Construction Of CYP450 2C9 Electrochemical Biosensors Based On Functionalized Carbon Nanomaterials And Its Mediated Drug Metabolism Research

Cytochrome P450(CYP450)are members of a family of heme proteins located on the membrane of the endoplasmic reticulum,which are responsible for the metabolism of almost all drugs and chemicals,and 15 species of CYP450 have been found in human liver.Among the human CYP members,CYP 2C9 is one of the most important CYP450 isoforms present in human liver(accounting for about 25% of the P450 s expressed in it).Substrates for CYP450 2C9 include about 10-15% of all drugs,including amititriptyline,fluoxetine,losartan,phenytion,and many nonsteroidal anirheumatics.CYP450 2C9 also catalyzes the metabolism of endogenous substrates,such as arachidonate and linoleate epoxygenase in human liver microsomes.CYP450-catalyzed monooxygenations are dependent on electron donation typically from NADPH catalyzed by NADPH-cytochrome P450 oxidoreductase.The consumption of the costly cofactor NADPH constitutes an economical obstacle for biotechnological in vitro applications of CYP450 s.This bottleneck has been overcome by the design of an electrochemical biosensors consisting of cytochrome P450 enzyme modified electrodes,which is able to carry out electron-driven P450 catalytic cycle.However,the active center of the CYP450 enzyme is embedded in the protein structure,which makes it more difficult to exchange electrons between the electrodes.Since a method for immobilizing CYP on an electrode surface has been one of the key technologies,Here,we constructed the third generation CYP2C9 biosensors based on various nanocarbon materials,which are NAD(P)H or mediator-free,and designed to measure the direct electron transfer(DET)between CYP and an modified electrode.Futher,we have also investigated the pharmacokinetics and metabolism of antidepressive drugs and evaluated the effect on CYP2C9 activity,using the simple and inexpensive sensing systems.Chapter 1: The concept of electrochemical sensor and CYP450 enzyme is briefly introduced,and the immobilization method of enzyme and various applications of enzyme electrochemical sensor are reviewed.Chapter 2: Here,we report a very simple electrochemically driven biosensor for detecting drug metabolism and its inhibition based on cytochrome P450 2C9(CYP2C9)and a nitrogen doped mesoporous carbon(N-MC)and AuNPs modified film electrode.Direct electron transfer(DET)between CYP2C9 and N-MC/AuNPs was observed at a formal potential of-0.472 V.The electrocatalytic reduction current increased with the addition of tolbutamide,further the corresponding kinetic parameters,namely Michaelis-Menten constant and heterogeneous reaction rate constant,were calculated to be 153.67 ?mol L-1and 0.26-5.35s-1 respectively.In contrast,the reduction current was greatly suppressed in the presence of chloramphenicol,which is a CYP2C9 inhibitor,and the IC50 value was calculated to 0.515 ?mol L-1.Overall,the N-MC material with high conductivity,a large surface area and sufficient edge planes provide a suitable microenvironment for achieving excellent DET and biocatalysis properties,indicating that our system is promising as a new bioelectronic platform for electrochemical biosensing.Chapter 3: In this paper,cationic polyacrylamide functionalized nitrogen-doped graphene(CPAM-NG)was prepared by modification of graphene using non-covalent modification and nitrogen atom doping.Then the P450 2C9 enzyme was immobilized on the glassy carbon electrode with this material,thus the electrochemical sensor of CYP2C9 catalyzing tolbutamide was successfully constructed.The electron transfer mechanism between CYP2C9 and electrode was studied by cyclic voltammetry and the direct electron transfer constant was calculated to be 1.84 s-1.The electrocatalytic reduction current increased with the addition of tolbutamide,further the Michaelis-Menten constant was 117.86 ?mol L-1.With the help of differential pulse voltammetry(DPV),the inhibitory effects of five antidepressants on CYP2C9-catalyzed metabolism were studied,and half of the inhibitory concentrations(IC50)were calculated.It was instructed to study clinical drugs and drug interactions.Chapter 4: Firstly,carboxylated multi-walled carbon nanotubes(COOH-MWCNTs)were prepared by carboxylation of MWCNTs in the traditional way of strong acid oxidation.The CYP2C9 was then successfully immobilized on COOH-MWCNTs modified glassy carbon electrode(GCE)by electrostatic interaction and covalent bonding.The electrochemical sensor of CYP2C9 catalyzing tolbutamide was successfully constructed due to good conductivity,biocompatibility,and dispersibility of the material.The electron transfer mechanism between electrode and CYP2C9 was studied by cyclic voltammetry(CV),and the direct electron transfer constant was calculated to be 1.62 s-1.The sensor had a good response to the substrate of tolbutamide and had a linear relationship in the range of 0-256.3 ?mol L-1,with an apparent Michaelis-Menten constant of 186.37 ?mol L-1.In addition,the inhibitory actions of fluconazole and cimetidine were determined by differential pulse voltammetry(DPV)with a half inhibitory concentration(IC50)of 4.2 and 17.6 ?mol L-1,as a moderate and weak inhibitoinhibitor of CYP2C9,respectively.