Microfluidic electrochemical array for detection of reactive metabolites formed by cytochrome P450 enzymes

A novel, simple, rapid microfluidic array using bioelectronically driven cytochrome P450 enzyme catalysis for reactive metabolite screening is reported for the first time. The device incorporates an eight-electrode screen-printed carbon array coated with thin films of DNA, [Ru(bpy)(2)(PVP)(10)](ClO(4)) {RuPVP}, and rat liver microsomes (RLM) as enzyme sources. Catalysis features electron donation to cyt P450 reductase in the RLMs and subsequent cyt P450 reduction while flowing an oxygenated substrate solution past sensor electrodes. Metabolites react with DNA in the film if they are able, and damaged DNA is detected by catalytic square wave voltammetry (SWV) utilizing the RuPVP polymer. The microfluidic device was tested for a set of common pollutants known to form DNA-reactive metabolites. Logarithmic turnover rates based on SWV responses gave excellent correlation with the rodent liver TD(50) toxicity metric, supporting the utility of the device for toxicity screening. The microfluidic array gave much better S/N and reproducibility than single-electrode sensors based on similar principles.     

Electrodeposited silicate films: importance of supporting electrolyte

Silica and hybrid organic-inorganic films, ca. 100-200 nm thick, can be grown on glassy carbon electrodes through reactions initiated by electrogenerated hydroxide or hydronium ions in water under reductive and oxidative conditions, respectively. A variety of different alkoxysilanes (tetramethoxysilane and organoalkoxysilanes) and supporting electrolytes were used to evaluate whether film formation takes place on glassy carbon electrodes. The results of the study indicate that the acid-base properties of the supporting electrolyte are an important factor in determining whether film formation will take place. For cathodic electrodeposition, thin films can be formed using supporting electrolytes that are close to neutral, such as KCl, KNO3, and NaClO4. For anodic electrodeposition, thin films can be formed using supporting electrolytes that are acidic, such as, KH2PO4, HNO3, H2SO4, etc. The acidity/basicity effects of the electrolytes arise in part from the strong dependence of the hydrolysis and condensation rates of the silicon alkoxide precursors on pH.     

Electroanalytical performance of carbon films with near-atomic flatness

Physicochemical and electrochemical characterization of carbon films obtained by pyrolyzing a commercially available photoresist has been performed. Photoresist spin-coated on to a silicon wafer was pyrolyzed at 1,000 degrees C in a reducing atmosphere (95% nitrogen and 5% hydrogen) to produce conducting carbon films. The pyrolyzed photoresist films (PPF) show unusual surface properties compared to other carbon electrodes. The surfaces are nearly atomically smooth with a root-mean-square roughness of <0.5 nm. PPF have a very low background current and oxygen/carbon atomic ratio compared to conventional glassy carbon and show relatively weak adsorption of methylene blue and anthraquinone-2,6-disulfonate. The low oxygen/carbon ratio and the relative stability of PPF indicate that surfaces may be partially hydrogen terminated. The pyrolyzed films were compared to glassy carbon (GC) heat treated under the same conditions as pyrolysis to evaluate the electroanalytical utility of PPF. Heterogeneous electron-transfer kinetics of various redox systems were evaluated. For Ru(NH3)6(3+/2+), Fe(CN)6(3-/4-), and chlorpromazine, fresh PPF surfaces show electron-transfer rates similar to those on GC, but for redox systems such as Fe3+/2+, ascorbic acid, dopamine, and oxygen, the kinetics on PPF are slower. Very weak interactions between the PPF surface and these redox systems lead to their slow electron-transfer kinetics. Electrochemical anodization results in a simultaneous increase in background current, adsorption, and electron-transfer kinetics. The PPF surfaces can be chemically modified via diazonium ion reduction to yield a covalently attached monolayer. Such a modification could help in the preparation of low-cost, high-volume analyte-specific electrodes for diverse electroanalytical applications. Overall, pyrolysis of the photoresist yields an electrode surface with properties similar to a very smooth version of glassy carbon, with some important differences in surface chemistry.     

