Nanostructured Materials Enhanced The Direct Electron Transfer Of The Immobilized Protein And Its Application For Biosensing

The electrochemical amperometric biosensors have been considered to be promising for determinations because of the advantages such as high sensitivity, low cost, easy miniaturization and automation. They have excellent selectivity, and can meet the challenges posed by complex samples, also can be applied to on-line and in vivo analysis. The key procedure in the electrochemical biosensor fabrication is the construction of a bionic interface on electrode surface, which is also called the immobilization of biomolecules. To using nanostructure materials biomolecule immobilization is a significant work for electrochemical biosensor development. Centering on this purpose, we carried out some researches as follow.First, we investigate the direct electron transfer reactivity of the immobilized hemoglobin (Hb) on polyurethane elastomer (PUE) film for biosensor design. The polyurethane elastomer film synthesized by an additional polymerization possesses good biocompatibility, good uniformity and conformability, and is ready for protein immobilization. Electrochemical and spectroscopic measurements show that the presence of multi-wall carbon nanotubes (MWNTs) increased the protein-PUE interaction, varied polymer morphology, improved the permeability and the conductivity of the PUE film, and thus facilitated the direct electron transfer between the immobilized Hb and the conductivity surface through the conducting tunnels of multi-wall carbon nanotubes. The immobilized Hb maintains its bioactivities and displays an excellent electrochemical behavior with a formal potential of -(334±7) mV. The addition of NaNO₂ leads to the increase of the electrocatalytic reduction current of nitrite at -0.7 V. This allows us to develop a nitrite sensor with a linear response range from 0.08 to 3.6 mM. The proposed method opens a way to develop biosensors by using nanostructured materials mixed with low electrical conductivity matrices.Second, the direct electron transfer between electrodes and glucose oxidase (GOD) immobilized in a matrix containing zirconium dioxide nanoparticles (ZrO₂) is described. The protein-nanoparticle assembly is stabilized by charged and uncharged compounds and the direct electron transfer is enhanced. The effects of different compositions on the electrochemical parameters, formal potential, surface loading, and constant heterogeneous electron transfer rate, is characterized with cyclic voltammetry. The fastest electron transfer rate with the smallest deviation of the E⁰' is obtained when GOD is immobilized with ZrO₂ nanoparticles, colloidal platinum and poly-Lysine (PLL). Incorporation of charged compounds for immobilization of GOD causes a larger positive shift of the formal potential. Electrochemical and spectroscopic measurements show that the GOD entrapped in ZrO₂/Pt-PLL or ZrO₂/Pt-PVA film retains its bioactivity efficiently and exhibits excellent electrocatalytic behaviour towards glucose. No enzymic activity of the immobilized GOD can be observed on ZrO₂/DMSO and ZrO₂/DDAB film.Third, we investigated the direct electrochemical and electrocatalytic behavior of the immobilized cytochrome P450 2B6 (CYP2B6) on zirconium dioxide nanoparticles (ZrO₂). Thin films of nano-structured ZrO₂ with incorporated cytochrome P450 2B6 (CYP2B6) with colloidal paltin stabled by poly-lysine (Pt-PLL) were prepared on glassy carbon electrodes. The modified electrodes were characterized by cyclic and square-wave voltammetric methods. The immobilized CYP2B6 could be reduced fast on ZrO₂-modified glassy carbon electrodes in the presence of Pt-PLL. In anaerobic solutions, the biosensor exhibited direct reversible electron transfer between the heme electroactive center of CYP2B6 and electrodes with a formal potential of -(0.449±0.004) V at pH 7.4. In air-saturated solution, an increased bioelectrocatalytic reduction current could be obtained with the CYP2B6-modified electrode on addition of anti-cancer drug such as lidocaine. This is potential to construct disposable biosensors for drugs by utilizing the electrochemical activity and catalytic reactions of the immobilized cytochrome P450.