Activity and lifetime of organophosphorous hydrolase (OPH) immobilized using layer-by-layer nano self-assembly on silicon microchannels

Abstract

Organophosphorous hydrolase (OPH) enzyme has been immobilized and layered onto the walls of silicon microchannels manufactured at Louisiana Tech University. Enzyme immobilization was performed using layer-by-layer (LbL) nano self-assembly. The polycation used was poly(ethylenimine) (PEI). The polyanion used was poly(styrenesulfonate) (PSS). Enzyme microreactors were constructed with one and two layers of enzyme to compare activity performance. The substrate utilized for this study was methyl-parathion (MPT) which is hydrolyzed by the OPH enzyme to para-nitrophenol (PNP). Reactor performance was characterized by feeding 100 μM MPT through the microreactors at a variety of flow-rates and measuring the concentration of PNP in the effluent using a UV/vis spectrometer. The reactor conversion versus residence time was used to determine the observed first order rate kinetics. The first order rate constant for the two bilayers of enzyme was double that observed for the single layer architecture, however the activity of both architectures dropped substantially over a period of 2 days. Microreactors with two channel dimensions were tested, one with 98 parallel channels 60 μm wide and another with 1000 parallel channels 5 μm wide. The microreactor with the smaller channel width demonstrated superior performance that was proportional to the increase in available surface area.