核酸探針電化學(xué)傳感技術(shù)用于酶活性和腺苷檢測(cè)的研究
發(fā)布時(shí)間：2018-11-05 11:19
【摘要】： 電化學(xué)生物傳感器,具有易于實(shí)現(xiàn)微型化、集成化、操作簡(jiǎn)單等優(yōu)點(diǎn),在化學(xué)、生物學(xué)基礎(chǔ)研究、臨床檢驗(yàn)、環(huán)境監(jiān)測(cè)等方面發(fā)揮著越來(lái)越重要的作用。核酸探針能夠與多種目標(biāo)物特異性結(jié)合,形成多種靈活的分子識(shí)別和信號(hào)轉(zhuǎn)換機(jī)制。本論文基于核酸探針的特點(diǎn),針對(duì)酶和生物小分子的檢測(cè),發(fā)展簡(jiǎn)便、快速、靈敏的電化學(xué)傳感器,具體完成了以下研究工作： 1.二茂鐵核酸探針的合成及電化學(xué)表征 對(duì)琥珀酰亞胺偶聯(lián)方法進(jìn)行改進(jìn),盡量消除水對(duì)反應(yīng)體系中活性中間體的影響,并采用高效液相色譜(HPLC)純化,得到了二茂鐵標(biāo)記的核酸探針。電化學(xué)測(cè)量證明二茂鐵已成功標(biāo)記到3’為NH2的核酸探針上。相比于一般的采用透析分離純化二茂鐵核酸探針的方法,HPLC分離純化的二茂鐵核酸探針具有純度高、信號(hào)強(qiáng)等優(yōu)點(diǎn)。為下一步的工作打下了基礎(chǔ)。 2.二茂鐵核酸探針用于APE1酶活性的電化學(xué)檢測(cè) APEl酶是一種重要的DNA損傷修復(fù)蛋白,經(jīng)典的放射性標(biāo)記活性分析雖然靈敏度高,但存在操作復(fù)雜、費(fèi)時(shí)、有放射性污染等缺陷。我們?cè)趲в蠥P位點(diǎn)的核酸探針上修飾二茂鐵,并與固定在金電極表面的捕獲探針雜交,形成帶AP位點(diǎn)的二茂鐵修飾的雙鏈DNA。利用APE1酶對(duì)AP位點(diǎn)切割產(chǎn)生的信號(hào)變化實(shí)現(xiàn)了酶活性的電化學(xué)檢測(cè)。該方法對(duì)APEl酶活性的檢測(cè)范圍是0.02-2 U／ml,檢測(cè)下限為0.02 U／ml,具有操作簡(jiǎn)單、靈敏度高等優(yōu)點(diǎn),有望用于生物樣品中DNA損傷修復(fù)的研究。 3.“裂開型”核酸適體結(jié)合金納米顆粒信號(hào)放大用于腺苷的高靈敏檢測(cè) 雖然核酸適體能夠?qū)崿F(xiàn)小分子的特異性識(shí)別,但由于結(jié)合常數(shù)一般相對(duì)較低,往往需要結(jié)合信號(hào)放大技術(shù)以提高檢測(cè)靈敏度。我們構(gòu)建了一種基于核酸適體和金納米顆粒信號(hào)放大的檢測(cè)小分子腺苷的電化學(xué)生物傳感器。該傳感器對(duì)腺苷檢測(cè)的線性范圍為0.1pM-1nM,檢測(cè)下限為0.1 pM,且特異性好,有望拓展到其他小分子的檢測(cè)中。
[Abstract]:Electrochemical biosensor, which is easy to realize miniaturization, integration, simple operation and so on, plays an increasingly important role in chemistry, biological basic research, clinical testing, environmental monitoring and so on. Nucleic acid probes can specifically bind to various targets and form a variety of flexible molecular recognition and signal conversion mechanisms. Based on the characteristics of nucleic acid probes, this paper develops a simple, rapid and sensitive electrochemical sensor for the detection of enzyme and biological small molecules. The following research work has been done: 1. The synthesis and electrochemical characterization of ferrocene nucleic acid probes improved the succinimide coupling method to eliminate the effect of water on the active intermediates in the reaction system and purified by high performance liquid chromatography (HPLC). Ferrocene labeled nucleic acid probes were obtained. Electrochemical measurements show that ferrocene has been successfully labeled into a 3 'NH2 nucleic acid probe. The ferrocene nucleic acid probe separated and purified by HPLC has the advantages of high purity and strong signal. It lays the foundation for the next work. 2. Ferrocene nucleic acid probe for electrochemical detection of APE1 enzyme activity APEl enzyme is an important DNA damage repair protein. Although the classical radiolabelling activity analysis is highly sensitive, its operation is complex and time-consuming. There are defects such as radioactive contamination. We modified ferrocene on a nucleic acid probe with AP site and hybridized with a trap probe fixed on the surface of gold electrode to form ferrocene modified double stranded DNA. with AP site. The electrochemical detection of enzyme activity was achieved by using APE1 enzyme to change the signal generated by AP site cleavage. The detection range of APEl enzyme activity is 0.02-2 U / ml, and the detection limit is 0.02 U / ml. This method has the advantages of simple operation and high sensitivity. It is expected to be used in the study of DNA damage repair in biological samples. 