Vertically integrated human P450 and oxygen sensing film for the assays of P450 metabolic activities

An assaying method of cytochrome P450 (P450 or CYP) monooxygenase activities for toxicological evaluation of drugs and environmental pollutants was developed by immobilizing P450 on an oxygen sensoring layer. Membrane fractions from E. coli expressing human P450 were entrapped in agarose or silica-based gels and immobilized on 96-well microarrays having an oxygen sensing film at the bottom. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). P450 activity toward the substrates was monitored through the fluorescence intensity enhancement due to the oxygen consumption by the metabolic reactions. For the metabolism of chlortoluron, a selective herbicide used to control grass weeds, CYP1A1 immobilized in agarose gel showed a higher activity and stability compared with those in silica gels and free suspensions. The luminescence changing rate evaluated by the dynamic transient method (DTM) could be correlated with the substrate concentration. We also compared the metabolic responses of human P450s (CYP1A1,CYP2C8, CYP2E1, CYP3A4) toward various substances. The use of immobilized P450 on an oxygen sensing layer provides a versatile assaying platform owing to the following features. First, the oxygen sensor can detect metabolic reactions of any P450 species, in contrast with assays using fluorogenic substrates. Second, vertical integration of the oxygen sensor and immobilized P450 enhanced the sensitivity because of the effective depletion of oxygen in the vicinity of the oxygen sensing layer. Third, immobilization enables repeated use of P450 by replacing the substrate solutions using a flow cell. Furthermore, the activity of immobilized P450 was retained at least for 3 weeks at 4 °C, suggesting its long-term stability, which is an additional attractive feature.     

Multiple microscale plasma CVD apparatuses on a substrate

Plasma chemical vapor depositions (CVD) of carbon films using a CH₄---H₂ gas system have been performed with a prototype 2×2 plasma chip which consists of 2×2 coplanar film electrodes (CFE). Using this chip, we could perform four plasma processing experiments simultaneously. A plasma chip on which plasma-processing apparatuses are integrated has advantages in plasma processing compared to conventional ones, such as high processing speed due to the high density of the microscale plasma, and high efficiency due to parallel processing. We have obtained various carbon films easily, such as glassy carbon and diamond-like carbon, with this simple prototype plasma chip. This is the first report on plasma processing with this innovative processing device, the plasma chip.     

Evaluating enzymes that generate genotoxic benzo[a]pyrene metabolites using sensor arrays

Arrays with individually addressable, demountable electrodes coated with ultrathin DNA/enzyme films were evaluated to estimate relative rates of genotoxic bioactivation of benzo[a]pyrene (BP) for several different enzymes simultaneously. Specifically, cytochrome (cyt) P450cam, cyt P40 1A2, and myoglobin in the array were activated with H2O2 to metabolize BP to genotoxic metabolites. DNA damage by the metabolites was detected by increases in square wave voltammetric oxidation peaks using Ru(bpy)3(2+) as catalyst. Cyt P450cam and cyt P450 1A2 showed 3-fold higher activity for genotoxic bioactivation of BP than myoglobin. The ability of the arrays to generate and detect metabolite-based DNA damage simultaneously for several enzymes is a rapid and promising approach to identify and characterize enzymes involved in genotoxicity of drugs and pollutants.     

ABTS-modified multiwalled carbon nanotubes as an effective mediating system for bioelectrocatalytic reduction of oxygen

The ability of such a common redox mediator as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) to undergo sorption on carbon surfaces is explored here to convert multiwalled carbon nanotubes (CNTs) into a stable colloidal solution of ABTS-modified carbon nanostructures, the diameters of which are approximately 10 nm (as determined by transmission electron microscopy). Subsequently, inks composed of fungal laccase (Cerrena unicolor) mixed with the dispersion of ABTS-modified CNTs and stabilized with Nafion, were deposited on glassy carbon and successfully employed to the reduction of oxygen in McIlvain buffer at pH 5.2. For comparison, the systems utilizing only ABTS-free CNTs and laccase as well as ABTS-modified CNTs did not show appreciable activity toward the oxygen reduction. The three-dimensionally distributed ABTS-modified CNTs are expected to improve the film's overall conductivity and to facilitate electrical connection between the electrode and the enzyme. The network film of ABTS-modified CNTs is rigid, and it is characterized by charge propagation capabilities comparable to the conventional redox polymers. The whole concept of utilization of CNTs modified with ultrathin films of redox mediators in the preparation of efficient bioelectrocatalytic films seems to be of general importance to electroanalytical chemistry and to the development of biosensors.     

