Cytochrome P450 electrochemical biosensors transforming in vitro metabolism testing - Opportunities and challenges

The ability of the living world to flourish in the face of constant exposure to dangerous chemicals depends on the management ability of a widespread group of enzymes known as heme-thiolate monooxygenases or cytochrome P450 superfamily. About three-quarters of all reactions determining the metabolism of endogenous compounds, of those carried in foods, of taken drugs, or even of synthetic chemicals discarded into the environment depend on their catalytic performance. The chromatographic and (photo)luminometric methods routinely used as predictive and analytical tools in laboratories have significant drawbacks ranging from limited shelf-life of reagents, use of synthetic substrates, laborious and tedious procedures for highly sensitive detection. In this review, alternative electrochemical biosensors using the cytochrome P450 enzymes as bio-element are emphasized in their main aspects as well regarding their implementation and usefulness. Despite the various schemes proposed for the implementation, reports on real applications are scant for several reasons, including low reaction rates, broad substrate specificity, uncoupling reactions occurrence, and the need for expensive electron transfer partners to promote electron transfer. Finally, the prospect for future developments is introduced, focusing on integrating miniaturized systems with electrochemical techniques, alongside optimizing enzyme immobilization methods and electrode modifications to improve enzymatic stability and enhance sensor reliability. This progress represents a crucial step towards the creation of portable biosensors that mimic human physiological responses, supporting the precision medicine approach.