溶膠-凝膠之陣列型免疫感測器於B型肝炎之檢測分析

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研究生：	林政鋒 Chen-Feng Lin
論文名稱：	溶膠-凝膠之陣列型免疫感測器於B型肝炎之檢測分析 Sol-Gel Derived Array-Based Immunosensor for the analysis of Hepatitis B virus
指導教授：	董瑞安 Ruey-An Doong
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：	2005
畢業學年度：	93
語文別：	英文
論文頁數：	80
中文關鍵詞：	陣列型免疫感測器、多重分析、B型肝炎、溶膠-凝膠
外文關鍵詞：	array-based immunosensor, multianalytes, Hepatitis B virus, sol-gel
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快速反應的陣列型生物感測器具有能快速篩檢、能大量且平行分析，容易操作，
敏感性佳和選擇性好的幾項優勢。然而，，將生物分子固化至固體表面上的固化技術是在發展陣列型生物感測器的過程□最主要的瓶頸。在這項研究過程中，利用添加劑的修飾和參數最佳化，使此溶膠凝膠技術可應用在被開發肝炎的檢測分析陣列型免疫分析感測器製程中抗體的固化。
TMOS(Tetramethylorthosilicate)溶膠溶液被使用來製備本實驗的溶膠凝膠溶液。甘油 (6 %)被加入於溶膠凝膠中而且能夠有效溶膠凝膠溶液的凝固時間， 0.6 %的PVA（Polyvinyl alcohol）能夠改善溶膠凝膠凝固時容易發生於表面的破裂情況。當溶膠和溶液的體積比率高於20 %時，被包埋的蛋白質從就不太容易從已凝固的溶膠凝膠中滲漏。另外，使用PVAc (Polyvinyl acetate )修飾的玻璃片有助於點印於玻璃上的溶膠凝膠的吸附。此生物感測器陣列可以應用於螢光免疫分析法中以偵測抗原及抗體。螢光免疫分析法中，老鼠免疫球蛋白 (Mouse IgG)被固化在溶膠凝膠中，並且用FITC標示的老鼠免疫球蛋抗體（Mouse IgG antibody）滲透穿過多孔性凝膠結構來和老鼠免疫球蛋白作親合性結合。此滲透至結合的過程需3小時的孕育時間。直接型螢光免疫分析 (direct fluorescence immunoassay) 的偵測極限在老鼠免疫球蛋白及羊免疫球蛋白的系統中分別是 5 ng/mL和 5 ~10 ng/mL。而在B型肝炎核心抗原及抗體反應的三明治型螢光免疫分析 (sandwich fluorescence-immonoassay)裡，偵測極限也接近30 ng / ml。在經過設計的同時偵測多種抗原或抗體的實驗裡，結果顯示抗原（或抗體）能在一次實驗中，辨識出相對應的抗體（或抗原）。此外，被凝膠固化的老鼠免疫球蛋白的活性在4oC 貯存條件中，其活性能能被維持超過兩周。此活性能被維持的時間較化學鍵結的固化技術的活性維持時間 (通常小於7天) 長。而連續7次重覆實驗的差異系數少於5%。基於上述的實驗結果，此實驗發展的溶膠凝膠之陣列型免疫感測器具有應用於多種抗體同時且平行分析潛力，故可應用此陣列型免疫分析感測器於B型肝炎的檢測分析。

