Preparation of reversibly immobilized Jack bean urease on microchannel surface and application for enzyme inhibition assay

Abstract

We have developed a sensitive enzyme inhibition assay on microfluidic system. The analysis was carried out by immobilizing enzyme through amide-bond or disulfide-bond formation with surface. Followed by detection of reaction product through fluorescent density were evaluated reusability, stability and sensitivity of microfluidic enzyme assay. The Michaelis–Menten parameters for free urease (K M = 1.027 μM) and for immobilized urease (K M = 1.528 μM of disulfide-bond immobilization; K M′ = 1.617 μM of amide-bond immobilization) showed reasonable activities maintained after immobilization with relative standard deviation (RSD) of 4.86 and 6.06 %, respectively. When compared enzyme activities of five repeated immobilization cycles through reversible disulfide-bond immobilization, we found that removal process and reversible immobilization did not affect efficiency of microreactor with RSD of 4.78 %. The IC50 value 368 μM of inhibitor acetohydroxamic acid determined on chip showed good agreement with reported data 375 μM; K i of 1.39 μM matched well with K i of 1.46 μM via the traditional 96-microplate. This microfluidic could be extended to screening of enzyme inhibitor and enzymatic reaction kinetics study, which may be useful for clinical diagnostics, biotechnological research, drug discovery and other bioassays.