Construction Of DNA Functional Nanomaterials Based On Rolling Circle Amplification Technology For Glucose Photo/electrochemical Sensing Analysis

DNA functional nanomaterials have been widely applied to the fields of biosensing and disease therapeutics due to their good biocompatibility,sequence programmability,controllable size and structure.Rolling circle amplification（RCA）as an isothermal enzymatic amplification reaction possesses several intrinsic advantages such as the facile operation and high amplification efficacy.In this work,a series of flower-like DNA functional nanomaterials have been successfully prepared via the RCA and applied to the construction of fluorescent,electrochemical and colorimetric biosensors,further achieving the ultrasensitive and selective detection of glucose,which provides new approaches and ideas for the development of DNA nanotechnology and glucose biosensors.This thesis contains the following three works:1.In situ synthesis of DNA flowers-encapsulated Agnanoclusters based on rolling circle amplification for fluorescent detection of glucoseIn this work,DNA flower-like nanostructure（DNA flowers,DFs）containing a large quantity of AgNCs-nucleation sequences has been successfully prepared via the rational design of RCA template.Processing good water-solubility and fluorescence stability,the fluorescent AgNCs with DFs as template（DFs/AgNCs）were prepared by in-situ synthesis method and systematically studied the morphology,composition and fluorescent properties.The property that H₂O₂ generating from the GOx-catalyzed glucose oxidation quenches the fluorescence of AgNCs makes DFs/AgNCs as fluorescent probe for the fast and sensitive detection of glucose.The linear range of this DFs/AgNCs-based fluorescence sensing platform for glucose detection was 50μM-1.0m M,and the detection limit was 20μM.Furthermore,this fluorescence sensing platform could also achieve the efficient and accurate analysis of human blood glucose,which was significantly meaningful to the analysis and monitoring of glucose in complex physiological enviroment.2.Electrochemical biosensor based on Ag⁺-induced conformational changes of DNA for the detection of glucoseHerein,a novel electrochemical biosensor based on the Ag⁺-induced conformational changes of DNA capture probes was constructed for the ultrasensitive and selective detection of glucose.The single-stranded DNA capture probes functized with electroactive ferrocene（Fc-DNA）were firstly immobilized on Au electrode via the Au-S bond and the resulting modified electrode was characterized with electrochemical methods.In the presence of GOx and glucose,DFs/AgNCs as the Ag⁺generators released Ag⁺,which could induce the linear capture probes to form hairpin structure via the cytosine（C）-Ag⁺-cytosine（C）coordination interaction.The Fc was brought to the surface of Au electrode resorting to the formation of hairpin structure in Fc-DNA capture probe,generating the electron transfer and enhancing the DPV current.Combining the high specificity of enzymatic reaction with C-Ag⁺-C coordination interaction,the sensitive and specific electrochemical biosensor for glucose detection could be accomplished with a limit of detection（LOD）of 0.26μM,whose linear range was from5 to 600μM.This work could provide the theoretical basis and application model for the application of AgNCs as well as the development of electrochemical biosensors.3.Construction of cascade catalysis system based on Cu²⁺-meidated RCA assembly for colorimetric detection of glucoseBased on the liquid crystallization of RCA product as well as the coordination interactions between Cu²⁺and DNA,a novel three-dimensional DNA nanostructure（GOx/Cu-DFs）with high yields and catalytic ability was successfully prepared in this work,achieving the co-encapsulation of GOx and Cu²⁺.Systematically studied the morphology,composition and catalytic activity,the GOx/Cu-DFs performs excellent GOx catalytic activity.Due to the addition of Cu²⁺in the self-assembly process,the self-assembly ability of RCA products could be efficiently enhanced,further improving the yields of nanomaterials.In addition,the attribute that Cu²⁺catalyzes Fenton-like reaction to produce hydroxyl radicals（·OH）could endow the nanomaterial with peroxidase-like activity.The cascade enzyme catalysis system has been successfully applied to the construction of colorimetric glucose biosensor,not only achieving the efficient and accurate detection of glucose with the detection limit of 0.45μM and the linear range over 2-400μM,but also allowing for the analysis and detection of glucose in serum,providing new approaches and ideas for the construction of glucose biosensors and development of DNA functional nanomaterials.