Amperometric Immunosensor And Hydrogen Peroxide Biosensor Based On Nanocomposites

With the increasing apply in food, medicine and environment areas, biosensor has been a subject area which has extremely potential in the future. Because of these factors, bisensors were required higher characteristics. The prospects for biosensor applications mainly in the following four areas:miniaturization, functional diversification, market-oriented, intelligent and integrated. However, the characteristics of the biosensor's sensitivity, detection limit and stability restrict the development of biosensors. In this thesis, the electrochemical biosensor based on nanocomposites was constructed to improve these characteristics.The main works and conclusions are included as follows:1. An immunosensor based on layer-by-layer assembly L-cysteine and nanosized prussian blue for determination of human chorionic gonadotrophin:A novel amperometric immunosensor based on L-cysteine/nanosized prussian blue bilayer films ({NPB/L-cys}₂) and gold nanoparticles (nano-Au) was fabricated for determination of human chorionic gonadotrophin (HCG). First, L-cys and NPB was self-assembed by layer-by-layer (LBL) technology to form {NPB/L-cys}₂ bilayer films on the gold electrode. Subsequently, nano-Au layer was immobilized on the {NPB/L-cys}₂ bilayer films by electrotodepositing gold chloride tetrahydrate and then anti-HCG was assembly on the nano-Au layer. Finally hemoglobin (Hb) was employed to block sites against nonspecific binding. In this paper, the {NPB/L-cys}₂ acted as redox in the immunoreaction, and the Hb instead of bovine serum albumin was used to block the sites against nonspecific binding. Because of the electrocatalytic ability of Hb and NPB for the reduction of H₂O₂, the current signal of the antigen-antibody reaction was amplified and the enhanced sensitivity was achieved. In this study, the assembly process and performance of the immunosensor were characterized by cyclic voltammetry (CV) and the morphology was researched by scanning electron microscopy (SEM). The immunosensor performed a high sensitivity and a wide linear response to HCG in two ranges from 0.5 to 10.0 mIU/mL and from 10.0 to 200.0 mIU/mL with a relatively low detection limit of 0.2 mIU/mL at 3 times the background noise, as well as good stability and long-term life.2. A novel immunosensor based on poly-(2,6-pyridinediamine)/multiwall carbon nanotubes composite for immunoassay of human chorionic gonadotrophin:A novel method based on gold nanoparticles (GNPs) and poly-(2,6-pyridinediamine) /multiwalled carbon nanotubes composite (pPA/MWNTs) was exploited to develop a highly sensitive amperometric immunosensor for detection of human chorionic gonadotrophin (HCG). In this method, pPA/MWNTs composite was prepared by electro-polymerization of PA onto MWNTs modified electrode, and GNPs were used as a linker to connect pPA/MWNTs composite with HCG antibody. Because the MWNTs modified electrode had high surface area, the amount of the pPA was increased. In addition, MWNTs had nanometer dimensions pores and good conductivity, the redox property of the pPA was improved. Several techniques, including cyclic voltammetry (CV) and scanning electron microscopy (SEM), were employed to characterize the assembly process and the performance of the immunosensor. Because of the synergistic augmentation of pPA and MWNTs to facilitate electron transfer, and the unique properties of GNPs, the obtained immunosensor exhibited a wide linear response to HCG in two range from 1.0 to 10.0 and 10.0 to 160.0 mIU/mL with a relatively low detection limit of 0.3 mIU/mL (S/N=3), as well as good stability and repeatability.3. Using flowerlike polymer-copper nanostructure composite to construct an amperometric biosensor for hydrogen peroxide:A new type of amperometric hydrogen peroxide biosensor was fabricated by entrapping horseradish peroxidase (HRP) into the organic-inorganic hybrid material composing of zirconia-chitosan sol-gel and Au nanoparticles (ZrO₂-CS-AuNPs). The sensitivity of the biosensor was inhanced by the fowerlike polymer-copper nanostructure composite (pPA-FCu) which was prepared from co-electrodeposition of the CuSO₄ solution and 2,6-pyridinediamine solution. Several techniques, including UV-vis absorption spectroscopy, scanning electron microscopy, cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy were employed to characterize the assembly process and performance of the biosensor. The results showed that this pPA-FCu nanostructure not only had the excellent good redox electrochemical activity, but also had the well catalytic efficiency to hydrogen peroxide. And the ZrO₂-CS-AuNPs had good film forming ability, high stability and well biocompatible to retain the bioactivity of immobilized enzyme. The resulting biosensors showed a linear range from 7.8×10^-7 to 3.7×10^-3 mol/L, with a detection limit of 3.2×10^-7 mol/L (S/N=3) under the optimized experimental conditions. The apparent Michaelis-Menten constant was determined to be 0.32 mmol/L, showing good affinity. In addition, the biosensor which exhibited good analytical performance, acceptable stability and well selectivity, would had a potential application in practical samples.