| Cytochrome P450 (CYP) is a family of redox proteins existed in most of the life. P450 enzymes possess excellent catalytic abilities, which are responsible for catalyzing various kinds of reaction. It is involved in metabolizing exogenous compounds, e.g. drugs, pesticides, environmental pollutants etc, and synthetizing of some endogenous substances such as fatty acids, steroid hormone, prostaglandins, etc. The electrochemistry of P450s attracted considerable attention recently because it can not only provide information of electrochemistry and electrocatalytic mechanism of P450s, but also is an effective way of fabricating of biosensors, bioreactors and biomedical devices.In this paper, several novel composite materials were used for immobilizing P450 to realize the direct electrochemistry and electrocatalysis of P450. These materials provide favorable micro-environment for P450s, and facilitate the direct electron transfer between P450 and the electrodes. P450s entrapped in these composites realized the direct electrochemistry, and showed good electrocatalytic performance towards theirs own substrates. The main work of this thesis is summarized as follows:1. Acetylene black (AB) was dispersed in surfactant sodium dodecyl sulfate (SDS), and a stable suspension solution SDS-AB was obtained. Cytochrome P450 6A1 (CYP6A1) was entrapped in this composite material. The results showed that CYP6A1 was immobilized in the SDS-AB composite film successfully. And this composite material can provide a favorable micro-environment for CYP6A1, and greatly promote the direct electron transfer between CYP6A1 and electrode surface. A pair of stable and quasi-reversible redox peaks can be obtained when SDS-AB/CYP6A1 modified electrode was scanned in 0.1 mol·L-1 phosphate buffer solutions (PBS). The SDS-AB/CYP6A1 modified electrode exhibited high electrocatalytic effects towards the reduction of oxygen and progesterone.2. Polyethylene glycol (PEG) is a material of growing importance for it is biocompatible and anti-fouling, and it was widely used in biomedical devices and biosensors. In this chapter, acetylene black (AB) was dispersed in polyethylene glycol (PEG2000) and a stable suspension was obtained. CYP3A4 was embedded in PEG2000-AB composite film. The results showed that PEG2000 could provide biocompatible microenvironment for CYP3A4 and AB could greatly enhance the direct electron transfer between CYP3A4 and electrode surface. The electrochemical behavior of CYP3A4 in PEG2000-AB composite film has been investigated, and a pair of well-defined quasi-reversible cyclic voltammetric peaks of heme Fe(Ⅱ/Ⅲ) redox couples were observed at -0.494 V (vs. SCE). This PEG2000-AB/CYP3A4 composite film exhibited excellent electrocatalytic activity to atrazine, which is an endocrine disruptor compound. These results indicated the potential applicability to detect the (EDCs) and develop a novel platform for immobilizing proteins.3. A novel epoxy polymer P (GMA-co-MPC) was prepared by copolymerization of glycidyl methacrylate (GMA) and 2-metharyloyloxyethyl phosphorylcholine (MPC). Acetylene black(AB) can dispersed evenly in this polymer solution, obtaining a stable suspension. CYP3A4 was fixed within the composite membrane successfully. The results showed that the polymers can fixed CYP3A4 stably and provide a bionic environment for CYP3A4, and AB was used as shuttle for electron transfer. The P(GMA-co-MPC)-AB/CYP3A4 modified electrode displayed stable reversible electrochemical response and the direct electron transfer between CYP3A4 and electrodes was realized. CYP3A4 immobilized in P(GMA-co-MPC)/AB film exhibited excellent electrocatalytical activity to diethylstilbestrol. The modified electrode was applied for the detection of environmental hormone diethylstilbestrol with a linear range from 2.0×10-7 to 2.8×10-6 mol·L-1 and the detection limit of 5.9×10-8 mol·L-1. |
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