全世界每年都有將近2500萬人因為心血管相關疾病而死亡，同時根據世界衛生組織的發布心血管疾病也是全世界十大死因之中最高的一項， 因此，能夠及時診斷心血管相關疾病為一重要的研究領域。C-反應蛋白是一個常被用作人體發炎和感染之生物標記，甚至可使用C-反應蛋白來做為評估心臟疾病風險的參考數值。在我們團隊的先前研究中曾結合微流體系統及一條對於C-反應蛋白具有高靈敏度和專一性的適合體來進行C-反應蛋白的偵測。除了可以自動化完成全部流程外，只需要少量的試劑和樣品即可完成偵測也是結合微流體系統的優點。另一方面，近年來也有許多研究指出以氮化鎵鋁/氮化鎵為基底的高電子移動性電晶體作為生物感測器之可行性，藉由這種電晶體之快速且高靈敏度的特性，可偵測如蛋白質、病毒等小的生物分子。在本篇研究裡，我們結合了微流體、適合體以及電晶體感測器的優點，來達成具有高靈敏度與高專一性的C-反應蛋白偵測。
在這篇一就當中，為了增強對C-反應蛋白專一性適體結合的專一性與靈敏度，我們使用了兩種不同的C-反應蛋白專一性適體去結合C反應蛋白。 由於使用了兩種不同的C-反應蛋白專一性適體，在實驗完成的最後會組合成像是一個三明治的結構，因此我們稱這個結構為雙適體的三明治檢測法。 這個雙適體的三明治檢測法首先會在磁珠的系統上進行驗證，經由螢光偵測的方式去檢測C-反應蛋白的濃度，而經由磁珠系統的檢測方式可以檢測的C反應蛋白濃度可以由1 mg/L 到 10 mg/L。 當磁珠系統上的螢光訊號可以被偵測時代表雙適體的三明治檢測法是可以完成的，因此我們在將他轉移到場效電晶體的系統上。
為了將場效電晶體晶片封裝至微流體系統裡，本研究發表了新的封裝技術去避免在我們實驗室過往的研究中場校電晶體的封裝問題造成液體從微流體系統中洩漏出來。此外，為了避免在氮化鎵鋁/氮化鎵為基底的高電子移動性電晶體的閘極上未有第一段適體的固化會被干擾物非專一性的黏附，導致場效電晶體在電訊號的偵測會有不問定與雜訊被增強的狀況，因此在本實驗中使用了乙醇胺作為封鎖閘極未被第一段適體固化的區塊，從實驗結果來看使用了乙醇胺作為封鎖的試劑比較適合本研究的實驗。
電晶體感測器之電子訊號結果顯示我們的系統可在具有可評估心血管疾病，並且從研究結果發現雙試體的三明治結構可以在場效電晶體上的閘極完成並偵測電訊號，可憑藉已歸一化之電流訊號結果，反推得知樣品C-反應蛋白的濃度。C-反應蛋白的濃度電訊號偵測可以從 0.625 mg/L 到10.000 mg/L，這個偵測範圍可以從低風險濃度到高風險濃度，而這個偵測範圍更適合心血管疾病的偵測。總結來說，我們發展了一套可以重複使用場效電晶體的整合微流體與場效電晶體感測系統可自動化完成檢測 C-反應蛋白的雙適軆三明治結構。

Cardiovascular diseases have leading causes of death for 17.3 million deaths worldwide on a yearly basis. They were one of the leading causes of death during the past decade as announced by World Health Organization (WHO). Early diagnosis of cardiovascular diseases has become an important research area. C-reactive protein (CRP) is a general biomarker when tissue has some inflammation and infections, and has become a good biomarker for evaluating risks of the cardiovascular diseases. In this work, dual CRP-specific aptamers (DNA-based) with high sensitivity and specificity screened from our previous works were used to detect CRP concentration by an integrated microfluidic system, which was capable of performing the CRP detection by an automated system while consuming minimal volumes of reagents and samples (10 microliters). Recently, AlGaN/GaN high-electron-mobility-transistor (HEMT)-based field-effect-transistor (FET) sensors have emerged as promising biosensors to detect small molecules, proteins and even viruses. They have demonstrated rapid and highly sensitive detection in a compact system. In this study, an integrated microfluidic system that combined the advantages of microfluidics, dual-aptamers and FET-based sensors was developed to achieve sensitive and specific CRP detection.
In this study, in order to increase the binding specificity and sensitivity of the CRP detection, we used two different CRP-specific aptamers to perform sandwich like assays. Due to the presence of two different CRP-specific aptamers, the dual-aptamer sandwich assay was the first to be demonstrated on the magnetic bead based microfluidic system to detect the concentration of CRP by using fluorescence signals with CRP concentrations ranging from 1 mg/L to 10 mg/L. The fluorescence signals confirm the realization of the dual-aptamer sandwich assay.
For the FET based system, the fabrication of the FET and microfluidic chip to an integrated microfluidic system is important. In order to package the FET device into the microfluidic system, this study presents a new method to prevent liquid leakage from the FET device. Besides, in order to prevent the interference material like proteins, cells and any nonspecific molecules from adhering onto the gate region of the FET device even after immobilization of 1st aptamer, we used the blocking agent named ethanolamine to prevent nonspecific adhesion, and the results confirmed that blocking using ethanolamine has a good congruence for this study.
Sensitive electric detection of CRP has also been demonstrated. It shows that the dual-aptamer sandwich assay is successful in detection of analytes on the gate region of the AlGaN/GaN HEMT-based FET. The concentration of CRP which was tested on FET ranges from 0.625 mg/L to 10.000 mg/L which is in congruence with the concentration of CRP in patients from low to higher risks of developing cardiovascular diseases. In summary, the dual-aptamer sandwich assay can be used for CRP concentration detection and automated by the integrated FET based microfluidic system with the FET sensor and the FET device could be reused.
Keyword: AlGaN/GaN HEMT-based FET, C-reactive protein, dual-aptamer,

致謝 II
Abstract III
摘要 VI
List of Figures X
List of Table XVII
Nomenclature and Abbreviations XVIII
Chapter 1 Introduction 1
1.1 C-reactive protein and its diagnostic methods 1
1.2 C-reactive protein specific aptamers 5
1.3 MEMS and Bio-MEMS 12
1.4 Field-effect transistor (FET) 14
1.5 Novelty and motivation 16
Chapter 2 Materials and Methods 20
2.1 Design of the integrated microfluidic chip 20
2.1.1 Design of magnetic beads based microfluidic chip for fluorescent signal analysis 20
2.1.2 Design of FET based microfluidic chip 24
2.1.3 Working principle of the microfluidic chip 31
2.2 Experimental process 33
2.2.1 FET chip design and its working principle 38
2.3 Integrated microfluidic chip and FET package 42
2.4 Preparation of the experimental materials 45
2.4.1 Preparation of the CRP-specific aptamers 45
2.4.2 Preparation of reagents 45
2.4.3 Preparation of the 1st aptamer conjugated magnetic beads 46
Chapter 3 Results and Discussion 48
3.1 Fluorescence signal of dual-aptamer sandwich assay 48
3.1.1 3’modified FAM and 5’ modified FAM 2nd aptamer 48
3.1.2 FET sensing area blocking 53
3.2 Electric signal of dual-aptamer sandwich assay 55
3.2.1 Dual-aptamer sandwich assay FET microfluidic system 55
3.2.2 Optimization of 1st aptamer immobilization time 57
3.2.3 Electrical signals of different CRP concentration 59
Chapter 4 Conclusions and Future Work 64
4.1 Conclusions 64
4.2 Future Works 67
References 68

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