「固定化酵素動力參數量測平台」之模型建構與計算

Abstract

有效整合top-down與bottom up製程技術於前瞻跨領域「奈米生醫電子」至為關鍵；其中如何評估局部固定化在元件無機介面上生物分子的工作性能就顯得十分重要，這些生物分子可以是DNA、RNA、蛋白質(特別是具有辨識與催化的酵素分子)。在先期測試中施以掃描電壓於固定化酵素，雖然其活性調控的現象可以被清楚地觀測到，然而卻無法量測固定化酵素動力參數的即時改變趨勢。因此，本研究團隊在執行94年度奈米國家型科技計畫學術卓越計畫─「生化感測與仿生調控功能的奈米結構與生物分子混成系統之研究」時，也規劃了生物調控器的研究子題；因應評估將酵素嵌入標準積體電路和微機電系統的平面化技術，我們著手開發了一套可靠合理的即時偵測與分析固定化酵素動力的量測平台。 在本研究中固定化於二氧化矽基材表面的酵素共有三株，它們分別是老鼠酚亞硫酸基轉移酵素(E.C.2.8.2.1)、醯亞胺水解酵素(E.C.3.5.2.2)、假絲酵母菌脂肪分解酵素(E.C.3.1.1.3)。兩項主要的貢獻與理論特徵是：(1)針對符合Michaelis-Menten動力學模式的酵素催化反應並考量質量傳送效應，以系統化、標準化建立表面反應限制的模型用以量測固定化於平面基材的酵素視動力參數值 K*m 和 V*max；(2)根據上述數學模型建構標準流程化的實驗操作方式，並對Michaelis-Menten參數估算提出新的線性圖解法，其斜率即為視參數K*m值，且縱座標與橫座標分別具有直觀的物理意義─縱軸表示在兩個極端基質濃度下反應產率的差值，橫軸在低基質濃度下則近似於基質濃度；此圖解法亦有利於數值分析求解。 我們以流道高度167微米的微流體反應器為實驗平台，成功地量測了老鼠酚亞硫酸基轉移酵素與假絲酵母菌脂肪分解酵素固定化在二氧化矽基材的及時視動力參數值 K*m 和 V*max；同時本研究也針對固定化酵素失活與基質溶解度限制的實務問題，提出對應的解決方法。整套量測系統將使我們有能力觀察到電訊號調控固定化酵素活性的動力參數改變的量值，進而研究相應的調控機制。

How to efficiently combine top-down and bottom-up approaches has become essential in the interdisciplinary field of nano-bio-electronics. It is also important to be able to evaluate the performance of working bio-molecules, like DNA, RNA, proteins (especially for enzymes), immobilized and localized onto the surface of inorganic devices. Although a response of modulated activity was clearly observed via applying a voltage scan onto immobilized enzymes in previous pilot testing, there was no available scheme used to characterize the intrinsic properties of the immobilized enzymes and the corresponding effect of in-situ stresses for further analyzing the modulated mechanism. For developing “ An Artificial-Bio Hybrid Nano-System Capable of Sensing and Regulation,” a reliable and reasonable analysis of immobilized enzyme in situ became a crucial step to embed enzyme onto the planar technology of standard IC and MEMS for the bioregulator subprogram, which belonged to National Research Program for Nanoscience and Technology in the period 2005-2008. In this study, we have successfully immobilized three enzymes, rat-phenol-sulfotransferase (rat-PST, E.C. 2.8.2.1), D-hydantoinase (E.C. 3.5.2.2), and Candida rugosa lipase (CRL, E.C. 3.1.1.3) onto the silicon dioxide surface. The main contributions and theoretical characteristics should be: (1) A surface reaction limited model, based on systematic and standardized approach, mathematically derived from mass transfer dynamics and Michaelis-Menten equation for measuring apparent K*m and V*max of immobilized enzyme on planar surface was developed. (2) A new linear plot proposed with a slope K*m, of which axes containing straightforward, meaningful parameter groups - the difference in reaction yield between two extreme substrate levels (y-axis) versus the reaction conversion fraction (x-axis), is simple to apply either graphically or numerically. The K*m and V*max of rat-PST and CRL immobilized on silicon oxide surface were successfully determined in situ. The issues of enzyme inactivation during activity assays and limit of substrate solubility were both concerned in developing measurement approach in this study. Based on this platform, we will be able to make quantitative analysis of electric signal regulation on enzyme activity, and to study its related fundamental mechanisms.