Stopped-Flow Enzyme Assays on a Chip Using a Microfabricated Mixer

This paper describes a microfabricated enzyme assay system including a micromixer that can be used to perform stopped-flow reactions. Samples and reagents were transported into the system by electroosmotic flow (EOF). Streams of reagents were merged and passed through the 100-pL micromixer in <1s. The objective of the work was to perform kinetically based enzyme assays in the stopped-flow mode using a system of roughly 6 nL volume. (beta)-Galactosidase ((beta)-Gal) was chosen as a model enzyme for these studies and was used to convert the substrate fluorescein mono-(beta)-D-galactopyranoside (FMG) into fluorescein. Results obtained with microfabricated systems using the micromixer compared well to those obtained with an external T mixing device. In contrast, assays performed in a microfabricated device by merging two streams and allowing mixing to occur by lateral diffusion did not compare well. Using the microfabricated mixer, Km and kcat values of 75 +- 13 (mu)M and 44 +- 3 s^-1 were determined. These values compare well to those obtained with the conventional stopped-flow apparatus for which Km was determined to be 60 +- 6 (mu)M and kcat was 47 +- 4 s^-1. Enzyme inhibition assays with phenylethyl-)beta)-D-thiogalactoside (PETG) were also comparable. It was concluded that kinetically based, stopped-flow enzyme assays can be performed in 60 s or less with a miniaturized system of roughly 6 nL liquid volume when mixing is assisted with the described device.