Real-time 96-well optoelectronic micro plate for kinetic and stability investigation of cytochrome P450 BM3

Simultaneous optical and electrochemical measurements are advantageous for biosensor technology. Although this is easy for single samples, parallelization is challenging, when ensuring spatial and temporal compatibility of measurement techniques, especially if the device is to be integrated into existing laboratory routines. Here, we developed an electrochemical-optical 96-well micro plate (eco-MP) to track the bioelectrocatalysis of cytochrome P450 BM3 fluorometrically and chronoamperometrically in a simultaneous and real-time manner. Fluorometric readout enables unbiased quantitative determination of the enzymatic product resorufin in parallel to the chronoamperometric data acquisition. We implemented 96 miniaturized three-electrode setups in the eco-MP with a 3D-printed divided cell architecture leading to 192 half-cells. Using a conventional fluorescence reader we demonstrate the potential of the developed eco-MP to control and monitor the active state of cytochrome P450 BM3 over hours. The benefit of reactive oxygen species scavenging enzymes on bioelectrocatalytic activity of P450 BM3 could be time-resolved assessed. We applied the eco-MP also to investigate the tolerance of P450 BM3 to organic solvent dimethyl sulfoxide, as an example of an area in bioelectrocatalysis where high-throughput measurement devices will kick in. Overall, this work stresses the demand for convenient multiparametric sensor technology in the growing field of electrified interfaces. •96-well electrochemical micro plate with horizontally divided cell architecture.•Real-time optoelectronic monitoring in compliance with common laboratory routines.•Stability investigation and On- and Off-switching of cytochrome P450 BM3.•Effect of reactive oxygen species scavenging enzymes on bioelectrocatalysis.•Organic solvent tolerance of bioelectrocatalytically activated P450 BM3.