A SERS-Based Analytical Strategy for Real-Time Quantitative Kinetic Analysis and Inhibitor Screening for Cytochrome P450-Mediated PAH Metabolism

Exploring the metabolic kinetics mediated by cytochrome P450 (CYP) enzymes is essential for understanding pharmacokinetic and toxicological mechanisms. However, real-time monitoring of CYP-mediated enzymatic reactions remains challenging for conventional techniques. Here, we report the first attempt to develop a surface-enhanced Raman scattering (SERS)-based analytical platform for the direct, real-time tracking of enzymatic metabolism and extraction of critical kinetic parameters. An in vitro CYP enzyme system was constructed to emulate hepatic microsomal activity, using pyrene (Pyr) as a model and polycyclic aromatic hydrocarbon (PAH) as the model substrate. Cetyltrimethylammonium bromide (CTAB)-induced Ag aggregates enabled simultaneous detection of Pyr (408 cm^–1) and its metabolite 1-hydroxypyrene (1-OHPyr; 426/617 cm^–1) directly within the reaction medium. The hydrophobic CTAB self-assembled monolayer enhanced analyte enrichment and suppressed protein interference, while the intrinsic Raman band of CTAB at 760 cm^–1 served as a reliable internal standard for quantitative kinetic assessment. This platform allowed real-time monitoring of CYP-catalyzed oxidation of Pyr, yielding pseudo-first-order kinetics (k = 0.00501 min^–1) and the Michaelis constant K_m of 13 μM. Furthermore, inhibition studies identified CYP1A2 as the principal isoform responsible for Pyr metabolism, corroborated by the potent inhibitory activity of α-naphthoflavone (with calculated IC₅₀). To our knowledge, this work represents the first instance of SERS for time-resolved monitoring of CYP-mediated metabolism, expanding its capability from static detection to dynamic biochemical analysis. The proposed strategy provides a simple, label-free, and time-resolved approach for enzyme kinetic studies, inhibitor screening, and metabolic analysis.