A digital microfluidic approach to homogeneous enzyme assays

摘要

A digital microfluidic device was applied to a variety of enzymatic analyses. The digital approach to microfluidics manipulates samples and reagents in the form of discrete droplets, as opposed to the streams of fluid used in channel microfluidics. This approach is more easily reconfigured than a channel device, and the flexibility of these devices makes them suitable for a wide variety of applications. Alkaline phosphatase was chosen as a model enzyme and used to convert fluorescein diphosphate into fluorescein. Droplets of alkaline phosphatase and fluorescein diphosphate were merged and mixed on the device, resulting in a 140-nL, stopped-flow reaction chamber in which the fluorescent product was detected by a fluorescence plate reader. Substrate quantitation was achieved with a linear range of 2 orders of magnitude and a detection limit of similar to 7.0 x 10(-20) mol. Addition of a small amount of a nonionic surfactant to the reaction buffer was shown to reduce the adsorption of enzyme to the device surface and extend the lifetime of the device without affecting the enzyme activity. Analyses of the enzyme kinetics and the effects of inhibition with inorganic phosphate were performed, and K-m and k(cat) values of 1.35 mu M and 120 s(-1), respectively, agreed with those obtained in a conventional 384-well plate under the same conditions (1.85 mu M and 155 s(-1)). A phototype device was also developed to perform multiplexed enzyme analyses. It was concluded that the digital microfluidic format is able to perform detailed and reproducible assays of substrate concentrations and enzyme activity in much smaller reaction volumes and with higher sensitivity than conventional methods.