基于银纳米簇和核酸探针生物传感新方法研究

发布时间：2018-04-11 18:41

  本文选题：杂交链式反应 + 信号放大　； 参考：《湖南大学》2016年硕士论文

【摘要】：生物传感器由于其具有成本低、选择性好、灵敏度高、分析速度快、能进行在线连续监测等优点,在实际分析检测和科学研究方面具有广阔的应用前景。银纳米簇和核酸探针在生物传感方面的广泛应用和取得的成果,为研究者提供了更多的传感设计思路。本论文基于银纳米簇和核酸探针在生物传感与生化分析应用方面的优势,同时结合巧妙的信号放大方式,致力于传感分析的研究热点,建立了多种生物传感和生化分析新方法用于mRNA的定量分析和小分子的高灵敏检测。本论文建立的分析方法操作简单,灵敏度高,特异性好且分析成本低,并初步显示了某些实际应用方面的检测能力。其具体内容如下：以寡聚核苷酸为模板合成的银纳米簇是常用的荧光纳米材料,但是由于银纳米的灵敏度不高限制了其在生物传感方面的发展。因此,我们利用杂交链式反应(HCR)来进行信号放大。在第1章,我们建立了基于HCR诱导银纳米簇荧光增强用于mRNA的检测。为了避免出现信号干扰,我们设计了四条发夹探针H1-H4,以及包含有银纳米簇DNA合成模版的NC探针,并对发夹探针H1的3’端设计了一段富G序列的拖尾、H3的5’端设计了能与NC探针的非银纳米簇模板部分互补的序列。目标物能够引发HCR反应,打开发夹H1,露出的H1能够打开H2,如此依次打开H3和H4,最后露出的H4的序列与mRNA引发链一致,因而能够再次打开新的H1,最后形成长链DNA聚合体结构,在该结构中不断的释放H1和H3的延长序列,由于H3能够与NC探针的一部分进行杂交,因而使得NC探针合成的银纳米簇与具有富G序列的H1靠近从而得到银纳米簇的荧光增强信号,而银纳米簇的荧光增强信号与目标物的浓度有关,因此实现对mRNA的定量分析。该方法灵敏度高,分析检测限为7 pM,能够实现对mRNA的突变分析。与传统的HCR放大方法相比,本章建立的方法操作简便,不需要任何化学标记。同时该方法能够用于复杂的细胞RNA提取物中mRNA的测定。裂开型核酸适配体是生物传感器的主要的机制之一。然而,目前的裂开型适配体传感设计由于缺乏耦合下游信号扩增方法而限制了分析方法的灵敏度。本论文第3章,我们发展了基于裂开型核酸适配体介导的内切酶扩增的适配体传感新方法(SAMEA)用于小分子的高灵敏检测。该设计思路依赖于我们的发现：以邻近杂交产生的DNA三通结构也能作为核酸内切酶IV (Endo IV)的底物,通过Endo IV循环剪切其中的检测探针,可以产生巨大的荧光激活式信号放大。基于此,通过对裂开型适配体的两条片段进行延长,利用分析物的诱导形成适配体片段夹心复合物,同时使得延长序列与检测探针邻近杂交,组装成DNA三通结构并结合EndoIV循环扩增,实现了分析物的高灵敏检测。利用可卡因作为该方法的模型检测对象,结果显示,基于裂开型核酸适配体介导的内切酶放大用于可卡因的检测限为7pM。相比于无放大的裂开型核适配体检测方法,该方法改善了大于105倍的灵敏度。同时,从血清样品的回收率结果可以看出,该方法在复杂体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.

【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3
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本文编号：1737203