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Microfluidic devices allow manipulation of reagents and fluids in a semi-automated fashion, ideal for performing multiple measurements or conditioning various reagents. Here, an enzyme assay has been performed in a multilayer poly(methyl methacrylate)-based microfluidic device, where the layers are fluidically connected via a nanocapillary array membrane serving as an effective injector and valve. As a model system, beta-glucuronidase from Escherichia coli and fluorescein di(beta-D-glucuronide) are used for the assay; offline mixing and online incubation of substrate and enzyme allow determination of the initial hydrolysis rates of the substrate under catalysis by beta-glucuronidase. The Michaelis constant Km was determined to be ~4.0 muM for the enzyme of 83 units/mL at ambient temperature. The 50% inhibitory concentration IC50 of D-saccharic acid-1,4-lactone to 167 units/mL was estimated to be 3.0 muM. These results demonstrate added functionality for a poly(methyl methacrylate)-based nanocapillary array membrane-containing microfluidic device for following enzyme reaction kinetics.