基于DNA自組裝的納米金比色檢測(cè)DNA新方法研究
發(fā)布時(shí)間：2019-01-09 11:17
【摘要】：DNA是生物體重要的遺傳物質(zhì),通過(guò)對(duì)DNA的檢測(cè)能實(shí)現(xiàn)對(duì)疾病的早期診斷和預(yù)防,不同物種的鑒定和環(huán)境的監(jiān)測(cè)保護(hù)等,因此,對(duì)DNA的檢測(cè)具有十分重要的意義。本論文以DNA檢測(cè)為主要目標(biāo),從DNA無(wú)酶自組裝信號(hào)放大出發(fā),提出了一種新型無(wú)酶的指數(shù)式發(fā)卡型DNA自組裝反應(yīng),并研究通過(guò)多種方式將該反應(yīng)應(yīng)用于DNA檢測(cè)中。首先,在雜交鏈?zhǔn)椒磻?yīng)的基礎(chǔ)上,本論文提出了一種新型的指數(shù)式發(fā)卡自組裝反應(yīng),該反應(yīng)只需要四條發(fā)卡型DNA,加入引發(fā)鏈之后,在自由能的驅(qū)動(dòng)下自發(fā)進(jìn)行組裝,在較短的時(shí)間內(nèi)生成一個(gè)較大的DNA聚合體。通過(guò)瓊脂糖凝膠電泳驗(yàn)證了該反應(yīng)的可行性；通過(guò)原子力顯微鏡進(jìn)一步表征了生成產(chǎn)物的形態(tài)。在優(yōu)化發(fā)卡型DNA的濃度和反應(yīng)時(shí)間后,形成了規(guī)則的納米微孔網(wǎng)狀結(jié)構(gòu),孔徑分布在200 nin到400 nm之間。該納米微孔網(wǎng)狀結(jié)構(gòu)可以作為一種新型的DNA納米材料。其次,以該納米材料作為信號(hào)放大的載體,結(jié)合熒光成像單分子檢測(cè)技術(shù)和納米金比色檢測(cè)技術(shù),將該反應(yīng)應(yīng)用于DNA的檢測(cè)中。實(shí)驗(yàn)結(jié)果顯示納米金比色檢測(cè)技術(shù)能有效的消除背景信號(hào)的影響,實(shí)現(xiàn)對(duì)DNA的靈敏檢測(cè)。在最佳反應(yīng)條件下,靶標(biāo)濃度在25-10000pM之間時(shí),比色檢測(cè)結(jié)果成良好的線(xiàn)性關(guān)系,該檢測(cè)方法能在15 min內(nèi)實(shí)現(xiàn)肉眼檢測(cè)25 pM的靶標(biāo)DNA,該檢測(cè)限比傳統(tǒng)的納米金比色檢測(cè)方法降低了約400倍,同時(shí)通過(guò)紫外可見(jiàn)光分光光度計(jì)可檢測(cè)11.2 pM的靶標(biāo)DNA。另外,通過(guò)對(duì)不同錯(cuò)配靶標(biāo)的檢測(cè),證明了該方法對(duì)單堿基突變具有良好的特異性。本論文通過(guò)DNA自組裝構(gòu)建了一種DNA納米材料,并將該納米材料應(yīng)用于納米金比色檢測(cè)DNA中,該檢測(cè)方法反應(yīng)迅速,耗時(shí)短,靈敏度高,反應(yīng)結(jié)果可通過(guò)肉眼直接觀(guān)察,操作簡(jiǎn)單,因此可潛在的應(yīng)用于實(shí)時(shí)現(xiàn)場(chǎng)快速檢測(cè)。
[Abstract]:DNA is an important genetic material of organism. The detection of DNA can realize the early diagnosis and prevention of disease, the identification of different species and the monitoring and protection of environment, etc. Therefore, the detection of DNA is of great significance. Based on the amplification of DNA self-assembly signal without enzyme, a new type of exponential DNA self-assembly reaction without enzyme was proposed in this paper, and the reaction was applied to DNA detection in many ways. Firstly, based on the hybrid chain reaction, a novel exponential hairpin self-assembly reaction is proposed. The reaction requires only four hairpin type DNA, to be added to the initiation chain, and then self-assembled under the driving of free energy. A large DNA polymer is formed in a short time. The feasibility of the reaction was verified by agarose gel electrophoresis, and the morphology of the product was further characterized by atomic force microscope (AFM). After optimizing the concentration and reaction time of card type DNA, a regular nano-pore network structure was formed, with pore size ranging from 200 nin to 400 nm. The nanoporous network structure can be used as a new type of DNA nanomaterials. Secondly, the nanomaterials were used as the carrier of signal amplification, combined with fluorescence imaging monolayer detection technology and nano-gold colorimetric detection technology, the reaction was applied to the detection of DNA. The experimental results show that the nanocrystalline gold colorimetry can effectively eliminate the influence of background signal and realize the sensitive detection of DNA. Under the optimum reaction conditions, the results of colorimetric detection show a good linear relationship when the concentration of target is between 25-10000pM. This detection method can detect 25 pM target DNA, with naked eyes within 15 min. The detection limit of this method is about 400 times lower than that of traditional nanocrystalline gold colorimetry, and the target DNA. of 11.2 pM can be detected