茲海默症（Alzheimer’s disease）是一種普遍性與年紀相關且會使腦部產生漸進式退化及痴呆的疾病。 目前全世界約有一千七百萬至兩千五百萬的人口得到阿茲海默症，有學者預測在21世紀中期時，全球阿茲海默症患者會成長達到六千萬人以上。 因為如此嚴重的情形被預期會發生，阿茲海默症在這近年來受到極大的重視。部分的研究指出，罹患阿茲海默症病人腦中的beta-amyloid, acetylcholine 和glutamate的含量會產生不正常的情況。因此發展可同時偵測 beta-amyloid, acetylcholine和 glutamate三種物質的生醫陣列型光學感測器，並將其應用於阿茲海默症上有其重要性。
此篇論文目的在利用溶膠凝膠法開發陣列型生醫感測器來測量beta-amyloid, glutamate 及 acetylcholine的含量加以應用於阿茲海默症偵測上。將不同的生物辨識酵素分子horseradish peroxidase (HRP), glutamate dehydrogenase (GLdh) 與acetylcholineasterase (AChE)包埋至溶膠凝膠系統中，並結合著螢光染劑amplex red 及 SNARF-1-dextran，利用其發光特性來分別得知beta-amyloid和glutamate， acetylcholine在系統中的含量。研究中也顯示此感測系統對beta-amyloid, glutamate 和 acetylcholine的偵測極限分別為 0.67 �慊/ml, 0.53�嵱 及1.02 �嵱。 當此生醫偵測系統應用於實際血清真實樣品時，對於beta-amyloid 的偵測極限為1.02 �慊/ml，對glutamate 和acetylcholine的偵測極限各為4.5�n�嵱及0.3�n�嵱。 另此感測器系統對於此三種感測物的偵測範圍可以都到4-5個層級（orders），顯示對於在偵測阿茲海默症的生醫應用上，此溶膠凝膠陣列型生醫感測器提供了一個極佳的成效。

Alzheimer’s disease is a progressive neurodegenerative disease of the brain which is the most common form of age-related dementia. Currently, approximately 17-25 million people worldwide suffer from Alzheimer’s disease, and by the middle of the 21st century, the number of patients with this form of dementia is expected to grow to at least 60 million. Due to that, previous studies have depicted that the beta-amyloid, acetylcholine and glutamate will change abnormally in AD patient brain. Therefore, to fabricate a biosensor for detecting Alzheimer’s disease, especially the amounts of beta-amyloid, acetylcholine and glutamate were is important.
In this study, sol-gel array-based optical biosensors for the detection of the Alzheimer’s disease were fabricated by determining of beta-amyloid, glutamate and acetylcholine. The sol-gel array-based optical biosensors were encapsulated with horseradish peroxidase (HRP), glutamate dehydrogenase (GLdh) and acetylcholineasterase (AChE) as biorecognization molecules. By incorporaing amplex red and SNARF-1-dextran into sol-gel matrices, the concentrations of beta-amyloid, glutamate and acetylcholine could be determined using the sol-gel array-based optical biosensor. The detection limits of beta-amyloid, glutamate and acetylcholine were 0.67 �慊/ml, 0.53�嵱 and 1.02 �嵱, respectively. The developed biosensors were also applied to real human serum samples and the detection limits were 1.02 �慊/ml to beta-amyloid, 4.5 �嵱 to glutamate and 0.3 �嵱 to acetylcholine, respectively. The array-based biosensors showed good analytical performance with dynamic range of 4-5 orders of magnitude. Results obtained cleanly show that the successful fabrication of sol-gel array-based optical biosensors that has excellent performances on determination of beat-amyloid, acetylcholine and glutamate in the Alzheimer’s disease.