The array-based biosensors contain several advantages including quick screening, massive parallel analysis, easy operation, good sensitivity and selectivity. The immobilization of biological compounds onto the solid surfaces is one of the bottlenecks in the development array-based biosensor array. In this study, a sol-gel technique with additives and optimized parameters that is suitable for the immobilization of antibody was developed to fabricate the array-based immunosensor for the determination of hepatitis. Tetramethylorthosilicate (TMOS) was selected as the sol solution. Glycerol (6 %) was added into the sol-gel solution to effectively extend the gelation time of sol-gel. The cracking condition in the gelation process could be improved by adding 0.6 % polyvinyl alcohol (PVA). When the ratio of sol/solution was higher than 20 %, leakage of biomolecules was not significant. In addition, protein was immobilized within sol-gel network in the presence of polyvinyl acetate (PVAc) on the glass slide. The optimized array biosensor was used for fluorescein-immunoassay to detect antigens or antibodies. The mouse IgG was entrapped within sol-gel, and anti-mouse IgG with FITC was allowed to diffuse through the pores of the micro-patterned gel structure. The antigen-antibody was observed after 3 hr incubation. The detection limits of direct fluorescein-immunoassay were 5 ng/ml and 10 ng/m, respectively for Mouse-IgG and Sheep-IgG systems. In the sandwich immunoassay of HBV antigen/antibody reaction, a detection limit of 30 ng/ml can be achieved. For multi-protein measurement experiment, results showed that the antibody could recognize or identify the specific corresponding receptor in one experiment even in sandwich immunoassay. In addition, the activity of the mouse-IgG could be retained for more than two weeks at 4oC. This storage period was longer than that using the normally covalent immobilization technique (less than 7 days). The relative standard deviation (RSD) of the array-based immunosensor was less than 5% (n = 7). Results obtained in this study clearly show that the developed sol-gel based array immunosensor has good potentials to perform the simultaneous and parallel analysis for the determination of multi-samples in the presence of multianalytes.

中文摘要………………………………………………………………………………    I
Abstract………………………………………………………………………………….    III Table Index……………………………………………………………………………    V
Figure Index……………………………………………………………………….…  VI

CHAPTER 1 Introduction and Motivation…………………………………     1
1    Motivation…………………………………………………………….…    1
2    Introduction of Sol-gel based immunosensor array ………………………    2
3    Objective..………………………………………………………..…….    4

CHAPTER 2 Background...…………………………………………………5
1    Introduction of Viral Hepatitis ……………………………………………    5
2    Microarray Technology..……..…………………………………………..    10
3    Sol-Gel Technique for Encapsulation of Biomolecules. …………………    11

Chapter 3 Material and Experimental Methods..…………………………...17
1    Reagents and Materials..………………………………………..……….    17
2    Apparats. ……………………………………………………………..…    18
3    Sol Solution Preparation…. …………….………………………….……    18
4    Preparing Sensor Array..………………………………………………..    19
5    Optimization of Sol-Gel Conditions.……………………………….……    20
5.1    Ratio of Sol Precursor to Solution…………………………..20
5.2    Prolongation of Gelation Time………………………………20
5.3    Delaying of Cracking Condition………...…………………...21
5.4    Addition of Fluorescence Modifying Agent…………………21
6    Procedure of Immunoassay Experiment…………………………………..22
6.1    Direct Immunoassay…………………………………………22
6.2    Sandwich Immunoassay……………………………………..    22
6.3    Direct immunoassay of analytes in serum……………………23
6.4    Sandwich Immunoassay of analytes in serum………………..24

Chapter 4 Results and Discussion………………………………………..    27
1    Automatic Pin-Printing and Optical Detection System...…….………...    27
1.1    Sol-Gel Pin-Printing System.…….……………………….    27
1.2    Fluorescent Detection System……………………………    29
1.3    LabviewTM Controlling Program….……………………..    32
2    Optimization of Sol condition…………………..…………..…….……    36
2.1    Optimization of Sol gel immobilization technique…...…….…...    36
2.1.1    Optimization of Sol solution content..……..……    36
2.1.2    Prolongation of the Gelation Time……………….    39
2.1.3    The Crack condition..……………………..……    41
2.1.4    Addition of fluorescence modifying agent………    42
2.1.5    The optimized condition of Sol-gel solution……    46
3    Direct Immunoassay. ………………………………………………….    47
3.1    Saturation test.…………………………………………………    47
3.2    Limit of detections.…………………………….………..…….    51
3.3    Repeatability.………………………………………….…….…    56
3.4    Stability..............................................…………….…………...     57
4    Sandwich immunoassay………………………………………………….    59
4.1    Saturation experiment……………………………………..    59
4.2    Limit of Detection…………………………………………    63
5    Multi-Protein Measurement………………………………………………    65
5.1    Direct Immunoassay……………………………………….    65
5.2    Sandwich Immunoassay……………………………………    67
6    Immunoassay of analytes in serum
6.1    Direct immunoassay……………………………………….    69
6.2    Sandwich immunoassay……………………………………    71
6.3    Multi-analytes measurement in serum……………………..    73

Conclusion………………………………………………………………..    75
Reference…………………………………….....………………...............76.

                                

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