Photopatterning Enzymes on Polymer Monoliths in Microfluidic Devices for Steady-State Kinetic Analysis and Spatially Separated Multi-Enzyme Reactions

Abstract

A method for photopatterning multiple enzymes on porous polymer monoliths within microfluidic devices has been developed and used to perform spatially separated multienzymatic reactions. To reduce nonspecific adsorption of enzymes on the monolith, its pore surface was modified by grafting poly(ethylene glycol), followed by surface photoactivation and enzyme immobilization in the presence of a nonionic surfactant. Characterization of bound horseradish peroxidase (HRP) was carried out using a reaction in which the steady-state profiles of the fluorescent reaction product could be measured in situ and then analyzed using a plug-flow bioreactor model to determine the observed maximum reaction rate and Michaelis constant. The Michaelis constant of 1.9 micro mol/L agrees with previously published values. Mass-transfer limitations were evident at relatively low flow rates but were absent at higher flow rates. Sequential multienzymatic reactions were demonstrated using the patternwise assembly of two- and three-enzyme systems. Glucose oxidase (GOX) and HRP were patterned in separate regions of a single channel, and product formation was analyzed as a function of flow direction. Significant product formation occurred only in the GOX to HRP direction. A three-enzyme sequential reaction was performed using invertase, GOX, and HRP. All possible arrangements of the three enzymes were tested, but significant product formation was only observed when the enzymes were in the correct sequential order. Photopatterning enzymes on polymer monoliths provides a simple technique for preparing spatially localized multiple-enzyme microreactors capable of directional synthesis.

Confinement effects on the morphology of photopatterned porous polymer monoliths for capillary and microchip electrophoresis of proteins

“…Single straight channel was etched in a series of depth (5,10,15,20, 50 mm) with the same mask feature width of 600 mm for investigation of confinement effects on monolith morphology. The etched channel plates, with holes drilled for external access, were bonded with 0.17-mm thick 0211 glass cover plates for LSCM observation.…”

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

“…As a post-modification method, the photografting technique [18,22] has been applied to improve hydrophilicity of monoliths for IMERs to reduce nonspecific adsorption of substrates. In contrast, a more straightforward method is to prepare monoliths via hydrophilic monomers [17,23] directly.…”

Monolithic porous polymer stationary phases in polyimide chips for the fast high-performance liquid chromatography separation of proteins and peptides

“…Numerous functional modules for different applications in microfluidic formats, such as pre-concentration and solid-phase extraction (SPE) [39,40], micro reactors [36,41], mixers [42], stationary phases [29] and valves [43] have already been reported.…”