Single-molecule enzymatic analysis in a droplet-based microfluidic system

The kinetic activity of individual enzyme molecules was determined in aqueous droplets generated in a nano- and microfluidic device. To avoid high background noise, the enzyme and substrate solution was confined into femtolitre carriers to achieve single-molecule encapsulation. The tiny droplets (f~2.5-3 μm) generated from this fluidic system were highly monodisperse, beneficial for an analysis of single enzyme activity. Single-enzyme kinetics has previously been demonstrated in the microfluidic format in PDMS containers [1], surface-immobilized droplets [2], or liposomes [3]. Single- enzyme analysis in the droplet-based microfluidics was reported before [4] by encapsulating highly-diluted enzyme solution (110 fM) into large droplets (f~40 μm). But to our knowledge this is the first demonstration of the direct method of single enzyme encapsulation and analysis at high enzyme concentration in tiny droplets in a microfluidic system.