by UV-Vis spectrophotometer. In addition, the detection of different mismatch targets proves that this method has good specificity for single base mutation. In this paper, a kind of DNA nanomaterials was constructed by DNA self-assembly, and the nanomaterials were applied to the determination of gold nanocrystalline colorimetric DNA. The method is rapid, time-consuming and sensitive, and the reaction results can be observed directly by naked eye. It is easy to operate, so it can potentially be used in real-time field fast detection.
【學(xué)位授予單位】：青島科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：O657.3;TB383.1
本文編號(hào)：2405550
[Abstract]:DNA is an important genetic material of organism. The detection of DNA can realize the early diagnosis and prevention of disease, the identification of different species and the monitoring and protection of environment, etc. Therefore, the detection of DNA is of great significance. Based on the amplification of DNA self-assembly signal without enzyme, a new type of exponential DNA self-assembly reaction without enzyme was proposed in this paper, and the reaction was applied to DNA detection in many ways. Firstly, based on the hybrid chain reaction, a novel exponential hairpin self-assembly reaction is proposed. The reaction requires only four hairpin type DNA, to be added to the initiation chain, and then self-assembled under the driving of free energy. A large DNA polymer is formed in a short time. The feasibility of the reaction was verified by agarose gel electrophoresis, and the morphology of the product was further characterized by atomic force microscope (AFM). After optimizing the concentration and reaction time of card type DNA, a regular nano-pore network structure was formed, with pore size ranging from 200 nin to 400 nm. The nanoporous network structure can be used as a new type of DNA nanomaterials. Secondly, the nanomaterials were used as the carrier of signal amplification, combined with fluorescence imaging monolayer detection technology and nano-gold colorimetric detection technology, the reaction was applied to the detection of DNA. The experimental results show that the nanocrystalline gold colorimetry can effectively eliminate the influence of background signal and realize the sensitive detection of DNA. Under the optimum reaction conditions, the results of colorimetric detection show a good linear relationship when the concentration of target is between 25-10000pM. This detection method can detect 25 pM target DNA, with naked eyes within 15 min. The detection limit of this method is about 400 times lower than that of traditional nanocrystalline gold colorimetry, and the target DNA. of 11.2 pM can be detected by UV-Vis spectrophotometer. In addition, the detection of different mismatch targets proves that this method has good specificity for single base mutation. In this paper, a kind of DNA nanomaterials was constructed by DNA self-assembly, and the nanomaterials were applied to the determination of gold nanocrystalline colorimetric DNA. The method is rapid, time-consuming and sensitive, and the reaction results can be observed directly by naked eye. It is easy to operate, so it can potentially be used in real-time field fast detection.
【學(xué)位授予單位】：青島科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：O657.3;TB383.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 陳德強(qiáng),夏安東;雙光子激光掃描熒光顯微鏡及其應(yīng)用[J];物理;2000年04期
，本文編號(hào)：2405550
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