Absorption based rapid acquisition of single enzyme kinetics in microdroplets

Single molecule catalysis reflects the heterogeneity of each molecule, providing a unique insight to the complex catalytic mechanism through the statistics of stochastic individuals. However, the present study methods for single molecule catalysis are either complicated or low throughput, limiting their rapid acquisition of single molecules reaction kinetics with statistic significance. Here, we developed a label-free imaging method 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 relative catalytic reaction rate constant value of 0.51-4.32 s-1 was observed from 37 single enzymes. Interestingly, a Boltzmann distribution of the enzyme activity can be clearly observed due to the rapid denaturation of the enzymes. The denaturation mechanism of HRP enzyme was clarified. It was 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, providing a indispensable route for the rapid statistical analysis of various single molecule catalytic reactions, especially when the enzymes is highly unstable.