本文選題：雜交鏈式反應 + 信號放大��； 參考：《湖南大學》2016年碩士論文

【摘要】：生物傳感器由于其具有成本低、選擇性好、靈敏度高、分析速度快、能進行在線連續(xù)監(jiān)測等優(yōu)點,在實際分析檢測和科學研究方面具有廣闊的應用前景。銀納米簇和核酸探針在生物傳感方面的廣泛應用和取得的成果,為研究者提供了更多的傳感設計思路。本論文基于銀納米簇和核酸探針在生物傳感與生化分析應用方面的優(yōu)勢,同時結(jié)合巧妙的信號放大方式,致力于傳感分析的研究熱點,建立了多種生物傳感和生化分析新方法用于mRNA的定量分析和小分子的高靈敏檢測。本論文建立的分析方法操作簡單,靈敏度高,特異性好且分析成本低,并初步顯示了某些實際應用方面的檢測能力。其具體內(nèi)容如下：以寡聚核苷酸為模板合成的銀納米簇是常用的熒光納米材料,但是由于銀納米的靈敏度不高限制了其在生物傳感方面的發(fā)展。因此,我們利用雜交鏈式反應(HCR)來進行信號放大。在第1章,我們建立了基于HCR誘導銀納米簇熒光增強用于mRNA的檢測。為了避免出現(xiàn)信號干擾,我們設計了四條發(fā)夾探針H1-H4,以及包含有銀納米簇DNA合成模版的NC探針,并對發(fā)夾探針H1的3’端設計了一段富G序列的拖尾、H3的5’端設計了能與NC探針的非銀納米簇模板部分互補的序列。目標物能夠引發(fā)HCR反應,打開發(fā)夾H1,露出的H1能夠打開H2,如此依次打開H3和H4,最后露出的H4的序列與mRNA引發(fā)鏈一致,因而能夠再次打開新的H1,最后形成長鏈DNA聚合體結(jié)構,在該結(jié)構中不斷的釋放H1和H3的延長序列,由于H3能夠與NC探針的一部分進行雜交,因而使得NC探針合成的銀納米簇與具有富G序列的H1靠近從而得到銀納米簇的熒光增強信號,而銀納米簇的熒光增強信號與目標物的濃度有關,因此實現(xiàn)對mRNA的定量分析。該方法靈敏度高,分析檢測限為7 pM,能夠?qū)崿F(xiàn)對mRNA的突變分析。與傳統(tǒng)的HCR放大方法相比,本章建立的方法操作簡便,不需要任何化學標記。同時該方法能夠用于復雜的細胞RNA提取物中mRNA的測定。裂開型核酸適配體是生物傳感器的主要的機制之一。然而,目前的裂開型適配體傳感設計由于缺乏耦合下游信號擴增方法而限制了分析方法的靈敏度。本論文第3章,我們發(fā)展了基于裂開型核酸適配體介導的內(nèi)切酶擴增的適配體傳感新方法(SAMEA)用于小分子的高靈敏檢測。該設計思路依賴于我們的發(fā)現(xiàn)：以鄰近雜交產(chǎn)生的DNA三通結(jié)構也能作為核酸內(nèi)切酶IV (Endo IV)的底物,通過Endo IV循環(huán)剪切其中的檢測探針,可以產(chǎn)生巨大的熒光激活式信號放大�；诖�,通過對裂開型適配體的兩條片段進行延長,利用分析物的誘導形成適配體片段夾心復合物,同時使得延長序列與檢測探針鄰近雜交,組裝成DNA三通結(jié)構并結(jié)合EndoIV循環(huán)擴增,實現(xiàn)了分析物的高靈敏檢測。利用可卡因作為該方法的模型檢測對象,結(jié)果顯示,基于裂開型核酸適配體介導的內(nèi)切酶放大用于可卡因的檢測限為7pM。相比于無放大的裂開型核適配體檢測方法,該方法改善了大于105倍的靈敏度。同時,從血清樣品的回收率結(jié)果可以看出,該方法在復雜體系中也具有良好的分析性能。
[Abstract]:The biosensor due to its low cost, good selectivity, high sensitivity, fast analysis, on-line continuous monitoring and other advantages, has broad application prospects in the research of detection and analysis of actual science. Silver nanoclusters and nucleic acid probe widely used in biosensors and achievements provide sensor design more and more researchers. The advantages of silver nanoclusters and nucleic acid probes in biological and biochemical sensing applications based on the analysis, combined with clever signal amplification, dedicated to sensing research, established a sensitive detection method for a variety of biological sensing and biochemical analysis for quantitative analysis and small molecules of mRNA. Analysis method established in this paper has the advantages of simple operation, high sensitivity, good specificity and low cost analysis, and showed the detection ability of some practical applications. The Content is as follows: to oligonucleotides as template for the synthesis of silver nanoclusters is fluorescent nano materials commonly used, but because of the sensitivity of silver nanoparticles is not high limit its in biosensor development. Therefore, we use the hybridization chain reaction (HCR) for signal amplification. In the first chapter, we established HCR induced Silver nanoclusters for enhanced fluorescence detection based on mRNA. In order to avoid signal interference, we designed four hairpin probe H1-H4, and contains NC probe silver nanoclusters DNA template synthesis, and the hairpin probe 3 'end of H1 design for a G rich sequence of tailing, 5' end of H3 design the NC probe and non silver nanoclusters template complementary sequences. The target can trigger the HCR reaction, open hairpin H1, exposing the H1 to open the H2, so in order to open the H3 and H4 sequences and mRNA finally exposed H4 caused by chain. To open a new H1 again, the last long chain DNA polymer structure formation, extended sequence in the structure to the release of H1 and H3, because the H3 can part with the NC probe hybridization, which makes the silver nanoclusters synthesized with NC probe with G rich sequences near H1 to obtain fluorescent silver nanoparticles cluster signal enhancement, and the enhancement of the concentration of fluorescent silver nanoclusters and the target signal, so the quantitative analysis of mRNA. The method has high sensitivity, detection limit was 7 pM, can realize the mutation analysis of mRNA. Compared with the traditional HCR amplification method, this chapter established the method is simple, do not need any chemical marker. At the same time, the method can be used for the determination of mRNA cell extract of RNA in complex. Based on split aptamer biosensor is one of the main mechanisms. However, ligand sensing Design by split current adaptation In the lack of coupling downstream signal amplification method limits the sensitivity analysis method. The third chapter of this thesis, we developed a new method of adaptive sensing based on split aptamer ligand mediated enzyme amplification based on (SAMEA) for highly sensitive detection of small molecules. The design depends on our findings generated by adjacent the DNA three hybrid structure can also be used as IV endonuclease (Endo IV) of the substrate, by which the Endo IV probe for the detection of cyclic shear, can produce a large fluorescence activated signal amplification. Based on this, was extended by two fragments of split type aptamer, using aptamer fragments induced the formation of sandwich complexes the analyte, and makes an extended sequence of probe and adjacent hybridization, assembled into DNA three structure combined with EndoIV cycle amplification, to achieve a high sensitive detection of analytes. As the use of cocaine The model results show that the method of detecting objects, based on split aptamer mediated enzyme amplification for cocaine detection limit is 7pM. compared with no amplification of the split type nuclear aptamer based detection method, this method improves the sensitivity is more than 105 times. At the same time, the rate of recovery from the serum samples of the results can be seen, the the analysis method has good performance in the complex system.

【學位授予單位】：湖南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：O657.3
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本文編號：1737203