【摘要】：隨著DNA納米技術(shù)的不斷發(fā)展和進(jìn)步,越來越多的方法出現(xiàn)并應(yīng)用于生物檢測(cè),臨床診斷,藥物治療,計(jì)算化學(xué)等領(lǐng)域。現(xiàn)代生物傳感器需要高靈敏度,極大的信號(hào)增強(qiáng)和顯著的選擇性來達(dá)到檢測(cè)和診斷的目的。傳統(tǒng)的分子信標(biāo)無法進(jìn)行信號(hào)放大,這極大的限制了它的適用范圍。利用酶,DNA酶和納米粒子的信號(hào)放大的過程通常是相當(dāng)復(fù)雜的,并限于特定的應(yīng)用范圍。在本文中,我們?cè)O(shè)計(jì)了自動(dòng)的DNA擴(kuò)增系統(tǒng)來構(gòu)建基于傳統(tǒng)分子信標(biāo)結(jié)構(gòu)的嶄新的和簡(jiǎn)便的生物傳感器。通過這種納米機(jī)器的雜交擴(kuò)增,這種經(jīng)過改進(jìn)的可擴(kuò)增分子信標(biāo)可以大大提高探測(cè)信號(hào)和檢出限,并能應(yīng)用于核酸檢測(cè),區(qū)分單堿基錯(cuò)配。通過引入適配體序列,這種可擴(kuò)增分子信標(biāo)也可以檢測(cè)蛋白質(zhì)和小分子,比如對(duì)β-肌動(dòng)蛋白基因和凝血酶的檢測(cè)。我們同時(shí)進(jìn)一步證實(shí)了可擴(kuò)增分子信標(biāo)具有較高的靈敏度和穩(wěn)定性,其擴(kuò)增能力為在活細(xì)胞中檢測(cè)miRNA提供了可能�？蓴U(kuò)增分子信標(biāo)的簡(jiǎn)單性和靈活性使它具有更廣泛的應(yīng)用前景。Toehold結(jié)構(gòu),作為鏈交換反應(yīng)的核心,具有舉足輕重的作用。研究人員針對(duì)toehold的長(zhǎng)度,與鏈交換區(qū)域的距離,位置等要素做了許多工作。不過,在大多數(shù)情況下,toehold結(jié)構(gòu)始終被默認(rèn)為雙鏈結(jié)構(gòu)。在本論文中,我們改變了它的基本構(gòu)型和鏈交換機(jī)理,設(shè)計(jì)了一個(gè)僅僅以DNA G-四鏈體為toehold結(jié)構(gòu)誘導(dǎo)的DNA鏈交換反應(yīng)。當(dāng)反應(yīng)環(huán)境中加入PEG時(shí),這種分子擁擠試劑使得反應(yīng)可以容易的發(fā)生,我們可以通過調(diào)節(jié)PEG的濃度和聚合度來控制反應(yīng),同時(shí)也可以通過改變G-四鏈體的結(jié)構(gòu)來進(jìn)行調(diào)節(jié)。同時(shí)我們還在遷移鏈中引入了錯(cuò)配的堿基,使鏈交換反應(yīng)在水溶液中也可以順利進(jìn)行。緊接著我們還設(shè)計(jì)了基于這種特殊鏈交換的DNA鏈?zhǔn)綌U(kuò)增反應(yīng),通過G-四鏈體這一結(jié)構(gòu)的特征實(shí)現(xiàn)無需熒光標(biāo)記的檢測(cè)體系,這種方法具有成本低,無需標(biāo)記,靈敏度高和選擇性強(qiáng)的特性,具有在DNA的納米機(jī)器,生物傳感和疾病診斷方面的應(yīng)用潛力。
[Abstract]:With the development and progress of DNA nanotechnology, more and more methods have been developed and applied in the fields of biological detection, clinical diagnosis, drug therapy, computational chemistry and so on. Modern biosensors require high sensitivity, great signal enhancement and significant selectivity for detection and diagnosis. Traditional molecular beacons can not amplify signals, which greatly limits its scope of application. The process of signal amplification using enzymes, DNA enzymes and nanoparticles is usually complex and limited to specific applications. In this paper, we designed an automatic DNA amplification system to construct novel and simple biosensors based on traditional molecular beacon structures. The improved amplification molecular beacon can greatly improve the detection signal and detection limit, and can be used to detect nucleic acid and distinguish single base mismatch. By introducing aptamer sequences, the amplified molecular beacons can also detect proteins and small molecules, such as 尾 -actin genes and thrombin. We also confirmed the high sensitivity and stability of the amplified molecular beacons, which provided the possibility for the detection of miRNA in living cells. The simplicity and flexibility of augmentable molecular beacons make it more widely used. Toehold structure, as the core of chain exchange reaction, plays an important role. Researchers have done a lot of work on the length of the toehold, the distance and location of the chain-exchange region. However, in most cases, the toehold structure is always default to a double-stranded structure. In this thesis, we have changed its basic configuration and chain switching mechanism, and designed a DNA strand exchange reaction induced only by DNA G- quadruplex as toehold structure. When PEG is added to the reaction environment, this kind of molecular crowding reagent makes the reaction happen easily. We can control the reaction by adjusting the concentration and degree of polymerization of PEG, and we can also adjust the structure of G- quad by changing the structure of PEG. At the same time, we also introduced mismatched bases into the transport chain, so that the chain exchange reaction can be carried out smoothly in aqueous solution. Then we also design the DNA chain amplification reaction based on this special chain exchange, and realize the detection system without fluorescent labeling by using the structure characteristic of G- quadruplex. This method has the advantages of low cost and no labeling. With high sensitivity and selectivity, it has potential applications in DNA nanomachines, biosensors and disease diagnosis.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78

【相似文獻(xiàn)】

相關(guān)期刊論文 前4條

1 馬麗娜;董亞非;張成;劉西奎;;基于DNA鏈置換與熒光標(biāo)記的0-1規(guī)劃問題的計(jì)算模型[J];數(shù)學(xué)的實(shí)踐與認(rèn)識(shí);2013年11期

2 ;以用DNA鏈造出納米機(jī)器人可注入活動(dòng)物體內(nèi)[J];黑龍江科技信息;2014年11期

3 張成;馬麗娜;董亞非;楊靜;許進(jìn);;自組裝DNA鏈置換分子邏輯計(jì)算模型[J];科學(xué)通報(bào);2012年31期

4 ;[J];;年期

相關(guān)會(huì)議論文 前5條

1 邢永政;劉冬生;;ATP誘導(dǎo)的DNA突環(huán)結(jié)構(gòu)的打開及其對(duì)DNA鏈替換反應(yīng)的調(diào)控[A];2011年全國(guó)高分子學(xué)術(shù)論文報(bào)告會(huì)論文摘要集[C];2011年

2 王蓓;梁好均;;基于DNA鏈替換反應(yīng)的DNA納米線與可調(diào)FRET信號(hào)[A];2014年兩岸三地高分子液晶態(tài)與超分子有序結(jié)構(gòu)學(xué)術(shù)研討會(huì)摘要集[C];2014年

3 鄧偉;丁偉;朱紅;梁好均;;金屬離子驅(qū)動(dòng)的DNA鏈替換反應(yīng)及其應(yīng)用[A];2014年兩岸三地高分子液晶態(tài)與超分子有序結(jié)構(gòu)學(xué)術(shù)研討會(huì)摘要集[C];2014年

4 任克維;吳潔;鞠q，

本文編號(hào)：2281746