Absorption‐Based Rapid Acquisition of Single‐Molecule Kinetics from Unstable Enzymes in Microdroplets

Abstract Single‐molecule catalysis reflects the heterogeneity of each molecule, providing a unique insight into the complex catalytic mechanism through the statistics of stochastic individuals. However, the present study methods for single‐molecule catalysis are either complicated or have low throughput, limiting their rapid acquisition of single‐molecule reaction kinetics with statistical significance. Here, a label‐free imaging method is developed for the study of single‐molecule catalysis in microdroplets with high throughput based on the absorption of the reaction molecules. A wide distribution of the catalytic reaction rate constant value of 238–2026 molecules s −1 is observed from 68 single enzymes. Interestingly, an exponential decayed distribution of the enzyme activity can be clearly observed due to the rapid denaturation of the enzymes. The denaturation mechanism of the Horse Radish Peroxidase (HRP) enzyme is clarified. It is revealed that the denaturation of each enzyme goes through a gradual decay rather than a truncated turn‐off process from a single molecule point of view. This absorption‐based method can be applied to most of the catalytic reactions with high throughput, which offers an indispensable route for the rapid statistical analysis of various single‐molecule catalytic reactions, making it particularly suitable for the acquisition of catalytic kinetics from highly unstable enzymes.