Effect of CYP2C9 genetic polymorphism on the metabolism of flurbiprofen in vitro

CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, and 57 cytochrome P450 2C9 alleles have been previously reported. To examine the enzymatic activity of the CYP2C9 alleles, kinetic parameters for 4′-hydroxyflurbiprofen were determined using recombinant human P450s CYP2C9 microsomes from insect cells Sf21 carrying wild-type CYP2C9*1 and other variants. The results showed that the enzyme activity of most of the variants decreased comparing with the wild type as the previous studies reported, while the enzyme activity of some of them increased, which were not in accordance with the previous researches. Of the 36 tested CYP2C9 allelic isoforms, two variants (CYP2C9*53 and CYP2C9*56) showed a higher intrinsic clearance value than the wild-type protein, especially for CYP2C9*56, exhibited much higher intrinsic clearance (197.3%) relative to wild-type CYP2C9*1, while the remaining 33 CYP2C9 allelic isoforms exhibited significantly decreased clearance values (from 0.6 to 83.8%) compared to CYP2C9*1. This study provided the most comprehensive data on the enzymatic activities of all reported CYP2C9 variants in the Chinese population with regard to the commonly used non-steroidal anti-inflammatory drug, flurbiprofen (FP). The results indicated that most of the tested rare alleles decreased the catalytic activity of CYP2C9 variants toward FP hydroxylation in vitro. This is the first report of all these rare alleles for FP metabolism providing fundamental data for further clinical studies on CYP2C9 alleles for FP metabolism in vivo.     

Electrodeposition and characterization of CdSe semiconductor thin films

The electrodeposition of cadmium selenide alloy on glassy carbon and gold electrodes has been studied by electrochemical techniques. Potentiostatic I-t transients were recorded to obtain the nucleation mechanism, while cyclic voltammetry was used to characterize the system. Structural information on the electrodeposited layers was obtained by X-ray diffraction. The experimental results clearly show that the deposition of cadmium selenide alloy on glassy carbon and gold electrodes is a diffusion-controlled process. The nucleation is progressive, and the number of nucleation sites decreases with increasing bath temperature. The deposition of CdSe alloy results in well-defined crystals with hexagonal shape. The films were characterized by optical absorption and electrical resistivity measurements. Films showed a direct band gap of 3.56 eV.     

Morphological and electrochemical properties of boron-doped diamond films on carbon cloths with enhanced surface area

The electrochemical properties of doped diamond electrodes (10¹⁷–10¹⁹ B cm^− 3) grown on carbon fiber cloths in H₂SO₄ 0.1 mol L^− 1 electrolyte were investigated. Cyclic voltammograms of B-doped diamond/carbon fiber cloth and carbon fiber cloth electrodes showed that both kinds of electrodes possess similar working potential windows of about 2.0 V. The electrode capacitance was determined by impedance spectroscopy and chronopotentiometry measurements and very close values were obtained. The capacitance values of the diamond film on carbon fiber cloths were 180 times higher than the ones of diamond films on Si. In this paper we have also discussed the capacitance frequency dependence of diamond/carbon cloth electrodes.     

Electrochemical performance of fixed-charge polymer films prepared on electrodes by plasma polymerization

Plasma-polymerized films were prepared from a 1:1 mixture of allylamine vapour and NH₃ on glassy carbon electrodes. The amine groups in the matrix were protonated at pH 2, producing a fixed-charge membrane. Electrochemical experiments were performed with these coated electrodes. Cyclic voltammograms showed that the redox system Fe(CN)₆^3-/Fe(CN)₆^4- was partitioned strongly into the films because of chemical interactions with the matrix.     

Ultraflat carbon film electrodes prepared by electron beam evaporation

A facile method for the preparation of thin-film carbon electrodes by electron beam evaporation onto highly doped silicon is presented. The physical and electrochemical properties of these films both before and after postdeposition pyrolysis are investigated. Raman spectroscopy establishes the amorphous structure of the nonpyrolyzed carbon films and confirms the formation of graphitic carbon after pyrolysis at 1000 degrees C. Scanning force microscopy reveals the root-mean-square roughness of nonpyrolyzed films to be approximately 1 A, while pyrolyzed films exhibit an increased roughness of approximately 4 A. The electrochemical behavior of the electrodes resembles glassy carbon, with measured heterogeneous electron-transfer rate constants among the highest measured for thin carbon films. These carbon film electrodes will potentially find applications in such fields as molecular electronics and scanning probe microscopy of adsorbed species.     

