Enzymatic activity of surface-immobilized horseradish peroxidase confined to micrometer- to nanometer-scale structures in nanocapillary array membranes.
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.
Wang
Zhen
Z
Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.
King
Travis L
TL
Branagan
Sean P
SP
Bohn
Paul W
PW
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
D004800:Enzymes, Immobilized; D010078:Oxazines; C470430:Amplex Red; C014180:resorufin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase