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An On-Chip Fluorogenic Enzyme Assay Using a Multilayer Microchip Interconnected With a Nanocapillary Array Membrane

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6 Author(s)
Maojun Gong ; Dept. of Chem., Univ. of Illinois at Urbana-Champaign, Urbana, IL ; Bo Young Kim ; Flachsbart, Bruce R. ; Shannon, M.A.
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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.

Published in:

Sensors Journal, IEEE  (Volume:8 ,  Issue: 5 )

Date of Publication:

May 2008

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