable the further development of varieties of polyfluorene derivatives with similar solubility features via 105 Chapter various polymerization protocols such as Suzuki, Heck or Yamamoto coupling reactions. Besides the ligand biotin, other receptors such as antigens, nucleic acids, or enzyme substrates can be conjugated to the CP chains for the detection of antibodies, nucleic acid complementary sequences and enzymes through specific host-receptor interactions. 106 Chapter CHAPTER CONCLUSION AND RECOMMENDATION One objective of this research study was to widen the applications of CPs and to develop novel CP based heparin sensors. Firstly, a cationic conjugated polymer containing 20 mol-% BT content was synthesized through a post-polymerization method. With a good water solubility (20 mg·mL-1), this polymer showed a low inherent BT emission in aqueous solution. Formation of interpolyelectrolyte complexes between the oppositely charged polymer and heparin facilitated energy transfer from fluorene segments to the BT unit within the polymer, leading to the intensity increase for the orange emission and the intensity decrease for the blue emission. By correlating the changes in BT emission with the heparin concentration, we obtained a practical calibration curve ranging from 0.02 to 8.0 U/mL. The significant advantage of this heparin macromolecular probe was that the calibration curve could cover the whole range of the therapeutic dosing level of heparin. Secondly, a water-soluble pyrene-based conjugated oligoelectrolyte (TFP) was synthesized and was integrated with GO for heparin detection. Efficient fluorescence quenching occurred between TFP and GO due to the strong electrostatic and π-π interactions, leading to nearly dark emission in the absence of analytes. Addition of heparin into TFP solution significantly minimized the fluorescence quenching of GO towards TFP, which was less effective for the heparin analogues such as HA and ChS. Light-up visual discrimination of heparin from its analogues was realized as the solution emitted strong yellow fluorescence only in the presence of heparin. A linear response of the TFP/GO integrated assay enabled heparin quantification in the range of to 1.76 U/mL, which was practical for heparin monitoring during post-operative and long term care. 107 Chapter We further demonstrated a strategy for the real time naked-eye detection and quantification of heparin in the biological medium by monitoring the absorbance change of a water-soluble cationic polythiophene. Electrostatic interactions between polymer and heparin led to polymer conformation and color change from yellow to orange in solution. Under optimized conditions, addition of heparin derivative HA or ChS to the same polymer solution led to less change in polymer conformation and solution color due to their lower charge density as compared to that of heparin. Increasing the detection temperature or adding some organic solvent to the aqueous media reduced the polymer-polymer interchain π stacking, and the polymer color change can be used to clearly differentiate heparin from its analogue in homogeneous solutions. By correlating the change in polymer absorbance to the heparin concentration, we obtained a linear calibration curve in the range of to 6.7 U/mL and to 2.2 U/mL in pure water and in FBS, respectively. Although we successfully demonstrated three CP based sensors for heparin detection and quantification, this study still have some limitations. (i) The crude heparin or even USP heparin usually contains a mixture of polysaccharides introduced from the raw materials or other possible semi-synthesized or synthesized economically motivated additives. Multiple orthogonal analytical techniques are required to identify the contaminants. In fact, few of the current colorimetric/fluorometric assays can clearly differentiate heparin from other contaminants, and more importantly, few of the current assays can differentiate heparin and contaminants from their mixtures or real samples. In this study, although we successfully demonstrate that these CPE-based assays show different responses towards different analytes studied, similar to previous studies, we no show that these assays can differentiate between mixtures of negatively charged polymers. In other words, these assays only show some potential applications. 108 Chapter (ii) Among the possible contaminants, the one of particular interest is oversulfated chondroitin sulfate A. OSCS is synthesized by chemical sulfonation of chondroitin sulfate A, an inexpensive substance for the treatment of osteoarthritis. Administration of heparin contaminated by OSCS led to nearly 100 deaths in USA.194 Therefore, the companies are required to provide the information about the amount of OCSC in heparin. In this study, we only chose HA and ChS, two polysaccharides with charge densities lower than that of heparin as analytes. We not study the CPE responses towards OCSC, a compound with negative charge density even higher than that of heparin. (iii) Blood samples containing heparin are detected clinically, however, only serum is used here as the biological media. Protamine is used to neutralize excess amount of heparin, and appropriate amount of protamine should be administrated because protamine its own is a toxic drug and is related to adverse responses.195 Fluorimetric and colorimetric responses after titration with protamine can also be studied. (iv) PFBT type polymer aggregates under high salt concentration and under high polymer concentration, which ultimately affect the performance of the assay. In addition, the