Microfluidic platforms for Enzymatic assays: advances, challenges, and clinical applications

Enzymatic assays are fundamental tools in clinical diagnostics and biomedical research owing to their high specificity and sensitivity for biomarker quantification. However, their translation from centralized laboratories to point-of-care (POC) and resource-limited settings remains limited by lengthy assay procedures, high reagent consumption, and reliance on conventional analytical instrumentation. Microfluidic technologies address many of these limitations by enabling precise fluid handling, reducing sample and reagent volumes, and integrating multiple analytical processes within miniaturized platforms. In this review, we critically examine recent advances in enzymatic microfluidic systems for biomarker analysis, with particular emphasis on their applicability to POC diagnostics. Rather than providing a descriptive overview, we systematically compare pressure-driven, centrifugal, droplet-based, and digital microfluidic platforms with respect to device architecture, reaction kinetics, detection strategies, analytical performance, system integration, and clinical applicability. We further discuss how microscale factors, including thermal regulation, mass transfer, surface interactions, and enzyme immobilization, influence the performance of enzymatic assays across different platforms. In addition, we critically assess the current level of experimental and clinical validation, highlighting persistent challenges related to reproducibility, standardization, scalability, and clinical translation. By linking microfluidic design principles with enzymatic assay performance, this review provides a conceptual framework for the rational development and future clinical implementation of next-generation microfluidic enzymatic assays.