Enzymatic biosensors: biocatalytic mechanisms, immobilization strategies, and signal amplification approaches

Enzymatic biosensors are a crucial component of modern analytical science, offering exceptional molecular specificity across clinical, environmental, and industrial applications. Despite their widespread use, the development of reliable biosensor platforms continues to face challenges, particularly regarding enzymatic stability, reproducibility, and long-term operational lifespan. This review critically examines the core principles of high-performance enzymatic biosensors, integrating biochemical fundamentals with advances in materials. Our analysis begins with the kinetic and thermodynamic factors that govern sensor sensitivity and dynamic range, focusing on how immobilization alters Michaelis–Menten parameters and how diffusional and mass-transport limitations impact traditional kinetic models. We also examine environmental factors such as pH, temperature, and cofactor regeneration. Special attention is given to interfacial engineering, comparing oriented and random immobilization, considering factors such as fabrication complexity, cost, enzyme-specific applicability, and activity losses due to modifications. The review evaluates various signal transduction mechanisms, including electrochemical, optical, semiconductor-based, wearable, and emerging in-sensor computing architectures. Using gas-phase biosensing of volatile organic compounds as a case study, we highlight how this niche stresses every design parameter, revealing it to be smaller and more chemically constrained than is often implied. Finally, we discuss major unresolved challenges, especially the lack of standardized benchmarking methods that would permit fair comparison between competing strategies.