博碩士論文 962411001 詳細資訊




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姓名 宋子承(Tzu‐Cheng Sung)  查詢紙本館藏   畢業系所 系統生物與生物資訊研究所
論文名稱 發展微脂粒核適體以應用於高通量生物感測
(Development of liposomal aptamers for the use in high throughput biosensing)
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摘要(中) 本研究,我們主要開發微脂粒核適體以應用於高通量生物感測。於本研究第一部分中,利用生物素作為主要模式偵測目標,開發微脂粒核適體作為競爭物以應用於置換分析。置換分析較係屬較有利應用於高通量生物檢測之分析形式。我們成功於在96孔盤系統中,開發一高通量樣本數生物感測平台。在第二部分中,我們利用微脂粒核適體與兔子抗體結合之技術,兔子抗體於生物感測中係屬於常見生物識別元件。因此,本研驗中能與兔抗體結合之的”抗兔抗體之微脂粒核適體”可利用於生物感測研究中,針對許多不同的分析物的同時檢測。我們證明於本研究中所使用之兩種微脂粒核適體具有相當潛力可用於最小化分析時間,並簡化了分析步驟與流程,有利於應用於高通量生物感測中。
本研究第一部分,我們開發微脂粒核適體作為生物素的競爭劑,於96孔盤系統中進行置換分析以測定生物素。本研究所使用之ST-21和ST-21M微脂粒核適體之檢測極限分別為1.32微克/ 80微升及0.47微克/ 80微升。該ST-21和ST-21M微脂粒核適體之動態測定範圍分別為七和四個數量級。成功證明了運用微脂粒核適體作為競爭劑於置換分析是可行的。同時也證實去除任何洗滌程序的置換分析法可提供快速並可應用於高通量生物感測之潛力。
在本研究的第二部分,我們應用微脂粒核適體於微陣列分析。我們利用微脂粒核適開發一可用於生物感測之通用試劑,R18微脂粒核適體可與兔抗體結合,吐抗體是在生物感測中常用之生物識別元件。在本研究中,R18微脂粒核適體可成功應用於96孔盤與晶片系統中。R18微脂粒核適體的檢測極限是80毫微克/毫升。動態檢測範圍在96孔盤系統中為三個數量級(80納克/毫升至50微克/毫升);在晶片上的檢測極限是18.2納克/毫升、在晶片上的動態檢測範圍是三個數量級(18.2毫微克/毫升至10微克/毫升)。我們並R18微脂粒核適體於高密度蛋白質體晶片之檢測,在晶片上可表現出高密度並相當清晰之信號。而R18微脂粒核適體在晶片上之檢測也呈現出明顯的濃度效應,並具有專一性,對其他非兔抗體則不會呈現訊號。因此,本研究成功證實微脂粒核適體可作為高通量生物感測所使用之通用試劑。
綜合本研究之成果,微脂粒核適體不僅能在置換生物感測分析中能作為競爭劑,並能作為免疫分析中之通用試劑。微脂粒核適體具有相當之潛力可應用於高通量生物感測中。
摘要(英) In this research, we developed liposomal aptamers for the use in high throughput biosensing. In the first part, the crucial vitamin biotin was used for model analytes for developing a liposomal aptamer as a competitor for replacement assay, which is an effective assay format for high throughput biosensing. We demonstrated the assay format in the 96-well plate, which provides high sample throughput for biosensing. In the second part, we developed a liposomal aptamer for binding to rabbit antibodies, which is common biorecognition elements in biosensing. Therefore, this anti-rabbit antibody liposomal aptamer can be used for multiplexed biosensing for simultaneous detections of many different analytes. We demonstrate that both liposomal aptamers have the potential to be used to minimize the assay time and simplified the assay protocol to be used in high throughput biosensing
In the first part, we developed a liposomal aptamer as the competitor of biotin for the detection of biotin in 96-wells plate format replacement assay. The limit of detection by using ST-21 and ST-21M liposomal aptamer were 1.32 pg/80 μl and 0.47 pg/80 μl, respectively. The dynamic ranges of our assays using ST-21 and ST-21M aptamers were seven and four orders of magnitude, respectively. This demonstrates the successful use of aptamer as a competitor of analyte in a replacement assay. It also shows the replacements assay without any washing procedure is fast enough and with a potential to use in the high throughput biosensing.
In the second part, we developed a liposomal aptamer as a secondary reagent for immunoassays. The R18 liposomal aptamer can bind with rabbit antibody, which is commonly used biorecognition elements in biosensing. In this research, the R18 liposomal aptamer can be used both on the 96-well plate and chips. LOD for R18 liposomal aptamer was 80 ng/ml, and the dynamic range was three orders of magnitude (80 ng/ml to 50 µg/ml) on the 96-well plate. The LOD for R18 liposomal aptamer on the chip was 18.2 ng/ml. The dynamic range was three orders of magnitude (18.2 ng/ml to 10 µg/ml) on the chip. We further used the R18 liposomal aptamer on high density proteome microarrays. We observed that the R18 liposomal aptamer showed high resolution and clear signals with dose response on the chip and also can R18 showed specific binding to the rabbit antibodies. There results demonstrate the R18 liposomal aptamer can be as a seconadry reagent for the use in high throughput immunoassays.
In summary, liposomal aptamers can be ideal competitors for the replacement biosensing and effective secondary reagents for immunoassays. They are both applicable for high-throughput biosensing.
關鍵字(中) ★ 核適體
★ 微脂粒
★ 高通量生物感測
★ 蛋白質體晶片
關鍵字(英) ★ aptamer
★ liposomes
★ high throughput biosensing
★ proteome microarray
論文目次 Table of contents
中文摘要 I
Abstract III
Acknowledgements V
I. Introduction 1
1. High-throughput biosensing 1
2. High-throughput biosensing techniques 3
2.1 DNA microarrays 3
2.2 Protein microarrays 3
2.3 Microplate-based assays 4
3. Replacement biosensing assay 4
3.1 Fluorescence polarization-based replacement assay 5
3.2 Fluorescent intercalator replacement assay 5
3.3 Target-induced replacement assay 5
3.4 Fluorescence replacement assay 6
4. Biotin detection 6
4.1 ELISA 6
4.2 Electrochemical immunosensor assay 7
4.3 Electrochemical magneto assay 7
4.4 HPLC/MS 7
4.5 Flow injection liposome immunoanalytical system 8
4.6 Immunoaffinity chromatographic assay 8
4.7 Isotope dilution assay 8
5. Purpose of developing liposomal aptamer for high-throughput biosensing assay 9
Reference 11
II. A replaceable liposomal aptamer for the ultrasensitive and rapid detection of biotin 15
Abstract 16
Introduction 16
EXPERIMENTAL SECTION 19
Results 21
Discussion 27
Conclusions 29
ACKNOWLEDGMENT 29
Reference 29
Supplementary Figure 32
III. Development of an anti-rabbit liposomal aptamer as a secondary reagent for immunoassays 35
Abstract 36
Introduction 36
EXPERIMENTAL SECTION 38
RESULTS 40
DISCUSSION 42
Conclusions 44
Reference 52
IV. Appendix 54
Published and under revision manuscript 55
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指導教授 陳建生(Chien‐Sheng Chen) 審核日期 2016-8-31
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