New Approaches For Biosensors’ Construction Based On Nanocomposites And Protein Application In Glucose And Toxin Analysis

Electrochemical analysis is an instrumental analysis methods based on the electrochemical properties of the material and its variation. It is widely used in food analysis, medical and environmental testing because of the advantages such as easy miniaturization, simplicity, high sensitivity and selectivity, flexibility and convenience. Nanomaterial has many advantages such as richly active sites on the surface, good biocompatibility, high specific surface area, high activity, catalytic efficiency and strong adsorption ability. In the field of electrochemical biosensors, nanomaterial is an ideal material for electrode modifidification and biomolecules immobilization, which can significantly improve the detection performance of the fabricated biosensors. Taking advantages of electrochemical analysis and unique properties of nanomaterial, protein electrochemical biosensor, for example, glucose oxidase (GOx) biosensors and deoxynivalenol (DON) antigen-antibody immunosensor with excellent performance, was built based on nano-materials modified glassy carbon electrode (GCE) for rapid sensing of glucose and DON.This paper mainly includes three parts as following:1. Preparation and characterization of nanocompositesA nanocomposite was prepared that consists of chitosan, reduced graphene oxide and gold nanoparticles by in-situ and simultaneous reduction of graphene oxide and hexachloroaurate whereby chitosan acts as a reducing and stabilizing agent.The structure and surface morphology of rGO-AuNPs were characterized by fourier transform infrared spectroscopy (FT-IR), UV-vis absorption spectra and transmission electron microscopy (FETEM). The results show that AuNPs was successfully decorated on reduced graphene oxide nanosheets. The prepared nanocomposite was also characterized by using electrochemical method. Compared to the bare glassy carbon electrode (GCE) and rGO or AuNPs modified electrode, rGO-AuNPs modified GCE has better conductivity than bare GCE and AuNPs modified GCE.Titanium phosphate nanospheres (TiP) with excellent ion-exchange properties was prepared by using the sodium dodecyl sulfate as a structure-directing agent. The as-prepared TiP nanoparticles offered promising templates for efficient incorporation of Ag+, which was then reduced by sodium borohydride forming AgNPs@TiP nanocomposites ultimately. The structure and surface morphology of AgNPs@TiP were characterized by UV-vis absorption spectra, X-ray photoelectron spectroscopy (XPS), field emissionscanning electron microscopy (FESEM) and X-ray spectrometer. The results show that AgNPs was successfully decorated on TiP surface. The average diameter of AgNPs@TiP was estimated to be 55 nm. The as-prepared AgNPs@TiP can be further used as labels for electrochemical detection of proteins due to their excellent ion-exchange property and abundant in active site.2. GOx-rGO-AuNPs modified electrode for rapid detection of glucose by amperometricAn amperometric GOx biosensor based on rGO-AuNPs has been developed for the glucose detection in beverages. The immobilized GOx displays fast electron transfer with a transfer rate constant of 2.80 s-1. Parameter optimization for biosensor performance such as enzyme concentration and pH value was investigated by cyclic voltammetry and chronoamperometry. The sensor gives a linear response to glucose in the 0.01 to 0.88 mM concentration range with a sensitivity of 22.54 μA mM-1 cm-2, and a 0.43μM detection limit. The apparent Michaelis-Menten constant is 0.497 mM. The constructed biosensor was successfully applied for glucose detection in beverages which was compared with glucose kit determination.3. AgNPs@TiP-based DON immunosensor for rapid detection of DONAn electrochemical competitive immunoassay for sensitive detection of DON was developed by using the as-prepared as signal amplification label. The immunosensing platform was developed based on AgNPs@TiP using competitive binding between conjugated antigen-AgNPs@TiP ((AgNPs@TiP-DONag)) and free antigen (DONag) with immobilized DON antibody on chitosan and electrochemically reduced graphene oxide film modified GCE. The differential pulse voltammetric signal produced by AgNPs in KC1 by performing was used to monitor the immunoreaction. Under the optimum conditions, the proposed method can detect DON antigen anging from 0.1 ng mL-1 to 1000 ng mL-1 with a detection limit of 0.059 ng mL-1 (signal to noise ratio S/N = 3). Moreover, the immunosensor exhibite excellent stability and acceptable reproducibility, attribute to excellent conductivity of rGO and good biocompatibility of chitosan.