A packed-bed immobilized enzyme microreactor with high-efficiency enzyme assay for rapid detection of nerve agent exposure

Organophosphorus nerve agents (OPNAs) can react with the tyrosine-411 residue of human serum albumin (HSA) in blood, producing phosphylated tyrosine adducts that can be used as long-lived biomarkers of nerve agent exposure. These adducts can be analyzed by liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS) after an enzymatic digestion step. The objective of this study was to develop an immobilized enzyme reactors (IMER) platform, in order to obtain a faster digestion of HSA than the conventional in-solution digestion (6–24 h). This study developed a packed-bed IMER platform based on γ‑Fe₂O₃ magnetic microbeads. The platform was first evaluated using α‑chymotrypsin (ChT) as a model enzyme. The resulting ChT@γ‑Fe₂O₃‑IMER showed enhanced catalytic activity and substrate affinity (Km = 0.25 mM) compared to the free enzyme (Km = 1.49 mM), along with good operational stability and repeatability. To demonstrate its specific practical utility in detection of nerve agent exposure, a pronase‑based IMER (Pronase@γ‑Fe₂O₃‑IMER) was fabricated for hydrolyzing phosphorylated albumin in exposed plasma. Multiple nerve agent adducts were successfully released from phosphorylated albumin within 1 h, which is six times faster than conventional solution digestion time. All the results show that the developed IMER platform offers a feasible method for rapid diagnostic analysis of protein biomarkers.