謝誌 Ⅰ
中文摘要 Ⅱ
Abstract Ⅲ
Content Index Ⅳ
Table Index Ⅶ
Figure Index Ⅷ
Chapter 1 Introduction 1
1-1 Introduction 1
1-2 Objectives and research plan 2
Chapter 2 Background and Theory 4
2-1 Alzheimer’s Disease 4
2-2 Biosensor 6
2-3 Enzyme-encapsulated biosensors 7
2-3-1 Glutamate biosensors 8
2-3-2 Acetylcholine biosensors 9
2-3-3 Beta-amyloid biosensors 10
2-4 Sol-gel techniques 11
2-3-1 Introduction to sol-gel techniques 11
2-5 Array-based Biosensors 15
2-6 Fluorescence detection 16
Chapter 3 Materials and Methods 20
3-1 Materials 20
3-2 Preparation of array-based biosensor 21
3-3 Substrate determinations 23
3-3-1 Solution form and real sample determination 24
3-3-2 Multi-analyte determinations 25
3-4 Fluorescence detections 25
Chapter 4 Results and Discussions 31
4-1 Fluorescence spectra of dye molecule at various pH values 31
4-2 Optimization of preparing array-based biosensor 34
4-2-1 Acetylcholine determination 34
4-2-1-1 Optimaliztion of pH value 34
4-2-1-2 Optimaliztion of buffer concentration 36
4-2-2 Glutamate determination 38
4-2-2-1 Optimaliztion of amount of NADP+ 38
4-2-2-2 Optimaliztion of buffer concentration 41
4-3 Real sample determination 42
4-4 Determination of enzyme activities 45
4-5 Optical beta-amyloid sensing systems 49
4-5-1 Determination of H2O2 using amplex red-HRP system 49
4-5-2 Optimization of beta-amyloid sensing system 50
4-5-2-1 Optimaliztion of Cu2+ concentration 50
4-2-2-2 Optimaliztion of pH value of Cu2+ solution 51
4-5-3 Optimaliztion of incubation time 52
4-5-4 Real samples determination 54
4-5-5 Multi-analytes determination 56
Chapter 5 Conclusion 60
References 62







Table Index
Table 5-1 The performance of the optical biosensor of detection for Alzheimer’s disease 62

















Figure Index
Figure 2-1. The formation of beta-amyloid in the amyloid cascade model 5
Figure 2-2. The structure of alanine, aspartate, glutamate and glutamine 8
Figure 2-3. The reaction scheme of sol-gel process 13
Figure 2-4. An array-based biosensors 16 Figure 2-5. Absorption spectra of carboxy SNARF-1 17
Figure 2-6. Emission spectra of carboxy SNARF-1 18
Figure 2-7. The structure of carboxy SNARF-1 19
Figure 3-1. The photograph of chambered coverslip with different wells 21
Figure 3-2. The pin-printing system for the fabrication of array-based biosensor 22
Figure 3-3. (A) The working principle of the total immersion 23
Figure 3-3. (B) The photograph of the total immersion system 24
Figure 3-4. The position of the optical chopper 27
Figure 3-5. (A) The developed LabVIEW program for the fluorescence detection system 27
Figure 3-5. (B) Illustration of the front panel and the block diagram of the program 28
Figure 3-6. The photograph of the developed fluorescence detection system 29
Figure 3-7. The schematic diagram of the developed fluorescence detection system 30
Figure 4-1.Fluorescence spectra of SNARF-1-dextran at variant pH values 32
Figure 4-2. Relative fluorescence intensity of SNARF-1-dextran at various pH 33
Figure 4-3. Relative fluorescence intensity of SNARF-1-dextran at various pH 34
Figure 4-4. Effect of pH values on the determination of various concentrations of ACh 36
Figure 4-5. ACh detection by array-based biosensor at various buffer concentrations. 37
Figure 4-6. The response of the array biosensor with respect to various Ach 38
Figure 4-7. L-glutamate determination by adding various concentrations of NADP+. 40
Figure 4-8. Effect of pH on the relative signal intensity of array-based biosensor 40
Figure 4-9. Effect of buffer concentration on the determination of glutamate. 41
Figure 4-10. The response of the biosensor array with respect to various L-glutamate. 42
Figure 4-11. The response of the biosensor array with respect to various Ach in PBS. 44
Figure 4-12. The response of the biosensor to various glu in human serum. 45
Figure 4-13. Response of the biosensor for the determine ach in the presence of glu 47
Figure 4-14. Response of the biosensor for the determine ach in the presence of glu 48
Figure 4-15. The intensity ratio of amplex red -FITC-dextran with various H2O2 50
Figure 4-16. Effect of Cu2+ conc on the determination of various concentrations of �毣 52
Figure 4-17. Effect of pH value of Cu 2+ solution on the determination of �毣 53
Figure 4-18. Effect of incubation time of 0.5 mg/ml �毣 react with 0.5mM Cu2+ 54
Figure 4-19. The response of the biosensor array with respect to various �毣 conc. 55
Figure 4-20. (A) The response of the biosensor in human serum solution 56
Figure 4-20. (B) The response of the biosensor in human serum solution 57
Figure 4-21. (A)Response of the biosensor on �毣 in the presence of glutamate 59
Figure 4-21. (B) Response of the biosensor on �毣 in the presence of Acetylcholine. 59
Figure 4-21. (C) Response of the biosensor on �毣 in the presence of Zn2+ 60
Figure 4-21. (D) Response of the biosensor on �毣 in the presence of Mn2+. 60

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