Study On Enzyme Detection And Logic Gate Construction Based On Novel Water - Soluble Conjugated Polymers

Water-soluble conjugated polymers (WSCPs) with unique structure have attracted much attention because of their good light-harvesting ability and signal amplification effect, which exhibits the broad applications in biological sensing, cell imaging, drug screening and delivery, disease diagnosis and treatment. In this thesis, the neutral poly(p-phenylenes) functionalized with oligo(oxyethylene) side chains (PPP-OR10) and cationic polyfluorene PFPB have been designed and synthesized. Based on these conjugated polymers, the new biosensing mechanism and analytical methods have been studied, and their applications in pH response, enzyme activity detection and reversible logic gates have been investigated, respectively. The results are as follows:1. Strategy for sensor based on fluorescence emission red shift of conjugated polymers:applications in pH response and enzymeA new strategy was developed and applied in monitoring pH response and enzyme activity based on fluorescence emission red shift (FERS) of the conjugated polymer PPP-OR10induced by the inner filter effect (IFE) of nitrobenzene derivatives. Neutral poly(p-phenylenes) functionalized with oligo(oxyethylene) side chains (PPP-OR10) was designed and synthesized by the Suzuki cross-coupling reaction. Nitrobenzene derivatives display different light absorption activities in the acidic or basic form due to adopting different electron-transition types. When environmental pH is higher than their pKa values, nitrobenzene derivatives exhibit strong absorbance around400nm, which is close to the maximal emission of polymer PPP-OR10. As a result, the maximal emission wavelength of PPP-OR10/nitrobenzene derivatives red shifts with the pH value increasing. Apparently, the IFE plays a very important role in this case. A new method has been designed that takes advantage of this pH-sensitive platform to sensor a-chymotrypsin (ChT) based on the IFE of p-nitroaniline, since the absorption spectrum of p-nitroaniline, the ChT-hydrolyzed product of N-benzoyl-L-tyrosine-p-nitroaniline (BTNA), overlaps with the emission spectrum of PPP-OR10. In addition, the present approach can detect a-chymotrypsin with a detection limit of0.1μM, which is lower than that of the corresponding absorption spectroscopy method. Furthermore, the pH response and enzyme detections can be carried out in10%serum, which makes this new FERS-based strategy promising in applications in more complex conditions and a broader field.2. Reversible logic gate modulated by nucleases based on cationic conjugated polymer/DNA assemblyReversible logic gates were designed and prepared by modulating the logic inputs based on the original system combining new cationic polyfluorene PFPB synthesized by Suzuki coupling reaction with labelled dsDNA. dsDNA was labeled with fluorescein (FAM) and TAMRA on each end, respectively. Once exciting PFPB, the two-step fluorescence resonance energy transfer (FRET) occurs from PFPB to FAM then from FAM to TAMRA. After addition of restriction endonucleases, the dsDNA was cleaved into small ssDNA with dyes, which leads to FRET being broken. In this system, addition of T4ligase can reset the PFPB/dsDNA self-assembly and then FRET recovers. Therefore, the NOT, YES, and NOR logic gates were built with T4ligase and restriction endonucleases such as HaeⅢ and PvuⅡ as the inputs and the emission intensity of dyes (FAM or TAMRA) as the output. Most importantly, the logic gates YES and NOT can switch back and forth by the addition of HaeⅢ and T4ligase. By a similar method, the logic gates NOR and YES also realized this recycling. This study is a promising starting point for constructing reusable systems for biosensors in the multiplex detection of nucleases.