An oil-well method for time-resolved microfluorescence assays

A simple fluorescence assay method has been devised for direct time-resolved recordings of NADH-coupled enzyme reactions in a microliter sample volume in an "oil-well". The apparatus allows practically instantaneous mixing and stirring of two droplets containing the enzyme and substrate, respectively, and which are viewed with a microscope spectrophotometer. This procedure can be used for instance for enzyme kinetic studies of ATPases on a microscale. Thus, by monitoring NADH-fluorescence changes we determined the time course of ATP splitting in nanogram quantities of myosin subfragment 1. In principle, the method can also be used to determine the time course of any other reaction causing fluorescence changes or luminescence changes.