interactions between CPEs and heparin are mainly nonspecific electrostatic interactions, interfering molecules in the sample may lower the sensitivity and selectivity of the assay. Based on the literature review of recent assays, results and discussion present and the conclusion drawn from this research work, we propose some possible modification methods to overcome above limitations. (i) In Chapter 4, we demonstrate that polythiophene show distinctive responses towards three analytes. Polysaccharides and additives exist in the heparin sample differ in chain length, backbone composition, charge density, charge arrangement, conformation and so on, and may lead to the different conformation and aggregation modes of polythiophene, which are 109 Chapter further reflected by the polythiophene optical properties. Polythiophene derivatives differ in ionic groups (cationic, anionic or zwitterionic), side chain (chain length, linear or branched chains, OEG chain), and backbone length (oligothiophene, polythiophene) can be developed and are expected to interact with analytes in different patterns. An array of these polythiophene derivatives can be formed for the detection of heparin samples in a microplate format. The data can be processed using techniques such as linear discriminant analysis (LDA) to classify and quantify the possible contaminants.196 It should be noted that this method cannot give chemical or structural information about the contaminant as NMR does, therefore, it may possibly fail if new types of economically motivated additives are introduced to heparin sample. (ii) Due to the high charge density nature of OCSC, discrimination between OCSC and heparin is expected to be difficult using currently developed CP based sensors. Heparinase, an enzyme which cleaves heparin into small fragments can be used to treat the sample prior the detection process. Short fragments produced are expected to trigger a sensor response different from that in the presence of heparin. (iii) Solubility of PFBT was obtained because oligo(ethylene glycol) side chain and ammonium groups show better affinity to water, and ammonium groups show electron repulsion force towards each other. Buffer ions screen the repulsion forces and lower the water-solubility of PFBT and lead to the aggregation of the polymer. Solubility of PFBT needs to be further improved by using longer oligo(ethylene glycol) chains or attaching side chains with higher charge densities. Nevertheless, close contact of segments or aggregation of PFBT after addition of heparin need to be guaranteed. Due to the intrinsic hydrophobic and charge nature of CPEs and interfering molecules, nonspecific interactions are unavoidable. Therefore, nonionic conjugated polymers based assays are preferred. Recently, our group197 developed a biocomponent FRET probe for the detection of Con A. The probe is composed a mannose-substituted cationic polyfluorene 110 Chapter (donor) and a mannose-substituted neutral conjugated oligomer (acceptor). Due to the nonionic nature of side chains and short hydrophobic backbone, the oligomer has only weak interactions with nonspecific molecules. FRET between polyfluorene and oligomer selectively occurs in the presence of Con A as the mannose-Con A specific interaction brings the two into close proximity. This biocomponent probe can also be applied for the selective detection of heparin by replacing mannose groups with boronic acid groups,106-108 which specifically interact with heparin even in serum. In addition, the fluorene unit carrying charged sides in the cationic polyfluorene can be replaced by the water-soluble nonionic fluorene unit to further reduce possible nonspecific interactions. Despite various limitations of aPTT method, it still continuous to be the widely used assay for heparin level in clinical practice due to its readily available and familiarities. More importantly, this method relies on the detection of heparin functional activities. The actual concentration of heparin determined by the chemical method may not be clinically relevant, because the percent of heparin active domains or pentasaccharide domains that bind with antithrombin may vary between different batches. Chemical method can therefore function as a complementary method to aPTT. Another objective of this research study was to develop new functionalized water-soluble nonionic conjugated polymers. Two fluorene monomers carrying two or four OEG side chains were synthesized. The nonionic PFVPs end capped with protected amine groups were then obtained through Heck coupling between monomers and 1,4-divinylbenzene followed by end capping step. Polymer with low OEG density (P1) was not readily soluble in water and had a quantum yield of 0.24, while polymer with higher OEG density (P2) showed excellent water solubility and had a high quantum yield (0.45). The emission intensity of P1 was greatly enhanced and was nearly 111 Chapter equal to that of P2 with the assistance of anionic surfactant SDS. Both polymers were optically stable under high salt concentrations and had minimal nonspecific interactions with biomolecules. Biotin was incorporated into polymer chain ends through conventional NHS/NH2 reaction. We showed that both biotinylated polymers can interact with streptavidin on solid surface and in solution in a highly specific manner. Although we successfully developed two functionalized water-soluble nonionic polymers and demonstrated their biorecognition abilities, this work still has some limitations. The following listed the limitations and some recommendations. (i) Purification of biotinylated polymers are needed to collect those polymers with one or two ends functionalized with biotin molecules. Collection of biotinylated molecules from the streptavidin resins are commonly realized using guanidine/HCl, pH = 1.5 or through boiling the beads in SDS-PAGE buffer.198 These two methods are not suitable for CPs because acid aqueous solutions may damage CPs199 and SDS can bind with CPs through hydrophobic interactions. Therefore, monomeric avidin resins with much gentle purification conditions (2mM biotin in PBS) are recommended. 