Insulin oxidation and determination at carbon electrodes

The redox chemistry of insulin was investigated at glassy carbon (GC) electrodes that were coated with films of chitosan (CHIT) and multiwalled carbon nanotubes (CNT). While bare electrodes deactivated quickly during insulin oxidation, the GC electrodes coated with CHIT and CHIT-CNT films generated stable insulin currents. The GC/CHIT-CNT electrodes were used for investigating the electrooxidation process of insulin and amperometric determination of insulin. The mass spectrometric, electron paramagnetic resonance, and separation studies of electrolyzed insulin solutions suggested that the loss of 4 mass units upon insulin oxidation at CNT could be accounted for by the formation of two dityrosine cross-links intramolecularly. At a potential of 0.700 V and physiological pH 7.40, the GC/CHIT-CNT electrodes displayed a detection limit of approximately 30 nM insulin (S/N = 3), sensitivity of 135 mA M(-1) cm(-2), linear dynamic range from 0.10 to 3.0 microM (R2 = 0.995), and superior operational and long-term stability. The CNT-based electrodes are promising new insulin detectors for diabetes-related studies such as fast chromatographic analysis of therapeutic insulin formulations or evaluation of quality of pancreatic islets prior to their transplantation.     

Electrochemiluminescent arrays for cytochrome P450-activated genotoxicity screening. DNA damage from benzo[a]pyrene metabolites

Arrays suitable for genotoxicity screening are reported that generate metabolites from cytochrome P450 enzymes (CYPs) in thin-film spots. Array spots containing DNA, various human cyt P450s, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy)2PVP10]2+ were exposed to H2O2 to activate the enzymes. ECL from all spots was visualized simultaneously using a CCD camera. Using benzo[a]pyrene as a test substrate, enzyme activity for producing DNA damage in the arrays was found in the order CYP1B1 > CYP1A2 > CYP1A1 > CYP2E1 > myoglobin, the same as the order of their metabolic activity. Thus, these arrays estimate the relative propensity of different enzymes to produce genotoxic metabolites. This is the first demonstration of ECL arrays for high-throughput in vitro genotoxicity screening.     

Fabrication of calix[4]pyrrole nanofilms at the glassy carbon surface and their characterization by spectroscopic, optic and electrochemical methods

meso-Octamethylcalix[4]pyrrole (CP) and meso-heptaethylcalix[4]pyrrole-meso-4-aminophenyl (4APCP) modified glassy carbon (GC) electrodes were prepared by the electrochemical oxidation in acetonitrile solution. Binding of the calix[4]pyrroles with the glassy carbon surface was investigated that it is through the etheric linkage revealed from the reflection-absorption infrared spectroscopy (RAIRS). Surface films of CP and 4APCP were investigated by cyclic voltammetry (CV), ellipsometry, X-ray photoelectron spectroscopy, RAIRS and the contact angle measurements. The thicknesses of the films were determined by ellipsometry which confirmed that the film was multilayer and homogeneous over the surface. Ellipsometric measurements also provided that the CP and 4APCP film thicknesses were 2.49 nm and 4.58 nm for 6 CV cycle modification, corresponding to 66 μF/cm² and 106 μF/cm² capacitances obtained by CV. The wetting behavior was examined by contact angle measurements and found that the hydrophobicity of the GC-4APCP surface was higher than that of GC-CP, probably due to the aromatic meso substituent present in the former.     

Effects of capsicine on rat cytochrome P450 isoforms CYP1A2, CYP2C19, and CYP3A4

Due to the frequent consumption of capsaicin (CAP) and its current therapeutic application, the correct assessment of this compound is important from a public health standpoint. The purpose of this study was to find out whether CAP affects rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5?mL/kg, which contained phenacetin (15?mg/kg), omeprazole (15?mg/kg), and midazolam (10?mg/kg), was given orally to rats treated for 7?d with oral administration of CAP. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS. The results showed that treatment with multiple doses of CAP had no significant effect on rat CYP1A2. However, CAP had a significant inhibitory effect on CYP2C19 and an inductive effect on CYP3A4. Therefore, caution is needed when CAP is co-administered with some CYP substrates clinically because of potential drug–CAP interactions.     

Inhibitory effects of curcumin on activity of cytochrome P450 2C9 enzyme in human and 2C11 in rat liver microsomes

