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.

Experimental and computational evaluation of area selectively immobilized horseradish peroxidase in a microfluidic device

“…x 10 4 M -1 cm -1 ) is shifted to 414 nm (ε 414 =3.6 x 10 4 M -1 cm -1 ) for its oxidized product ABTS •+ , from which the activity of immobilized HRP can be determined [41]. Kinetic parameters of immobilized HRP have been studied previously in a microfluidic packed bed reactor [18], monolith structures [42] or on planar surfaces [43]. It has been…”

Lab-on-a-Chip and Fluid Manipulation Applications

“…Applications employing nanochannelimmobilized enzymes are particularly attractive. In another study, reaction-diffusion modeling was combined with measurements of enzymatic reaction velocities during injection-relaxation cycles across an NCAM to show that the enzymatic activity of immobilized horseradish peroxidase in NCAMs is up to 100 times higher than in free solution [51,52]. Figure 5.17 from [72] illustrates the immobilization of the enzyme, invertase, onto a nanoporous membrane for the conversion of sucrose into glucose and fructose.…”

Enzyme Technology of Peroxidases: Immobilization, Chemical and Genetic Modification

“…The stability on acetonitrile mixtures was enhanced [13] (continued) The HRP was immobilized on NCAMs planar surfaces and also inside its nanopores. Reaction velocities were estimated to be $10-fold higher in the nanopores than for the enzyme bound to the planar surfaces [14] Coimmobilized lignin peroxidase (LiP) and GOx on nanoporous gold…”