陳逸聰Chen, Yit-Tsong臺灣大學:化學研究所曾坤長Tseng, Kun-ChangKun-ChangTseng2010-06-302018-07-102010-06-302018-07-102009U0001-2107200909553400http://ntur.lib.ntu.edu.tw//handle/246246/187527隨著奈米技術的演進，矽奈米線場效電晶體（silicon nanowire field-effect transistor, SiNW-FET）因其高靈敏度、免標定、可即時偵測等特性，被大量應用在研發為奈米生物感測器，近年來是許多學術團隊的熱門研究課題。此類生物感測器已成功的量測多種生物與醫學上的重要物質，包括：蛋白質、DNA、癌症標誌物、單一病毒的偵測，以及神經傳導物質等多項重要指標物，將來不論是在藥物開發上、還是生化物質快速偵測分析上，都非常具有研究潛力與應用價值。統上，大部分此類的生物晶片，在修飾特定蛋白分子或是抗體時，都是以共價鍵結的方式，缺點有兩個：如果被修飾分子（probe）與被偵測分子（target）的作用是不可逆的，如抗體與抗原，那這個晶片只能使用一次；也因為probe是化學鍵結在晶片上，所以此類晶片一旦修飾後，只能做單一種類的分子偵測，兩種結果都會造成晶片的浪費。論文的研究主題為，建立一個可重複使用的生物晶片，因此，我們導入，一般在純化蛋白的技術上常用的穀胱甘肽（glutathione, GSH）與穀胱甘肽硫基轉移酶（glutathione S-transferase, GST）的系統。由於GSH與GST之間具有適中的作用力（Kd ~106），可進行重複的結合（association）與分離（dissociation），將其應用於SiNW-FET，便能作為可重複修飾使用蛋白分子的生物感測器。經由化學分析電子光譜、螢光實驗、原子力顯微鏡與電性量測的實驗，我們的確成功在單一晶片上，進行蛋白分子的重複修飾，而達到重複使用的目的，且能更進一步地偵測蛋白質與蛋白質之間的作用，將來極具有潛力與應用價值，發展為可快速篩選可能作用蛋白的系統平台，或是應用在偵測蛋白與DNA、RNA、醣類等其他生化分子的作用。Over past years, the silicon nanowire field-effect transistors (SiNW-FETs) as chemical and biological sensors have attracted wide attention because of their merits of free labeling, ultrasensitivity, and real-time recording. There are many applications of SiNW-FETs, such as immuno-protein detection, DNA hybridization, virus detection, etc. However, most of applications are based on an irreversible functionalization of receptor molecules on the surface of SiNW-FETs. Consequently, one chip that could only be used for a single time to detect one protein is very wasteful in chip-consuming. lutathione-S-transferase (GST) is widely applied to fuse with a target protein by genetic engineering in protein purification. The GST-fused protein can be separated from a lysate by the glutathione-coated beads via its high affinity for glutathione (GSH).ased on the reversible association-dissociation of GSH-GST, we provide a novel strategy of using a reusable GSH modified SiNW-FET to immobilize a GST-fusion protein and then to screen possible interacting proteins. The reversible surface-functionalization method is proven by ESCA, fluorescence imaging, atomic force microscopic imaging, and electrical measurements. The reversible GSH-GST functionality on the SiNW-FET has made this device consecutively reusable, allowing for quantitative analysis. In this work, we have successfully demonstrated that the reusability of GSH-functionalized device should be a very useful and economic biosensor. his reversible surface functionalized biosensor can potentially serve as a fast high-throughput screening platform for detecting proteins which could possibly interact with a particular GST-fused protein, and also be further applied to study biomolecular associations, such as protein-protein interactions, protein-DNA interactions, protein-carbohydrate interactions, and other similar interactions.口試委員審定書……………………………………………………………i辭………………………………………………………………………...ii文摘要…………………………………………………………...……...iv文摘要…………………………………………………………………..v錄……………………………………………………………………….vii目錄………..…………………………………………………………x目錄……………………………………………………………………xii一章 序論…………………………………………………………..1-1. 場效應電晶體簡介…………………………………………..……1-1.1. 發展簡史…..…………………………………………………1-1.2. 金氧半場效應電晶體………………………………………..1-1.3. 矽奈米線場效應電晶體……………………………………..3-2. 實驗目的…………………………………………………………..7-2.1. 實驗動機……………………………………………………..7-2.2. 實驗構思………………………………….………………….9-2.3. 實驗目的……………………………………………………10二章 實驗方法…………………..…………………………………..13-1. 矽奈米線場效應電晶體製作……………………………………13-2. 緩衝溶液配製……………………………………………………15-3. 晶片表面修飾方法………………………………………………16-3.1. 清潔晶片……………………………………………………16-3.2. 修飾連接分子與穀胱甘肽（GSH）…………………………16-4. 修飾證明方法……………………………………………………17-4.1. 化學分析電子光譜術………………………………………17-4.2. 螢光影像實驗..……………………………………………..18-4.3. 原子力顯微鏡………………………………………………20-5. 電性量測實驗……………………………………………………21三章 實驗結果與討論………………………………………………23-1. 表面修飾證明……………………………………………………23-1.1. 化學分析電子光譜術.……………………………………..23-1.2. 螢光影像實驗………………………………………………28-1.3. 原子力顯微鏡………………………………………………29-2. 電性量測實驗……………………………………………………30-2.1. 離子強度對蛋白質偵測之訊號的影響……………………30-2.2. 專一性實驗…………………………………………………30-2.3. 可重複使用性………………………………………………35-2.4. 蛋白質間作用偵測…………………………………………36-2.5. 檢量性………………………………………………………38四章 結論與未來展望………………………………………………41考文獻………………………………………………………………….42獻發表………………………………………………………………….454798596 bytesapplication/pdfen-US場效應電晶體重複使用穀胱甘肽生物晶片silicon nanowirefield-effect transistorreusableglutathionebiosensorthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/187527/1/ntu-98-R96223155-1.pdf