Lee Cheng-Che, Chiang Han-Ping, Li Kun-Lin, Ko Fu-Hsiang, Su Chien-Ying, Yang Yuh-Shyong
Institute of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan.
Anal Chem. 2009 Apr 1;81(7):2737-44. doi: 10.1021/ac802650k.
Analysis of immobilized enzyme in situ is a crucial step to embed an enzyme onto the planar technology of standard integrated circuit (IC) and microelectromechanical systems (MEMS) for a bioreactor or enzyme-coupled biosensor. A surface reaction limited model, based on a systematized and standardized approach, mathematically derived from mass transfer dynamics and the Michaelis-Menten equation for the measuring the apparent K*(m) (Michaelis-Menten constant) and V*(max) (maximum reaction rate per unit surface area of catalyst) of an immobilized enzyme on a planar surface was developed. The derived equations for the kinetic model were simulated and experimentally confirmed. A platform of a microflow bioreactor with a one-sided planar catalytic surface that contained immobilized enzyme was constructed. The microfluidic bioreactor was designed to possess a channel height less than that of the diffusion layer thickness in a semi-infinite diffusion process, and K*(m) and V*(max) of rat phenol sulfotransferase (PST) immobilized on the silicon oxide surface were successfully determined in situ. Variation in kinetic constants and the possible differences in performance between free and immobilized PST are discussed.
原位分析固定化酶是将酶嵌入用于生物反应器或酶联生物传感器的标准集成电路（IC）和微机电系统（MEMS）平面技术的关键步骤。基于系统化和标准化方法，从传质动力学和米氏方程数学推导得出一个表面反应受限模型，用于测量平面表面上固定化酶的表观K*（m）（米氏常数）和V*（max）（单位催化剂表面积的最大反应速率）。对动力学模型的推导方程进行了模拟并通过实验得到证实。构建了一个具有单面平面催化表面且包含固定化酶的微流生物反应器平台。该微流生物反应器设计为其通道高度小于半无限扩散过程中扩散层厚度，并且成功原位测定了固定在氧化硅表面的大鼠酚磺基转移酶（PST）的K*（m）和V*（max）。讨论了动力学常数的变化以及游离PST和固定化PST之间性能可能存在的差异。
