Method of conducting a droplet based enzymatic assay

A method of conducting a droplet-based enzymatic assay, the method comprising:

• (a) providing a droplet actuator;
• (b) providing on the droplet actuator:
• (i) a droplet comprising an enzyme of interest;
• (ii) a droplet comprising a substrate which is potentially modified in the presence of the enzyme;
• (c) executing droplet operations on the droplet actuator to combine the droplet of (b)(i) with the droplet of (b)(ii), thereby yielding an assay droplet, wherein the droplet operations are electrowetting mediated;
• (d) detecting modification of the substrate by the enzyme in the assay droplet on the droplet actuator;
• and
• wherein each of the droplet of (b)(i), the droplet of (b)(ii) and the assay droplet is at least partially surrounded by a silicone oil filler fluid comprising an nonionic low hydrophile-lipophile balanced (HLB) surfactant.