Cytochrome P450 2C9 (CYP2C9), one of the most important phase I drug metabolizing enzymes, could catalyze the reactions that convert diclofenanc into diclofenac 4′-hydroxylation. Evaluation of the inhibitory effects of compounds on CYP2C9 is clinically important because inhibition of CYP2C9 could result in serious drug–drug interactions. The objective of this work was to investigate the effects of curcumin on CYP2C9 in human and cytochrome P450 2C11 (CYP2C11) in rat liver microsomes. The results showed that curcumin inhibited CYP2C9 activity (10?µmol?L^–1 diclofenac) with half-maximal inhibition or a half-maximal inhibitory concentration (IC50) of 15.25?µmol?L^–1 and Ki?=?4.473?µmol?L^–1 in human liver microsomes. Curcumin’s mode of action on CYP2C9 activity was noncompetitive for the substrate diclofenanc and uncompetitive for the cofactor NADPH. In contrast to its potent inhibition of CYP2C9 in human, diclofenanc had lesser effects on CYP2C11 in rat, with an IC50 ≥100?µmol L^–1. The observations imply that curcumin has the inhibitory effects on CYP2C9 activity in human. These in vitro findings suggest that more attention should be paid to special clinical caution when intake of curcumin combined with other drugs in treatment.     

Electron transfer mediation characteristics of conducting Polymer Films of 4(5-chloro-2-pyridyl)-1,3-diaminobenzene cobalt complex

Stable electroactive polymer films of cobalt complex of 4(5-chloro-2-pyridylazo)-1,3-diaminobenzene (abbreviated as PCoL₂) were prepared on glassy carbon electrodes by the oxidative polymerization of the complex in acetonitrile. Two pairs of reversible redox peaks for PCoL₂ films were observed. The first pair is attributed to the electroactivity of the skeleton of the polymer and the other is attributed to the redox processes of the CO^3+/2+ couple. The surface concentration of the electroactive Co sites was found to increase with increasing the film thickness. Mediation of reduction of [Fe(CN)₆]^3? by PCoL₂ films was investigated by steady state measurements. The rate of the mediated reaction was found to be controlled by the rate of the charge propagation within the film & the rate of diffusion of the substrate through the film beside the rate of diffusion of the substrate to the electrode. Thin films were found to be potentially more efficient for the mediation than thicker films.     

Sample prefractionation for mass spectrometry quantification of low-abundance membrane proteins

Use of stable isotope-labeled full-length proteins as an internal standard prior to multiple reaction monitoring (MRM) analysis enables prefractionation of the target proteins and quantification of those low-abundance proteins, which cannot be reached without biological sample enrichment. In terms of membrane proteins, this benefit can be used if a sample processing workflow allows entire solubilization of membrane proteins. We have developed a universal workflow for sample processing and enrichment by optimizing washing and solubilization conditions and implementing sample fractionation by Whole Gel Eluter. The optimized protocol was applied to various membrane-bound cytochromes P450 (CYPs) and their electron transferring protein partners, cytochrome P450 reductase (CPR), ferredoxin reductase (FdR), and ferredoxin (Fdx), all important proteins for cholesterol elimination from different organs. Both, weakly associated (CPR and FdR) and tightly associated (CYP7B1, CYP11A1, CYP27A1, and CYP46A1) membrane proteins were quantified. Measurements were performed on three human tissues (temporal lobe of the brain, retina, and retinal pigment epithelium) obtained from multiple donors. The biological implications of our quantitative measurements are also discussed.     

Chip-based P450 drug metabolism coupled to electrospray ionization-mass spectrometry detection

A chip-based P450 in vitro metabolism assay coupled with ESI-MS and ESI-MS/MS detection is described in this paper. The chips were made of a cyclic olefin polymer using a hot embossing process. The introduction of reagent solutions into the chip was carried out using fused-silica capillaries coupled to two syringes with the flow rate controlled by a syringe pump. Initial experiments described here employed a small commercial guard column in an off-chip format to desalt and concentrate the products of the enzymatic reaction prior to ESI-MS analysis. The system was used both to yield the Michaelis constant (K(m)) of the P450 biotransformation of imipramine into desipramine and to determine the IC50 value of a chemical inhibitor (tranylcypromine) for this CYP2C19-mediated reaction. The results demonstrated that the kinetics of the reaction inside the 4-microL volume within the channels of the cyclic olefin polymer chip provided results in agreement with those reported in the literature using conventional assays. The above reactions were carried out using human liver microsomes, and the metabolites were detected by ESI-MS showing the potential of the chip-based P450 reaction for metabolite screening studies as well as for P450 inhibition assays. A porous monolithic column was subsequently integrated into the chip to perform the reaction mixture cleanup process in an integrated fashion on the chip that is necessary for ESI-MS detection. The miniature monolithic SPE column was prepared in situ inside the chip via UV-initiated polymerization. The results obtained using the integrated system demonstrated the possibility of performing P450 enzymatic reactions in a microvolume reaction chamber coupled directly to ESI-MS detection and required less than 4 microg of HLM protein.