Constructions Of Nano-Biointerface And The Applications In Electrochemical Immunosensor

Electrochemical biosensor has been greatly developed and widely used in a great amount of fields like environment, clinic diagnosis and biology. Additionally, nanoparticles have some prominent advantages and own serials of interesting physical and chemical properties. They can enlarge the geometrical area that greatly enhance the active surface suitable for biomolecules binding, and at the same time provide a more friend microenvironment to maintain the biological activity. This thesis mainly focuses on the investigations of nanomaterials like colloid gold nanoparticles and SiO₂, and more details are presented below:In the first study, we describe an improved system to study the two-electron delivery reaction pathway of cytochrome P450, family 2, subfamily B, polypeptide 6 (CYP2B6) in vitro. In particular, a biocompatible film containing colloidal gold nanoparticles and chitosan was used to encapsulate CYP2B6 on an electrode. The electrochemical behaviors of CYP2B6 towards common drugs in the absence of NADHP-cytochrome P450 reductase as electron donor were studied and the results confirmed that the immobilized CYP2B6 retained most of its bioactivity. In an anaerobic solution, direct and reversible electron transfer between the electroactive heme center of CYP2B6 and the electrode was observed with a formal potential of -(0.454±0.006) V at pH 7.4. In an air-saturated solution, an increase in the bioelectrocatalytic reduction current was observed after drug addition. The bioelectrocatalytic products were analyzed using high performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS). Both results confirmed that C-hydroxylation and heteroatom release were the main pathways for CYP2B6-mediated drug oxidation, similar to what occurred in vivo. The use of immobilized proteins in nanoparticle-containing films in drug biosensing wasalso demonstrated.In the second study, we produced a novel separation-free electrochemical immunosensor for AFP detection. SiO₂ nanopartilces, chemically modified with NH2-on the surface, were used to immobilize the AFP through the linkage reagent of glutaric dialdehyde (GDI). The modified surface provided a larger number of active sites for covalently binding of AFP, and the orientation of AFP through step by step modification offered a more congenial space for the immuno-reaction between AFP antigen and AFP antibody. Besides, the polyurethane elastomer (PUE) film is used as a immobilization regent. It is reported that PUE film has been already applied into the protein immobilization due to its good biocompatibility, good uniformity and conformability. According to the results obtained previously, the permeability and conductivity of PUE could be significantly improved by adding some nanoparticles like multi-wall carbon nanotubes. Hereon, SiO₂ nanoparticles modified with AFP are chosen to increase there qualities. A competitive immunoassay approach was utilized to detect AFP antigen with horseradish peroxidase (HRP) labeled AFP antibody as tracer, thionine and hydrogen peroxide as enzyme substrates. After the immunosensor was incubated with a mixture of HRP labeled AFP antibody and AFP antigen at 37*C for 45 min, the amperometric response decreased with an increasing AFP concentration in the incubation solution. The immunosensor showed good stability, acceptable accuracy, and would be applicable to clinical detection for AFP.