| Electrochemical biosensors based on the specific recognition of biomaterials with the magnification function of electrochemical determination present some outstanding advantages including high sensitivity, nice selectivity, low cost and easy miniaturization. The key aspect of the development of the biosensors is the methodology of immobilization of biomolecules on the electrode surface. This dissertation focuses on developing new immobilization strategies of biomaterials for the purpose of improving the performance and long-term stability of biosensors. The major contents are as follows:1. Au nanoparticles modified with electroactive Prussian blue (PB) were for the first time synthesized by a simple chemical method. Transmission electronic microscopy showed that the average size of the Prussian blue shell/Au core hybrid composite (PB@Au) was about 50 nm, and Fourier transform IR, UV-Vis spectra and cyclic voltmmetry confirmed the existence of PB on the surface of Au nanoparticles. Using the LbL technique, multilayer thin films of PB@Au nanoparticles were prepared by the alternate adsorption of oppositely charged linear polyelectrolyte poly(allylamine hydrochloride) (PAH) onto ITO glass for construction of a hydrogen peroxide sensor. The novel multilayer films were characterized by SEM, cyclic voltammetry and UV-Visible absorption spectroscopy. The {PAH/PB@Au}n multilayer-modified electrode showed a well-defined pair of redox peaks and dramatic catalytic activity towards the reduction of hydrogen peroxide.2. Ordered 3D interconnected macroporous Prussian blue (PB) films were electrochemically fabricated by using colloidal crystals of polystyrene beads as sacrificial templates. The prepared PB film electrodes have excellent catalytic activity towards the reduction of hydrogen peroxide. The PB structure was further used as functional interface for fabricating an enzyme-based glucose sensor by using surface modification technique based on the electrostatic interactions. The resulted sensor has higher functional density, and larger surface area. The interconnected macroporous structure allows enhanced mass transport. These characteristics of the sensor enable us to detect glucose with high sensitivity. Therefore, the present 3D ordered macroporous film sensor exhibits wide linear detection range towards glucose, acceptable reproducibility and operational and storage stability. The present approach is promising for the generation of high-enzyme-content thin films with tailored bioactivity.3. A novel three dimensional porous chitosan membrane material was prepared as a matrix to encapsulate hepatitis B surface antibody (HBsAb) for fabrication of immunosensors. The porous chitosan matrix was prepared by electrodepositing a designer nanocomposites solution of chitosan encapsulated silica nanoparticles hybrid film on ITO electrode, and then removing silica nanoparticles with HF solution. Using HBsAb as a model, the potentiometric immunosensor was constructed by linking HBsAb molecules to the three dimensional porous chitosan film using glutaraldehyde as a cross-linker. Scanning electron microscopy was used to investigate the surface morphologie of the three dimensional porous chitosan films. Cyclic voltammograms and electrochemical impedance spectroscopy were used to probe the interfacial properties of the immunosensor. Results showed that the fabricated immunosensor with three dimensional porous structure possessed high surface area, good mechanical stability and well hydrophilicity, which provided a biocompatible microenvironment for maintaining the bioactivity of the immobilized protein and increased the protein loading. Therefore, the present immunosensor exhibits a wide linear range from 6.85-708 ng·mL-1 with a low detection limit of 3.89 ng·mL-1 for the detection of hepatitis B surface antigen (HBsAg). This work implied that the biocompatible and controllable three dimensional porous chitosan membrane possessed potential applications for biosensing.4. A novel method for fabrication of hepatitis B surface antibody (HBsAb) immunosensor has been developed by self-assembling gold nanoparticles to a thiol-containing network CHIT-GA-Cys composites which were prepared based on chemical binding approach. A cleaned gold electrode was first immersed in a network CHIT-GA-Cys composites, and then gold nanoparticles were chemisorbed onto the thiol groups of the network composites. Finally, HBsAb was adsorbed onto the surface of the gold nanoparticles. The performance and factors influencing the performance of the resulting immunosensor were studied in detail. The present immunosensor exhibits a wide linear range from 3.0-1066 pg·mL-1 with a low detection limit of 1 pg·mL-1. Moreover, the studied biosensor exhibited high sensitivity, good reproducibility and long-term stability. |
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