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  • 學位論文

利用超靈敏矽奈米線場效應電晶體偵測活細胞釋放之神經傳導物

An Ultra-sensitive Nanowire-Transistor Biosensor for Detecting Neurotransmitters Release from Living cells under Stimulation

指導教授 : 陳逸聰
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摘要


過去十年來,由於矽奈米線場效應電晶體 (silicon nanowire field-effect transistor , SiNW-FET) 具有可調控的導電性與生物相容性,其作為生物感測器,在生物學研究的應用上廣受矚目。將矽奈米線表面修飾受體,SiNW-FET 即可即時性並且選擇性偵測特定的目標分析物,同時 SiNW-FET 亦具有高靈敏度與免標記等特性。到目前為止,SiNW-FET 已經成功地被應用於偵測蛋白質、DNA、癌症標記物、病毒以及其他生化分子。 本論文中有兩個研究主題,第一個研究主題是以超靈敏奈米線電晶體生物感測器偵測缺氧刺激下的 PC12 細胞釋放的多巴胺。多巴胺 (dopamine, DA) 是重要的神經傳導物,負責神經細胞的訊號傳遞,並且與許多疾病有關。然而,在這些病患血液或尿液中的 DA 濃度非常低,而現有的電化學感測器對 DA 的偵測極限約為 10-9 M,要用於檢測這些疾病是非常困難的。在此研究中,我們藉由在網路式(multiple-parallel-connected, MPC) 矽奈米線場效應電晶體上修飾去氧核醣核酸適體 (DNA-aptamer) ,建構出一個具有高靈敏度與選擇性 DA 感測器,稱之為 (MPC DA-specific aptamer/SiNW-FET)。實驗結果證明 MPC DA-specific aptamer/SiNW-FET 對 DA 的偵測極限可低於 10-11 M 且其可區分開其他化學相似物 (包含抗壞血酸、鄰苯二酚、苯乙胺、酪胺酸、腎上腺素和正腎上腺素),選擇性地偵測 DA。MPC DA-specific aptamer/SiNW-FET 更進一步應用於即時偵測缺氧刺激下的 PC12 細胞釋放的 DA,實驗結果顯示以缺氧刺激使 PC12 細胞釋放 DA 的機制中,細胞內鈣離子的上升主要是細胞外的鈣離子經由鈣離子通道運送到細胞內而所導致的,而並非儲存在細胞內胞器的鈣離子釋放出來所引起。 第二個研究主題是以修飾 DNA-aptamer 之矽奈米線場效應電晶體偵測神經肽 Y。神經肽 Y (neuropeptide Y, NPY) 是重要的神經傳導物,負責神經細胞的訊號傳遞且與許多疾病有關。目前常用於偵測與定量多巴胺的方法有酵素結合免疫吸附法 (Enzyme-linked immunosorbent assay, ELISA) 與液相層析質譜儀 (Liquid chromatography–mass spectrometry, LC-MS/MS),然而這兩種方法皆無法進行即時偵測,因此在細胞釋放神經傳導物研究的應用上有其限制。在本研究中,我們藉由在 SiNW-FET 上修飾 DNA-aptamer 作為受體,建構出一個可即時性與選擇性偵測 NPY 的生物感測器,稱之為 NPY-specific aptamer/SiNW-FET。實驗結果顯示 NPY-specific aptamer/SiNW-FET 偵測 NPY 的線性工作範圍為 10-7~10-5 M且偵測極限為 24 nM,且其亦成功地被應用於即時偵測缺氧刺激下的 PC12 細胞釋放之多巴胺。 在本研究中,本實驗室的李博仁博士與劉佳融研究助理負責製作 MPC SiNW-FET;陳彥溪研究助理與潘建源教授實驗室的葉乃馨同學負責培養 PC12 細胞;我負責量測 MPC SiNW-FET 的電性,確認其品質,並且將其應用於偵測多巴胺和神經肽Y等生化分子。

並列摘要


Over the past decade, silicon nanowire field-effect transistors (SiNW-FETs) with their tunable conductivity and biocompatibility have become significant biosensors. Moreover, the thin silicon oxide sheathes on SiNW-FETs are easily functionalized with selected receptors prior to bio-detections, providing strong and specific binding affinity for biomolecules. Because of strong interaction between the receptors and biomolecules, modified SiNW-FETs have been employed for the detections of protein, DNA, cancer markers, viruses, and other biochemical species. The first part of this thesis focuses on the detection of dopamine (DA) released from living PC12 cells under hypoxic stimulation using an ultrasensitive SiNW-FET biosensor. DA is an important neurotransmitter which plays crucial roles in neuronal signal transduction and causes several critical illnesses. However, it is difficult to detect the extremely low DA content in patients using existing electrochemical biosensors with detection limits typically around nanomolar levels (∼10−9 M). This thesis describes a DNA-aptamer modified multiple-parallel-connected (MPC) SiNW-FET (referred as MPC DA-specific aptamer/SiNW-FET) device for ultrasensitive and selective DA detection. The MPC DA-specific aptamer/SiNW-FET has been employed to improve the detection limit of DA to <10−11 M and the specifically distinguishing ability from other chemical analogues, such as ascorbic acid, catechol, phenethylamine, tyrosine, epinephrine, and norepinephrine. The MPC DA-specific aptamer/SiNW-FET was also employed to monitor DA release under hypoxic stimulation from living PC12 cells in real time. The experimental results revealed that the increase in intracellular Ca2+ that is required to trigger DA secretion is dominated by an extracellular Ca2+ influx, rather than the release of intracellular Ca2+ stores. The second project of the thesis focuses on the detection of neuropeptide Y (NPY) using a DNA-aptamer modified SiNW-FET. NPY is an important neurotransmitter and is related with several critical diseases. Two most common detection and quantification techniques for NPY are enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–mass spectrometry (LC-MS/MS). However, both techniques lack the real-time detection ability for NPY and are not suitable for cellular investigation. Herein, we report a DNA-aptamer modified SiNW-FET (referred to as NPY-specific aptamer/SiNW-FET) applicable for the real-time and selective detection for NPY. The linear working range of the NPY-specific aptamer/SiNW-FET for NPY detection spanned from 10-7 to 10-5 M and the limit of NPY detection is 24 nM. The NPY-specific aptamer/SiNW-FET has shown a detection specificity that is able to distinguish NPY from other neurotransmitters, such as dopamine. Furthermore, this NPY-specific aptamer/SiNW-FET has been successfully applied for real-time monitoring DA release from living PC12 cells under hypoxic stimulation.

並列關鍵字

SiNW-FET dopamine neuropeptide Y aptamer biosensor

參考文獻


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