偵測阿茲海默症光學陣列生醫感測器之製備｜國立清華大學博碩士論文庫

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研究生：	李平善
論文名稱：	偵測阿茲海默症光學陣列生醫感測器之製備 Fabrication of Sol-gel-based Optical Array Biosensor for Detection of Alzheimer's Disease
指導教授：	董瑞安
口試委員:
學位類別：	碩士 Master
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2007
畢業學年度：	95
語文別：	中文
論文頁數：	64
中文關鍵詞：	阿茲海默症、溶膠凝膠法、生醫感測器
相關次數：	點閱：2 下載：0
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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 □g/ml, 0.53□M 及1.02 □M。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 □g/ml，對glutamate 和acetylcholine的偵測極限各為4.5□□M及0.3□□M。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 □g/ml, 0.53□M and 1.02 □M, respectively. The developed biosensors were also applied to real human serum samples and the detection limits were 1.02 □g/ml to beta-amyloid, 4.5 □M to glutamate and 0.3 □M 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 □A    52
Figure 4-17. Effect of pH value of Cu 2+ solution on the determination of □A            53
Figure 4-18. Effect of incubation time of 0.5 mg/ml □A react with 0.5mM Cu2+          54
Figure 4-19. The response of the biosensor array with respect to various □A 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 □A in the presence of glutamate          59
Figure 4-21. (B) Response of the biosensor on □A in the presence of Acetylcholine.      59
Figure 4-21. (C) Response of the biosensor on □A in the presence of Zn2+              60
Figure 4-21. (D) Response of the biosensor on □A in the presence of Mn2+.             60

                                

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