Rational Design of a New Class of Versatile Enzyme-Based Biosensors

The majority of enzyme-based sensors rely on electrochemical approaches for continuous monitoring. For example, commercially available glucometers are electrochemical-based sensors. However, these sensors are not suitable for contactless monitoring as electron signals require direct conduction from the enzyme reaction site to the signal analyzing unit. Fluorescent dyes, on the other hand, emit photons that can penetrate certain barriers, making them ideal candidates for contactless monitoring. In this study, we investigated the design and functionality of a new class of biosensors based on the conjugation of enzymes with pH-sensitive fluorophores, creating a novel single-molecule biosensor capable of versatile, contactless detection of different disease- and treatment-related biomarkers. We conjugated various enzymes (glucose oxidase, phenylalanine ammonia-lyase, and β-lactamase) with pH-sensitive fluorophores (FITC and pH-sensitive Cy7 derivatives) and tuned linkers’ properties to modulate the distance between the enzyme and fluorophore, as well as the hydrophilicity of the linker. The experimental data demonstrate that fluorophore-conjugated enzymes exhibit substrate-dependent fluorescence responses under physiologically relevant buffered conditions, enabling the quantitative analysis of substrate concentrations through fluorescent signal detection. This innovative sensor design not only provides critical insights into enzyme-based fluorescent detection mechanisms but also represents a promising candidate for the development of next-generation contactless biosensing platforms.