| 摘要: | 根據世界衛生組織報告：2030年時，全世界每年將會有兩千三百萬人死於心肌梗塞(常被稱為心臟病)。心臟病發後，心肌會隨即釋放出心血管疾病相關的生物標誌物，眾多的生物標誌物中，心肌肌鈣蛋白I(cardiac troponin I, cTnI)與心肌損傷有著高度相關，而心肌梗塞也會伴隨著心肌損傷，因此在心肌梗塞的診斷中，心肌肌鈣蛋白I也被歐洲心臟學會當做檢測的黃金標準。然而，我們需要時間(約1小時)去檢測到顯著的cTnI濃度變化(troponin盲區間)，為了解決上述問題，許多研究團隊致力於發展高靈敏度和高解析度的cTnI檢測方法。而矽奈米線場效應電晶體(silicon nanowire field effect transistor, SiNW FET)具有高靈敏度、免標定檢測以及即時檢測的優勢，被視為極具發展潛力檢測平台。
本實驗室先前發展以3-Aminopropyltriethoxysilane (APTES)固定化於SiNW FET表面，再改質戊二醛(Glutaraldehyde, GA)以及生物探針以進行實驗。本研究初期以延續先前改質方式進行實驗，後期更加入其他兩種不同的改質方法於SiNW FET的效果進行探討，分別是1-(3-Aminopropyl)silatrane(APS)以及?silane-PEG-NH?_2：silane-PEG-OH =1：10 (mM/mM)的混合自組裝單層膜(mixed self-assembled monolayers, mixed SAMs)
首先利用原子力顯微鏡(AFM)分析樣品表面粗糙度以及模擬樣品表面形貌；再利用X光光電子能譜儀(XPS)進行樣品表面元素分析，確認每一改質步驟的正確性；最後比較利用各種表面改質在SiNW FET上進行對cTnI專一的核酸適體固定化後，進行人體血清環境下的cTnI檢測之檢測結果。實驗結果顯示，改質APTES可能會有聚合的問題，讓整體表面較為粗糙，也有可能使胺基被遮蔽而降低改質的成效；而改質APS則是可以控制矽烷化的過程，胺基較不容易被遮蔽使得表面改質的成效較好，然而，在FET上檢測結果顯示APS的抗非專一性吸附能力較差；最終選擇mixed SAMs的原因是因為透過不同SAMs的比例可以製造出核酸適體與目標分子接合的空間，使捕獲目標物更有效率。除此之外，PEG更有著抗非專一性吸附的能力，能降低複雜環境下的檢測誤差。FET的實驗結果顯示，改質mixed SAMs的FET在未稀釋的人體血清環境下檢測cTnI，所得到的LOD為0.2pg/mL，且檢測時間約為30分鐘。本裝置之LOD不但比臨床檢測方法的LOD低，而且時間也比臨床檢測方法來得短，這也凸顯我們的裝置對於解決上述臨床醫護人員所遇到的問題是很有潛力的。
;According to WHO, it is expected that about 23 million people will die from MI (known as heart attack) annually by 2030. Cardiac biomarkers are released from myocytes immediately after a heart attack. Among of the biomarkers, cardiac troponin I (cTnI) is highly correlated with myocardial damage. Myocardial infarction is also accompanied by myocardial damage; thus, in the diagnosis of MI, cTnI is also regarded as the gold standard for the detection by European Society of Cardiology. However, it takes time (>1hr) to detect a measurable troponin concentration and a significant concentration rise (troponin blind interval). To solve the problem above, there are many researcher teams committed to the develop a detection method with high sensitivity and good resolution. At the same time, silicon nanowire field effect transistor (SiNW FET) has many advantages, such as, high sensitivity, label-free detection and real-time detection; therefore, it is regarded as a detection platform with great development potential.
Our laboratory previously developed 3-aminopropyltriethoxysilane (APTES) immobilized on the surface of SiNW FET, and then modified glutaraldehyde (GA) and bioprobes for experiments. In the early stage of this research, experiments were carried out by continuing the previous surface modification method. Later, we discussed about the effects of two different surface modification methods on SiNW FET, namely, 1-(3-Aminopropyl)silatrane(APS) and ?silane-PEG-NH?_2：silane-PEG-OH =1：10 (mM/mM) mixed self-assembled monolayers (mixed SAMs).
In the thesis, firstly, we use atomic force microscope (AFM) to analyze the surface roughness and morphology of samples. Furthermore, we analyze the surface elements by X-ray photoelectron spectroscopy (XPS). By doing these two experiments, we could claim that the surface modification is successful. Finally, we compare the results of cTnI detection in human serum after immobilizing anti-cTnI aptamers on SiNW FET by using different surface modification methods.
Results reveal that modifying APTES on the surface probably causes the problem of aggregation; it may make the surface rougher and it may also mask the amine group; consequently, reducing the effectiveness of modification. Modified APS could control the silylation process; the amine groups are less likely to be masked; thus, it makes the effect of surface modification better. However, the results of the detection on FET show that APS has worse anti-non-specific adsorption ability. Our final choose is mixed SAM, the reason why is it could provide an enough space for the binding between aptamer and target molecule to make the target capturing more efficiency by regulating the ratio of mixed SAM. What is more, PEG has the ability of anti-fouling, decreasing the detection errors in complex environments. Results of detection on FET shows that LOD obtained is 0.2 pg/mL under the condition that the FET is modified by mixed SAM and cTnI is spiked in undiluted human serum. Besides, the detection time is about 30 minutes.
In brief, our device has a great potential to solve the above problem (troponin blind interval), since not only LOD of our device is lower than clinical use, but the detection time of our device is shorter than clinical detection. |
