Microfluidic Electrochemical Array for Detection of Reactive Metabolites Formed by Cytochrome P450 Enzymes

Abstract

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 8-electrode screen printed carbon array coated with thin films of DNA, [Ru(bpy)2(PVP)10](ClO4) {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 TD50 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.

High-throughput metabolic genotoxicity screening with a fluidic microwell chip and electrochemiluminescence

“…15,32–34 Following the enzyme reactions, washing with anaerobic 50 mM phosphate buffer + 0.1 M NaCl of pH 7.4 was done for 3 min at 500 µL min −1 . Then, 1.25 V vs. Ag/AgCl was applied to the array for 180 s with a CHI 1232 electrochemical analyzer (CH Instruments Inc.) to generate ECL acquisition which is detected by the CCD camera.…”

“…In the reaction step, metabolites produced by these enzymes are formed in films with high concentrations of DNA, favouring reaction with DNA bases if possible. 5,10,12 In the detection step, relative amounts and rates of DNA damage are measured by voltammetry, 13–15 or electrochemiluminescence (ECL) 11,16,17 by including metallopolymer [Ru(bpy) 2 (PVP) 10 ] 2+ {(PVP = poly(4-vinylpyridine)} (Ru II PVP) in the films as a electrochemical ECL-producing catalyst.…”

State-of-the-Art Metabolic Toxicity Screening and Pathway Evaluation

“…80 The device incorporates an eight-electrode screen-printed carbon sensor array coated with the DNA/RuPVP/RLM films. Cyt P450s in RLMs are activated by the electrochemical approach described above.…”

Electrochemistry-mass spectrometry to study reactive drug metabolites and CYP450 simulations

“…There are a few strategies describing the modification of several electrode materials with CYP450 [64][65][66], but some of these are often confronted by the method used for the electrochemical regeneration of the enzymatically active form of CYP450. Another advantage of modified CYP450-electrodes is that they can be appropriate for the routine generation of various metabolites [67,68]. Both mediated and unmediated processes have been reported, but the direct electrochemical regeneration of the active form of CYP450 still remains inefficient at this stage and displays low conversion rates.…”