膽道閉鎖屬於新生兒的疾病的其中一種，為先天膽道系統發育異常所致，如果沒有得到即時的治療，最壞的情況會引發肝衰竭進而導致死亡。雖然這是不常見的疾病，但由於其難以於早期診斷出來，父母仍無法忽視此疾病恐造成的嚴重性。目前臨床上是依賴血液生化檢查或是一些手術方式來做診斷，但準確性的不足還是無法及時給予最為正確的治療，因此一個可以快速鑑別的方式變得極為重要。目前研究對於許多特殊疾病均已找到相應的生物性指標。近幾年中，基質金屬蛋白酶7 (MMP7) 也被廣泛討論並將之視為是一個新穎的生物性指標，其價值在於患有膽道閉鎖症狀的新生兒中 MMP7 會明顯提升。在此研究中，我們將設計並開發一個平台來檢測 MMP7，此機制的原理為應用電雙層 (EDL) 場效電晶體 (FET)，而檢測的靈敏度來自於當蛋白與探針結合時產生的閘極電壓變化。透過開發此平台，膽道閉鎖於早期診斷變為可能，而此平台的優點不僅是可以偵測 MMP7 蛋白，對於一般人也是方便使用。在未來應用中，期望此生物感測器可以在醫院操作以促進臨床研究並提升新生兒福祉。

Biliary Atresia (BA) is a severe disease would occur in newborns. If newborns do not get timely treatment, the worst consequence finally results in liver failure (needs liver transplantation) and even death. Even if this disease is rare, parents still consider this disease serious because BA is hard to diagnose early. The clinical method relies on blood testing or some invasive surgical way, but the accuracy is too low to treat correctly. It is necessary to detect BA quickly for newborns to address these challenges. As the biomarker for a specific disease is researched widely, some studies also reported that Matrix metalloproteinase-7 (MMP7) protein that would increase under the BA disease is valuable to be a biomarker. MMP7 has been regarded as a novel indicator for BA in recent years. In this study, a detection platform was developed to detect the MMP7 protein. The mechanism is based on an electric-double-layer (EDL) field-effect-transistor (FET), and the sensitivity of the detection is from probe bind with protein and caused different gate voltage. With this EDL-gated FET biosensor, the early diagnosis for BA is possible. The advantage of this platform not only can use for MMP7 detection and is easy to operate in general. This biosensor will be deployed in hospitals to benefit clinical studies and improve newborn welfares in future applications.

Contents
摘要 i
Abstract ii
Chapter 1 Introduction 1
1.1 Motivation 1
1.2 Objective 2
Chapter 2 Literature Review 4
2.1 Biliary Atresia (BA) 4
2.2 Diagnosis methods for BA 6
2.3 MMP7 as the biomarker 7
2.4 Field-effect transistor (FET) based biosensor 8
2.5 Electrical double layer (EDL) model 10
2.6 Charge screening effect 12
Chapter 3 Experimental Design 14
3.1 Fabrication of sensor chip 14
3.2 Surface clean 16
3.3 Surface functionalization 16
3.3.1 Traut’s reagent functionalization 16
3.3.2 PEG linker functionalization 18
3.3.3 TCEP functionalization 19
3.4 Electrical signal measurement 22
3.6 Detection antibody 24
3.7 Dot blot 24
Chapter 4 Results and Discussion 26
4.1 Characteristic curve of MOSFET LND 150 26
4.2 Surface functionalization 27
4.3 The operation of FET sensor 29
4.4 MMP7 protein detection using the EDL-gated FET biosensor with antibody functionalization 30
4.5 The function of antibody when reacting with Traut’s reagent 32
4.6 Testing MMP7 protein with new antibody (only in PBS) 40
4.7 The type of FET shift from LND150 to VN10LP 42
4.8 Antibody react with different fold of Traut’s reagent 43
4.9 Immobilizing antibody on sensor with PEG linker 51
4.10 The concept of competitive ELISA 53
4.11 Optimize the condition of antibody to detect protein immobilized on sensor 54
4.12 Optimization of the immobilized MMP7 protein on sensors 57
4.13 The competitive ELISA principle applies to this sensor 59
4.14 The immobilization probe changing to Aptamer 61
4.15 The sensor surface changed to evaporated gold deposition 62
4.16 Optimize the concentration of aptamer immobilization 64
4.17 MMP7 protein detection limit 66
4.18 The range of MMP7 protein detection 67
4.19 Investigation of the sensor selectivity 68
4.20 Salt concentration will affect the ability of the aptamer to capture protein 70
Chapter 5 Summary and Future Work 75
Chapter 6 Reference 81

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