Liu Ai-Lin, Zhou Ting, He Feng-Yun, Xu Jing-Juan, Lu Yu, Chen Hong-Yuan, Xia Xing-Hua
Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093, P. R. China.
Lab Chip. 2006 Jun;6(6):811-8. doi: 10.1039/b514612g. Epub 2006 Mar 20.
We firstly transformed the traditional Michaelis-Menten equation into an off-line form which can be used for evaluating the Michaelis-Menten constant after the enzymatic reaction. For experimental estimation of the kinetics of enzymatic reactions, we have developed a facile and effective method by integrating an enzyme microreactor into direct-printing polymer microchips. Strong nonspecific adsorption of proteins was utilized to effectively immobilize enzymes onto the microchannel wall, forming the integrated on-column enzyme microreactor in a microchip. The properties of the integrated enzyme microreactor were evaluated by using the enzymatic reaction of glucose oxidase (GOx) with its substrate glucose as a model system. The reaction product, hydrogen peroxide, was electrochemically (EC) analyzed using a Pt microelectrode. The data for enzyme kinetics using our off-line form of the Michaelis-Menten equation was obtained (K(m) = 2.64 mM), which is much smaller than that reported in solution (K(m) = 6.0 mM). Due to the hydrophobic property and the native mesoscopic structure of the poly(ethylene terephthalate) film, the immobilized enzyme in the microreactor shows good stability and bioactivity under the flowing conditions.
我们首先将传统的米氏方程转化为一种离线形式，该形式可用于在酶促反应后评估米氏常数。为了对酶促反应动力学进行实验估计，我们通过将酶微反应器集成到直接打印的聚合物微芯片中，开发了一种简便有效的方法。利用蛋白质的强非特异性吸附将酶有效地固定在微通道壁上，在微芯片中形成集成的柱上酶微反应器。以葡萄糖氧化酶（GOx）与其底物葡萄糖的酶促反应为模型系统，对集成酶微反应器的性能进行了评估。使用铂微电极对反应产物过氧化氢进行电化学（EC）分析。获得了使用我们的米氏方程离线形式的酶动力学数据（K(m) = 2.64 mM），该数据远小于溶液中报道的数据（K(m) = 6.0 mM）。由于聚对苯二甲酸乙二酯薄膜的疏水性和天然介观结构，微反应器中固定化的酶在流动条件下表现出良好的稳定性和生物活性。
