Fabrication Of Electrochemical Biosensor Based On Nanoparticle Signal Amplification And Its Application Of MicroRNA And Thrombin

Electrochemical methods and electrochemiluminescence methods are two popular analytical methods in recent years.Their high sensitivity,wide detection range,low background signal,and simple operation make them develop rapidly.In this thesis,different kinds of nanoparticles were prepared,biorecognition probes were prepared combined with DNAzyme signal amplification technology,surface enhancement methods,anti-fouling techniques,intramolecular self-enhancement methods.Three novel electrochemical biosensors were designed to realize the highly sensitive and highly selective detection of thrombin and microRNA.The content was mainly described as follows:?1?Electrochemical detection of microRNA was proposed by using the dual-amplification effect of the nanoparticles and DSN.Due to the characteristics of DSN enzyme cleaving strands of DNA,the experiment reacted only one step in a centrifuge tube,achieving sensitive detection of microRNA.By attaching DNA probes modified with CdS quantum dots on the magnetic beads,a double-stranded structure was formed by hybridizing with the target microRNA.When DSN enzyme was in the presence,it specifically recognized and cleaved DNA in the double strand,and the microRNA was released into the solution for the next hybridization and cleaving,achieving exponential amplification of the signal through multiple cycles.At the same time,the signal molecule CdS QDs could release a large amount of Cd²⁺after dissolution,which further amplified the response signal.Finally the Cd²⁺was detected by anodic stripping voltammetry.The obtained electrochemical signal had a linear relationship with the concentration of the target microRNA and the cell extracting solution.The detection range was between 0.001 to 10000 pM L^-1,with an ultralow detection limit of 0.48 fM L^-1.?2?An electrochemiluminescence aptasensor based on Ru?bpy?₃²⁺-TPA was constructed to detect thrombin.The surface of the electrode was modified with Au@SiO₂ as reaction substrate,and the capture probe was attached through the silanization of SiO₂.Thrombin aptamer and assistant DNA were partially hybridized on magnetic beads.When thrombin was present in the system,the assistant DNA was replaced and formed a rigid"T"structure with the captured DNA on the reaction substrate and the signal DNA modified with Ru-SiO₂,so that Ru-SiO₂ was closer to the surface of the electrode,and the ECL signal of Ru?bpy?₃²⁺was enhanced through the Au@SiO₂ to detect the concentration of substituted DNA,achieving the detection of thrombin in PBS and serum.The detection range was between 0.1 to 10000 pM L^-1,with an ultralow detection limit of 0.052 pM L^-1.?3?A self-enhanced luminescent reagent Ru-PAMAM was formed by amidation using PAMAM and Ru?dcbpy?₃²⁺.At the same time,3,4-ethylenedioxythiophene?PEDOT?/ethylene glycol?PEG?composites were polymerized on the electrode which coated with gold nanoparticles?AuNPs?.Through the specific binding of aptamer to thrombin,we took advantages of high conductivity,stability of PEDOT and good biocompatibility,anti-fouling of PEG to fabricate the surface anti-fouling thrombin ECL biosensor.The obtained ECL signal had a linear relationship with the concentration of the thrombin in PBS and the complex real samples such as serum.The detection range was between 0.1 to 10000 pM L^-1,with an ultralow detection limit of0.034 pM L^-1.