3. "split-type" aptamer combined with gold nanoparticles signal amplification for high sensitive detection of adenosine although nucleic acid aptamers can achieve specific recognition of small molecules, the binding constants are generally relatively low. Signal amplification is often needed to improve detection sensitivity. We have constructed an electrochemical biosensor for the detection of small molecule adenosine based on aptamer of nucleic acid and amplification of gold nanoparticles. The linear range of the sensor for adenosine detection is 0.1pM-1nM.The detection limit is 0.1 pM, and the specificity is good. It is expected to be extended to the detection of other small molecules.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341
本文編號(hào)：2311898
[Abstract]:Electrochemical biosensor, which is easy to realize miniaturization, integration, simple operation and so on, plays an increasingly important role in chemistry, biological basic research, clinical testing, environmental monitoring and so on. Nucleic acid probes can specifically bind to various targets and form a variety of flexible molecular recognition and signal conversion mechanisms. Based on the characteristics of nucleic acid probes, this paper develops a simple, rapid and sensitive electrochemical sensor for the detection of enzyme and biological small molecules. The following research work has been done: 1. The synthesis and electrochemical characterization of ferrocene nucleic acid probes improved the succinimide coupling method to eliminate the effect of water on the active intermediates in the reaction system and purified by high performance liquid chromatography (HPLC). Ferrocene labeled nucleic acid probes were obtained. Electrochemical measurements show that ferrocene has been successfully labeled into a 3 'NH2 nucleic acid probe. The ferrocene nucleic acid probe separated and purified by HPLC has the advantages of high purity and strong signal. It lays the foundation for the next work. 2. Ferrocene nucleic acid probe for electrochemical detection of APE1 enzyme activity APEl enzyme is an important DNA damage repair protein. Although the classical radiolabelling activity analysis is highly sensitive, its operation is complex and time-consuming. There are defects such as radioactive contamination. We modified ferrocene on a nucleic acid probe with AP site and hybridized with a trap probe fixed on the surface of gold electrode to form ferrocene modified double stranded DNA. with AP site. The electrochemical detection of enzyme activity was achieved by using APE1 enzyme to change the signal generated by AP site cleavage. The detection range of APEl enzyme activity is 0.02-2 U / ml, and the detection limit is 0.02 U / ml. This method has the advantages of simple operation and high sensitivity. It is expected to be used in the study of DNA damage repair in biological samples. 3. "split-type" aptamer combined with gold nanoparticles signal amplification for high sensitive detection of adenosine although nucleic acid aptamers can achieve specific recognition of small molecules, the binding constants are generally relatively low. Signal amplification is often needed to improve detection sensitivity. We have constructed an electrochemical biosensor for the detection of small molecule adenosine based on aptamer of nucleic acid and amplification of gold nanoparticles. The linear range of the sensor for adenosine detection is 0.1pM-1nM.The detection limit is 0.1 pM, and the specificity is good. It is expected to be extended to the detection of other small molecules.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341
【參考文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 劉斌;熒光分子探針技術(shù)在基因表達(dá)產(chǎn)物研究中的應(yīng)用[D];湖南大學(xué);2007年
，本文編號(hào)：2311898
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