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Issue 5, 2009
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Enzymatic activity of surface-immobilized horseradish peroxidase confined to micrometer- to nanometer-scale structures in nanocapillary array membranes

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Abstract

Horseradish peroxidase (HRP) was immobilized on the planar surfaces and inside the cylindrical nanopores of nanocapillary array membranes (NCAMs) to study how the enzyme-catalyzed oxidation of a fluorigenic substrate, Amplex Red (AR), to fluorescent resorufin by hydrogen peroxide is influenced by confinement. Because AR was also found to be converted to resorufin photolytically at high laser fluences, a modified laser-induced fluorescence protocol was developed to characterize the enzyme-catalyzed reaction in the absence of interference from the photolytic reaction. Surface-immobilized HRP was studied in two environments: bound to the surface of a microfluidic channel, and bound to the interior of cylindrical nanopores in NCAMs connecting crossed microfluidic channels. HRP was immobilized through reaction of solvent-accessible primary amines with the epoxy group of the methyl methacrylate–glycidyl methacrylate copolymer synthesized in either planar or annular geometries to construct the test structures for enzymatic activity. HRP immobilized on planar surfaces shows high activity (∼10 µM min−1) meaning that the copolymer membrane exhibits good potential for immobilizing the enzyme, especially since active structures are obtained in a one-step reaction. HRP was also immobilized inside nanopores via physisorption. Enzymatic reactions inside the nanopores were characterized and compared to finite element simulations of a modified Eley–Rideal mechanism to bracket the value of the overall rate constant for the confined enzyme. Reaction velocities were estimated to be ∼10-fold higher in the nanopores than for the same enzyme bound to a planar microfluidic surface.

Graphical abstract: Enzymatic activity of surface-immobilized horseradish peroxidase confined to micrometer- to nanometer-scale structures in nanocapillary array membranes

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Publication details

The article was received on 05 Sep 2008, accepted on 05 Feb 2009 and first published on 27 Feb 2009


Article type: Paper
DOI: 10.1039/B815590A
Citation: Analyst, 2009,134, 851-859
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    Enzymatic activity of surface-immobilized horseradish peroxidase confined to micrometer- to nanometer-scale structures in nanocapillary array membranes

    Z. Wang, T. L. King, S. P. Branagan and P. W. Bohn, Analyst, 2009, 134, 851
    DOI: 10.1039/B815590A

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