title: Enzymatic activity of surface-immobilized horseradish peroxidase confined to micrometer- to nanometer-scale structures in nanocapillary array membranes.
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 (approximately 10 microM 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 approximately 10-fold higher in the nanopores than for the same enzyme bound to a planar microfluidic surface.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.forename: Travis L
authors.initials: TL
authors.lastname: King
authors.forename: Sean P
authors.initials: SP
authors.lastname: Branagan
authors.forename: Paul W
authors.initials: PW
authors.lastname: Bohn
mesh_terms: D004800:Enzymes, Immobilized; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide; D046210:Microfluidic Analytical Techniques; D049329:Nanostructures; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010782:Photolysis
chemical_list: D004800:Enzymes, Immobilized; D010078:Oxazines; C470430:Amplex Red; C014180:resorufin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase