攜鈣素修飾之矽奈米線場效應電晶體偵測蛋白質間之作用

Label-free detection of protein-protein interactions using a calmodulin-modified nanowire transistor

謝博任(Po-Jen Hsieh)

指導教授 ： 陳逸聰

國立臺灣大學/理學院/化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00132

摘要

本論文藉由高靈敏度、可重複使用的矽奈米線場效應電晶體(silicon nanowire field effect transistor, SiNW-FET)生物感測器，偵測心肌肌鈣蛋白（troponin I）、與N型電壓閘門鈣離子通道（n-type voltage-gated Ca2+ channel），作為SiNW-FET可運用在偵測蛋白質間相互作用的指標。
　我們藉由穀胱甘肽（glutathione）與穀胱甘肽轉移酶（glutathione s-tranferase）可逆結合特性，成功地讓SiNW-FET擁有可重複使用的特性，並將GST融合之攜鈣素（calmodulin）修飾在SiNW-FET上，使其可檢測與攜鈣素有相互作用之蛋白質。我們先選擇純化後的心肌肌鈣蛋白當作目標，在有Ca2+的環境中，我們能夠量測到7 nM的偵測極限，並可藉由調控Ca2+濃度，作出濃度對電導度的線性回歸，得到10 nM ~ 1 μM的線性範圍。除了偵測蛋白間相互作用外，我們亦可決定其作用機制，推斷當Ca2+濃度為10-6 M時，才足以活化攜鈣素與心肌肌鈣蛋白相互作用，符合細胞生理濃度。
　更進一步地我們將實驗拓展至生物系統上，將修飾有攜鈣素的SiNW-FET偵測以293T細胞表達的N-型電壓閘門鈣離子通道，儘管細胞萃取液較為複雜，我們仍可即時並選擇性的感測出鈣離子通道，並藉由調控不同濃度的Ca2+，推斷出無論有無Ca2+的存在，N-型電壓閘門鈣離子通道均會與攜鈣素作用。此結果代表著我們成功地將SiNW-FET生物感測器的技術實際應用在細胞生物學中，我們也相信將來SiNW-FET可應用於高通量、快速篩選蛋白質間相互作用。

關鍵字

矽奈米線場效應電晶體 ； 蛋白質間作用 ； 鈣離子 ； 攜鈣素 ； 心肌蛋白 ； N-型鈣離子通道

並列摘要

We demonstrate a highly sensitive, label-free, and reusable silicon nanowire field-effect transistor to detect protein-protein interactions. This reusable sensorial device was made possible by the reversible association-dissociation of glutathione S-transferase-tagged calmodulin with a glutathione modified nanowire transistor. The calmodulin-modified nanowire transistor showed an electrical response selectively to Ca2+ (1 ｕM) and a purified cardiac troponin I (~7 nM) with its conductance change having a linear dependence on the concentration of troponin I in the range between 10 nM and 1 ｕM, consistent with the previously reported concentration range where the dissociation constant for the troponin I-calmodulin complex was determined. The minimum concentration of Ca2+ required to activate the interaction between troponin I and calmodulin was determined around 1 ｕM. We have also successfully demonstrated that the N-type Ca2+ channels expressed in cultured 293T cells could be recognized specifically by the calmodulin-modified nanowire transistor. This highly sensitive and selective nanowire transistor can serve as a high-throughput biosensor to fast screen interacting proteins under physiological conditions.

並列關鍵字

silicon nanowire field-effect transistor ； protein-protein interaction ； calcium ion ； calmodulin ； cardiac troponin I ； N-type calcium channel.

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