氧化銦鎵鋅薄膜電晶體生物感測器應用於蛋白質與配體之動態反應分析

Abstract

這篇論文介紹以氧化銦鎵鋅薄膜電晶體與感測金屬電極組成之生物感測器偵測生物分子擴散與混合狀態並探討蛋白質與配體之動態反應，此研究分兩部分： 第一部分，分析生物素與鏈親和素的混合情況，我們採用薄膜電晶體生物感測器外接Y型微流道。此外，使用聚二甲基矽氧烷來密封微流道系統以避免待測溶液蒸發。接著分別量測生物素與鏈親和素的電流訊號，定義待測物的擴散時間。進行一系列同時與時間差的混合實驗。藉觀察即時的電流變化，分析待測物在流道中的混合狀況。最後我們使用牛血清白蛋白作為對照組，驗證薄膜電晶體生物感測器之非特異性結合情況。 第二部分，以溶菌酶及其適體三乙醯殼三糖作為動態反應分析標的物，改使用直線型微流道作為感測平台。溶菌酶以及三乙醯殼三糖溶液注入微流道感測電流訊號。首先，單獨通入溶菌酶溶液至流道中建立溶菌酶濃度與電流變化關係。將三種濃度比例之溶菌酶以及三乙酰殼三糖混合在離心管中，控制兩者的反應時間；對擷取之電流變化，考量屏蔽效應進行修正後可藉已建立之溶菌酶濃度與電流變化關係將電流變化轉為剩餘溶菌酶濃度。以此，可建立剩餘溶菌酶濃度與反應時間擬合曲線。曲線提供之資訊，可助我們藉化學公式得到反應級數、結合速率常數與分解常數。其中，分解常數之結果為39.10μM，與其他團隊提出之數值十分接近。

In this thesis, a biosensor consists of an Indium-Gallium-Zinc-Oxide (IGZO) thin film transistor (TFT) and a gold sensing electrode is demonstrated for diffusion and mixing properties detection of biomolecules. The protein-ligand kinetic reaction is further investigated. The thesis includes two parts. In the first part, in order to analyze the streptavidin-biotin mixing condition, a Y-type external microfluidic channel is integrated with the TFT biosensor. In addition, the channels are sealed with polydimethylsiloxane to avoid evaporation of the target analyte solutions. The current signals of streptavidin and biotin are measured separately to define the diffusion time. Then, a series of mixing and time delay experiments are conducted. By observing the real-time current change of the TFT biosensor, the mixture condition of the reaction can be analyzed. Finally, bovine serum albumin (BSA) is used for a control experiment to verify the specificity and reliability. In the second part, kinetic reaction of lysozyme and tri-N-Acetylglucosamine (NAG3) are investigated and applied to a TFT biosensor integrated with a linear shape microfluidic channel. First, lysozyme solutions of several concentrations are introduced into the microfluidic channel to construct the relation between lysozyme concentration and drain current variation. Then, three mixing ratios of lysozyme and NAG3 solution are incubated in the micro-centrifuge for different periods of reaction time. Considering the screen effect, the extracted drain current variations are calibrated by the revision factor and the revised current variations are converted into remained lysozyme concentration by the correlation of current variation and lysozyme concentration. Based on the converted information, the fitting curves of remained lysozyme concentration versus reaction time are illustrated. The curves provide the information that can be utilized to calculate the partial orders, association rate constant, and dissociation constant by biochemical formulas. It is noteworthy that the derived dissociation constant is 39.10 μM, which is close to the results reported by previous researches.