Development Of Water-Soluble Thiophene Conjugated Polymer-Based Optical Analytical Method And Application

Water-soluble conjugated polymers (WSCPs), a new class of polyelectrolytes consisting of both polyions and electronically active conjugated backbones, have been applied to biosensors due to their unique signal amplification effects and good biocompatibility. Hitherto, most WSCPs-based biosensors have been applied to biomacromolecules detection due to the non-specific interaction between biological molecules and WSCPs, such as electrostatic and hydrophobic interaction. The detection schemes of these sensors are mainly employing the efficient light harvesting properties or the conformation sensitive optical properties of the WSCPs. Hence, the presence of biomolecules or biological events can be detected through fluorescence resonance energy transfer (FRET) between the WSCPs and an acceptor molecule, or through their impact on the conformation of the conjugated backbone, which is seen as an alteration of the optical properties of the WSCPs. We have found another special characteristic of WSCPs, the catalytic properties, and then this catalytic propertiey of WSCPs has been applied to chemiluminescence analysis and photometric analysis system in order to achieve a higher sensitivity of detection. Specifically, this paper would focus on the application of polythiophene conjugated polymers with intrinsic peroxidase-like activity to establish a homogeneous and label-free detection of some biological important biomolecules (such as nucleic acid and protein). The main contents and results are summarized in the following five chapters:In Chapter1, on the basis of a brief introduction of WSCPs, the sensing mechanisms and the recent developments of WSCPs-based optical analysis were emphatically reviewed.In Chapter2, it was found that water-soluble conjugated polymer, poly [3-(3-N,N,N-triethylamino-1-propyloxy)-4-methyl-2,5-thiophenehydrochloride](PMNT)(a kind of cationic WSCPs) could initiate the strong chemiluminescence (CL) emission of luminol-H2O2in weak basic media. The catalytic mechanism of PMNT on luminol-H2O2CL was discussed based on CL spectra, ultraviolet visible light spectra, fluorescence spectra and electron spin resonance (ESR) spectra. Furthermore, it was found that the catalytic activity of PMNT on the luminol-H2O2CL system was closely relevant to the conformation of PMNT. The CL intensity of luminol-H2O2-PMNT system in the presence of single-stranded DNA was much larger than that in the presence of double-stranded DNA. By taking advantage of this phenomenon, a label-free and homogeneous CL detection of DNA hybridization is proposed. The detection limit of target DNA (3σ) was estimated to be as low as3.7×10-13M. The present CL method for DNA hybridization detection offers the advantages of being simple, cheap, rapid and sensitive.In Chapter3, in alkaline medium, bicarbonate reacts with hydrogen peroxide to form peroxymonocarbonate ion (HCO4-), which is unstable compound. During the decomposition of HCO4-, a weak CL was observed. In this work, we found that the CL emission intensity of NaHCO3-H2O2was significantly enhanced by anionic polythiophene conjugated polymer poly[2-(3-thienyl)ethyloxy-4-butylsulfonate](PTE-BS), and the enhanced effect was closely relevant to the conformation of PTE-BS. The catalytic mechanism of PTE-BS on NaHCO3-H2O2CL was discussed based on CL spectra, ultraviolet visible light spectra, fluorescence spectra and electron spin resonance spectra. Protamine with positive charge can bind with PTE-BS by electrostatic interactions, as a result of occurring red shift of the PTE-BS absorption, can inhibit this enhanced CL signal and the CL intensity is dependent of protamine concentration. Based on the phenomenon, a new, simple, sensitive and selectivity CL method is proposed for determination of protamine.In Chapter4, thiophene water-soluble conjugated polymers possesses intrinsic peroxidase-like activity, and can catalyze the reaction of peroxidase substrate3,3’,5,5’-tetramethylbenzidine (TMB) in the presence of H2O2to produce a blue color reaction, which provides colorimetric detection of H2O2. PTE-BS exhibits several advantages such as high catalytic efficiency, good stability, and rapid response over horseradish peroxidase (HRP). By coupling the oxidation of glucose catalyzed by glucose oxidase, a simple, inexpensive, highly sensitive and selective colorimetric method for glucose detection has been developed. The absorbance was proportional to the concentration of glucose in the range from0.01to0.5mM with a detection limit of0.004mM. This work is not only of importance for a better understanding of the unique properties of polythiophenes derivative but also of great potential for medical diagnostics and biotechnology.In Chapter5, in previous research work, we have found that the polythiophene derivative PMNT possesses intrinsic peroxidase-like activity that can catalyze the reaction of TMB in the presence of H2O2to produce a blue color reaction, and also the catalytic activity of cationic polymer PMNT was closely related with its conformation. In this work, we found the catalytic effect of PMNT on chromogenic reaction of TMB-H2O2can change with the alteration of its conformation induced by thrombin-binding aptamer (TBA) and TBA/thrombin complex, respectively. PMNT show the higher catalytical activity when combining with TBA forming the planarization conformation than when combining with TBA/thrombin complex forming the quadruplex conformation. Based on the different electrostatic interactions and conformational structures between a cationic polythiophene derivative PMNT and TBA, a sensitive and lable-free aptamer-based biosensing for thrombin was developed.