博碩士論文 102324030 詳細資訊




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姓名 張育銘(Yu-ming Chang)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 探討抗體之RNA核適體作為免疫分析及檢測平台訊號放大之研究
(Universal Signal Amplifying Development in ELISA by using RNA Aptamer on Detection Platform)
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摘要(中) 生物感測器在目前醫學領域尤其是轉譯醫學中扮演著十分重要的角色,生物感測技術為基礎醫療研究以及臨床診斷治療提供了一種快速簡便的檢測方法,並且其具有專一、靈敏、反應時間短等特性,運用在檢測微量生物指標(Biomarker),期望能夠達到快速的疾病診斷之感測、少量檢體檢驗感測及高靈敏生醫感測等目標,以便提供醫療人員能夠快速方便使用的檢測平台,進而提升檢測效率,創造病人福祉。

自人類基因體計畫(Human Genome Project)啟動以來,基因資訊的快速獲得極充分利用,將人們朝向精確醫學(Precision Medicine)所強調之個人化預防醫學與治療邁進,核酸類似物的發展促使基因能更準確極方便的定序,從生命體最根源的基因表現來達到疾病的預防及治療,更進一步地將每個人的基因進行定序,規劃屬於個人專屬的健康照顧,將醫療推向另一個新的層級。

近年發展的核酸適體(aptamer)做為生物感測,其優點為方便取得,且能夠方便的改質上不同的官能基,使其應用面更為廣闊。

在本研究中運用的核酸適體(aptamer)為RNA-18核酸適體,可發展出一種具有辨識能力且放大訊號的檢測平台。

實驗分為三個階段,第一階段為核酸適體之合成;而後經由粒徑分析儀(DLS)來判定合成之大小。

第二階段為核酸適體的專一性結合測試,判定aptamer的結合能力是否正常。

第三階段在檢測平台上利用aptamer,運用於實際檢測物之檢測。

本實驗中藉由檢測平台來證實此核酸適體可成為新一代通用之訊號放大器,其靈敏性及專一性極佳,擁有便宜、合成程序少、穩定度高等優點,可預測是否罹患病症,發展快速簡便的檢測方法,在未來醫學領域必能發展一項便利又具可行性的檢測方法。

另一部分是本實驗室建立不帶電核酸於寡核苷酸(nDNA)合成改質之原則。此原則之建立以協助本實驗室於中性核酸探針改質以調整與目標核酸雜交之溶解溫度(Tm)與熱穩定性為主。利用不同的量測平台,能夠幫助我們了解nDNA與目標分子的Tm值,並且歸納出相關的設計原則,以利往後能夠發展高專一性、高靈敏性的探針能夠使用於檢測平台上,得到高效率的檢測。

摘要(英) This research is separate into two part of experiments, First part is R18 RNA aptamer for a universal signal amplifying platform on ELISA, and second part is design nDNA in the sequence for improve melting temperature and have higher specificity, selectivity, and heat stability.

Enzyme-linked immunosorbent assay (ELISA) system is a useful detection technique for antigen or other biomolecules by using antibodies, but there also have some difficulties. For example, antibody is time-consuming in produce, expensive, difficult to label, and more background signal by labeling on secondary antibody, etc. However, development with the SELEX method which can select the nucleic acid based probe-aptamer. This molecule has more advantages than antibody. Aptamer is an artificial product which has high affinity and high specificity with target molecular. We find that R-18 RNA aptamer can become a signal amplifying platform. Finally, we use RNA aptamer to create a universal signal amplifying platform.

At the second part, we develope a neutralized DNA called “nDNA”, it backbone has no charge because of a methyl modified at the phosphate side. It can increase the specificity and selectivity with the target sequence, because of no charge backbone can help it to hybridize with target sequence in low salt solution. We design the position of the nDNA in the sequence and use many instrument to measure the melting temperature, afterthat we find nDNA can increase the melting temperature and heat stability with different position and numbers. Finally, we have some rules for how to design nDNA in the sequence, for high specificity and heat stability. We can use these rules to control melting temperature on the same platform afterthat we can use nDNA for more efficiency in diagnosis or therapeutic applications.

關鍵字(中) ★ 核適體
★ 兔子抗體
★ 免疫分析
★ 訊號放大檢測平台
★ 微脂粒
★ 核酸類似物
★ 熔點溫度
關鍵字(英) ★ Aptamer
★ Rabbit antibody
★ Enzyme-linked immunosorbent assay
★ Signal amplifying detection platform
★ Liposome
★ Nucleic acid analogue
★ Melting temperature
論文目次 目錄

摘要 i

Abstract iii

致謝 iv

圖目錄 vii

表目錄 ix

第一章 緒論 - 1 -

1.1 研究背景與動機 - 1 -

1.2 研究目的 - 2 -

第二章 文獻回顧 - 3 -

2.1 核適體(aptamer) - 3 -

2.1.1 核適體簡介 - 3 -

2.1.2 核適體在生物感測器上的應用 - 5 -

2.2 微脂粒(liposome) - 6 -

2.2.1 微脂粒簡介 - 6 -

2.2.2微脂粒組成 - 6 -

2.2.3微脂粒結構 - 7 -

2.2.4微脂粒訊號放大應用 - 8 -

2.3 ELISA (Enzyme-linked immunosorbent assay) - 9 -

2.3.1 ELISA簡介 - 9 -

2.4 FET - 10 -

2.4.1 FET簡介 - 10 -

2.4.2 FET於生物感測應用 - 12 -

2.5 核酸類似物 - 13 -

第三章 材料及實驗方法 - 17 -

3.1藥品與材料 - 17 -

3.1.1藥品 - 17 -

3.1.2溶液 - 18 -

3.2 實驗儀器 - 19 -

3.2.1一般儀器 - 19 -

3.2.2主要儀器 - 19 -

3.3實驗方法 - 20 -

3.3.1微脂粒(liposome)之合成 - 20 -

3.3.2 R-18 aptamer liposome與 Rabbit antibody結合測試(binding test) - 21 -

3.3.3 Dose response測試 (on 96 well-plate) - 21 -

3.3.4 Dose response 測試 (on chip) - 22 -

3.3.5 Real sample E. Coli proteome 測試 (E. Coli proteome chip) - 22 -

3.3.6 RNA aptamer 合成 - 23 -

3.3.7 量測RNA aptamer 在FET系統中訊號放大效果 - 25 -

3.3.8 nDNA溶點溫度測試 - 26 -

第四章 結果與討論 - 30 -

4.1 合成R18 RNA 核適體螢光標定微脂粒 - 30 -

4.2 aptamer conjugate liposome對於rabbit antibody濃度效應 - 34 -

4.3 aptamer在Field Effect Transistor作為訊號放大器 - 39 -

4.4 nDNA 引子設計原則實驗 - 45 -

4.4.1 SYBR Green量測Tm值 - 47 -

4.4.2 恆溫滴定微卡計熱變化之探討 - 49 -

4.4.3 紫外光/可見光光譜儀之Tm值量測 - 51 -

4.4.4 圓二色光譜儀之Tm值量測 - 56 -

4.4.5示差掃描量熱儀之Tm值量測 - 58 -

4.4.6 nDNA設計原則於不同序列之驗證 - 61 -

第五章 結論與未來展望 - 65 -

參考文獻 - 69 -



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指導教授 陳文逸(Wen-yih Chen) 審核日期 2015-8-25
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