Preparation Of Fluorescent Probes Based On Water-Soluble Polythiophene Derivatives And Their Application In Biological Analysis

The use of conjugated polyelectrolytes?CPE?as optical platforms in biosensors has attained significant interest because their good biocompatibility and fluorescence signal amplification property by a collective optical response.Adenosine triphosphate?ATP?and heparin are two important biological molecules,and abnormal levels of them often directly associated with certain human diseases.Thus,the development of the probe materials for the purpose of the detection of these biologically-relevant molecules is required,which will promote the the research of the life process,and possess potential applications in wide fields such as clinical diagnosis,drug analysis and the development of new drugs.Water-soluble polythiophene derivatives have been confirmed to be sensitive colorimetric and fluorescent probes for biosensors due to their unique chain conformations sensitive to external stimuli.In this dissertation,two polythiophene derivatives PT-1 and PT-2 were designed and synthesized,and their interactions with biomolecules substrates such as ATP and heparin had been studied in detail by various analytical methods.Further,the sensing mechanism was also discussed.The main contents and results are summarized as follows:?1?Two water-soluble polythiophene derivatives,PT-1 and PT-2,were prepared by traditional oxidative polymerization using FeCl₃ as oxidant.Their chemical structure and average molecular weights were determined by nuclear magnetic resonance spectroscopy?NMR?,elemental analysis?EA?and gel permeation chromatography?GPC?.?2?The sensing properties of PT-1 for ATP was investigated by UV-vis and florescent spectra.The results indicated that the sensing mechanism can be attributed to the analyte-induced aggregating/disaggregating of polymer backbones,under the synergetic effects of electrostatic,hydrophobic and aromatic stacking interactions.Upon adding ATP into the tris-HCl buffer solution?10 mM,pH = 7.4?of PT-1,UV-vis absorption spectra of PT-1becomes red shift accompanied by the solution color changed from yellow to dark purple,and its fluorescence is almost completely quenched with quenching constant of 4.4 × 10⁶ M^-1 and the detection limit is about 9.5 × 10_-7M.Job's-plot experiment shows the stoichiometric ratio between PT-1 and ATP is 1: 1 and the the binding constant is of 6050 M^-1.?3?The sensing properties of PT-2 for protamine and heparin was investigated by UV-vis and florescent spectra.The complex of PT-2 with protamine reduces the fluorescent intensity of PT-2,subsequently,the addition of heparin,which can specifically interact with protamine,restores the PT-2 fluorescence.Based on this displacement strategy,a fluorescent turn-on biosensor is developed for the detection of heparin.The results shows that the binding constant and quenching constant of PT-2 and protamine is about 4.5 × 10⁷ M^-1 and 1.65 × 10⁷M^-1,respectively.The changes of fluorescence intensity is linearly proportional to the concentration of heparin in the concentration ranges from 0-14 ?M.