Study On The Novel Nano-modified Electrode Based Electrochemical Biosensors

Biosensor is a novel analytical technique and developed on the multidisplineintercross of life, chemistry, medicine and information sciences. By using thebioactive substance such as enzyme and antibody as the recognition unit of biosensor,it can transform the weak change caused by the biorecognition reaction intomeasurable physical or chemical signal, thus achieving the quantitative detection ofthe bio or chemical substances. Electrochemical biosensors based on variouselectroanalytical methods possess the advantages of high sensitivity, convenientoperation and fast analytical speed. Recently, various electrochemical biosensors haveshown great potentials in the analytical fields of medicine, food, environment, and soon.The key issue for the development of electrochemical biosensors is to immoblizehigh-content biomacromolecules on the electrode surface with good stability and highbioactivity. Nanomaterials possess the advantages such as large specific surface area,strong adsorption capacity, good biocompatibility and fast electron transfer rate. Whennanomaterials are used for the electrode modification, it can greatly enhance theimmobilization content, stability and bioactivity of the biomacromolecules, thusimproving the analytical performance of biosensors.By combination the nanomaterial-modified electrodes and the electrochemicalbiosensing technology, in this thesis three work has been carried out and listed asfollows:1A hydrogen peroxide biosensor based on the direct electrochemical behavior ofthe porous magnetic microsphere/peroxidase modified electrodeA novel porous-magnetic chitosan microsphere （PMMS） was prepared for themodification of carbon paste electrode （CPE）. Peroxidase （POD） was furtherimmobilized on the surface of PMMS modified electrode successfully through thecross-linking of glutaraldehyde. This modified electrode was characterized byelectrochemical impedance spectroscopy, cyclic voltammetry and steady-state amperometry. The effects of the experimental variables such as solution pH and scanrate were investigated. The diffusion coefficient of H2O2substrate and the apparentMichaelis-Menten constant of POD were estimated to be5.03×10^-10cm2·s^-1and0.069mmol·L^-1, respectively. Under the optimum conditions, this biosensor had a fastresponse towards hydrogen peroxide less than10s. A good linear relationship wasobtained in the concentration range from0.1mmol·L^-1to0.8mmol·L^-1with adetection limit of0.03mmol·L^-1（S/N=3）.2Label-free electrochemical immunosensing based on the graphene/polythionine/gold nanoparticles modified electrodeA label-free electrochemical immunosensor for the accurate determination of humanIgG was developed based on the polymerization of thionine and further assembly ofnanoparticles （AuNPs） at the PDDA-functionalized graphene modified electrode. Thestudy showed a layer of uniform, stable and high-content polythionine film could bewell formed on the surface of PDDA-functionalized graphene modified electrode. Bycombination with the direct immunoassay, differential pulse voltammetry （DPV） wasemployed to monitor the electrochemical signal decrease of polythionine which wascaused by the immuno-recognition of different concentrations of antigen at thedeveloped immunosensor, thus a novel label-free electrochemical immunoassaymethod was constructed. Under the optimum conditions, the DPV current decreaseshowed a good linear relationship with the logarithm of the concentration of humanIgG in the range from0.2to200ng·mL^-1. The limit of detection was estimated as0.07ng·mL^-1（S/N=3）.3In situ preparation of graphene-thionine nanocomposite and its application forhighly sensitive electrochemical immunosensingUsing thionine as reduction and protection agents for graphene oxide, a graphene-thionine nanocomposite with good electrochemical activity was prepared by aone-step, in-situ method for the modification of glassy carbon electrode by a one-step,in-situ method. Based on the assembly of AuNPs at the modified electrode and thesubsequent antibody immobilization, an immunosensor was thus constructed for sandwich-type immunoassay. On the one hand, the electrochemical signal response ofthionine was inhibited by the antigen-antibody immnocomplexes formed on theproposed immunosensor. On the other hand, the antibody functionalized silicananospheres captured by the sandwich immunoreaction was used to amplify the signalinhibition, thus resulting the great enhancement of the detection sensitivity and thedevelopment of a novel ultrasensitive electrochemical immunosensor method withlow cost and convenient operability. The linear concentration range of this methodwas from0.01to10ng·mL^-1, and its limit of detection was estimated as7pg·mL^-1...