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Y.; Liu, B., Surfactant Effect on Energy Transfer between Cationic Conjugated Polymer and Dye-Labeled Oligonucleotide. Macromolecular Symposia 2009, 279, 48-51. 11. Shi, J. B.; Pu, K. Y.; Zhan, R. Y.; Liu, B., Cationic Conjugated Polymer/Heparin Interpolyelectrolyte Complexes for Heparin Quantification. Macromolecular Chemistry and Physics 2009, 210 (15), 1195-1200. 120 [...]... oligo(ethylene glycol) PA polyacetylene PAE poly(arylene ethynylene) PANI polyaniline PAV poly(arylene vinylene) PBS phosphate buffered saline PF polyfluorene xv List of abbreviations PFVP poly(fluorene-divinylene-phenylene) pI isoelectric point PL photoluminescence PLC phospholipase c pNA p- nitroanilide PNA peptide nucleic acid PPP poly(para-phenylene) PPy polypyrrole PT polythiophene QTL quencher-tether-ligand... shows the backbone structures of some representative CPs, including polyacetylenes (PAs), poly(arylene vinylene)s (PAVs), poly(para-phenylene)s (PPPs), polyfluorenes (PFs), poly(arylene ethynylene)s (PAEs), polyanilines (PANIs), polypyrroles (PPys) and polythiophenes (PTs) 3 Chapter 1 Scheme 1.1 Chemical structures of some common CPs Traditional applications of CPs range from early anti-static coating,... engineering, as the performance of optical biosensors is highly relied on the properties of sensory materials In particular, polymeric functional materials such as conjugated polymers (CPs) have emerged as a versatile building block for the construction of optical biosensors.6-21 Water- solubility is a prerequisite for CPs in biological applications, conjugated polyelectrolytes (CPEs) with water- soluble ionic... absorption (black) and PL spectra (red) of TFP in 10 mM PBS at pH = 7.4, λex = 380 nm Figure 3.2 (A) PL spectra of TFP and TFP/GO in 10 mM PBS at pH = 7.4 [TFP] = 1 µM, [GO] = 3.5 μg/mL, λex = 380 nm (B) Stern-Volmer plot of TFP quenched by GO [TFP] = 1 µM, [GO] = 0-0.62 μg/mL, λex = 380 nm Figure 3.3 (A) PL spectra of TFP, TFP/Hep, TFP/ChS and TFP/HA in 10 mM PBS at pH = 7.4 [TFP] = 1 μM, [Hep] =... 1 of peptide bonds Fluorescence turn-on and turn-off approaches were proposed for peptidase and papain activity detection, respectively In the turn-on approach, quencher p- nitroanilide (pNA) labeled lysine (K-pNA, Scheme 1.5A) was used as the substrate Columbic interactions brought K-pNA and PPE-SO3- into close proximity and the polymer fluorescence was significantly quenched Introduction of peptidase... Introduction of PLC into the 10CPC/BpPPE-SO3- complex induced the catalytic hydrolysis of 10CPC, and the originally amphiphilic 10CPC broke into two parts: zwitterionic head group phosphorylcholine (Scheme 1.6A) with a net negative charge and the neutral hydrophobic tail DAG (Scheme 1.6A) Neither of these two products could disrupt polymer- polymer 11 Chapter 1 interactions and polymer aggregates were formed... illustration of PPE based “turn on” and “turn off” assays for protease activity study.46 Schanze and co-workers49 developed a real time, fluorescence turn-off assay for phospholipase C (PLC) activity detection, based on the aggregation induced quenching and lipid induced dequenching phenomena Polymer BpPPE-SO3- (Scheme 1.2) was used in this assay BpPPE-SO3- emitted weak fluorescence and its emission appeared... reported NCPs and their applications Although highly polar groups were introduced, their solubility and quantum yield may still be not satisfying In addition, only few types of NCPs have ever been developed and few have bioapplications Therefore, another main objective of this Ph D project is to develop new NCP materials with desirable features Through this Ph D project, we anticipate (i) developing... (CCPs), anionic CPs (ACPs), zwitterionic CPs (ZCPs), and nonionic CPs (NCPs) The first three types (CCPs, ACPs and ZCPs), being characterized as CPs with water- soluble ionic side chains, are also named conjugated polyelectrolytes (CPEs, Scheme 1.2) The charged CPE side chains and the hydrophobic CPE aromatic backbones enable CPEs to interact with other molecules through electrostatic and hydrophobic interactions... Rho-Arg (B) Schematic illustration of PPE based “turn on” and “turn off” assays for protease activity study Scheme 1.6 (A) Scheme for the hydrolysis of substrate 10CPC by PLC into DAG and phosphorylcholine (B) Mechanism of PLC turn off assay Scheme 1.7 Schematic representation for the use of cationic water- soluble CP with specific PNA-C* optical reporter probe to detect complementary ss-DNA sequence Scheme . WATER- SOLUBLE π -CONJUGATED POLYMER FOR BIOSENSOR APPLICATIONS ZHAN RUO YU NATIONAL UNIVERSITY OF SINGAPORE 2012 WATER- SOLUBLE π -CONJUGATED POLYMER FOR. phosphate buffered saline PF polyfluorene List of abbreviations xvi PFVP poly(fluorene-divinylene-phenylene) pI isoelectric point PL photoluminescence PLC phospholipase c pNA p- nitroanilide. p- nitroanilide PNA peptide nucleic acid PPP poly(para-phenylene) PPy polypyrrole PT polythiophene QTL quencher-tether-ligand Rho-Arg 2 bis-arginine derivative of Rhodamine-110 RU repeat unit