基于酶扩增合成铜纳米颗粒及其在生物传感上的应用

发布时间：2018-03-02 08:20

本文关键词： 铜纳米颗粒末端脱氧核苷酸转移酶限制性内切酶碱性磷酸酶核酸内切酶V 放大检测　出处：《湖南大学》2015年硕士论文　论文类型：学位论文

【摘要】：纳米技术的提出为纳米材料科学的发展及应用开辟了新的领域。现代科学研究中,纳米技术结合物理、化学、生物和医学成像和超灵敏检测的方法在分析化学和生命科学中的地位越来越重要,是一个极具前景的领域。由于粒径微小(1-100 nm),金属纳米材料在化学、物理和电子性质非常独特,可用作构建新型的传感设备,特别在生化传感和电化学传感等方面。金属纳米材料的运用可以降低检测限,提高传感器的灵敏性,开拓一些已有材料不能完成的新的工作。近年来,以DNA为模板来合成铜纳米颗粒(DNA-CuNPs)在生物成像及传感应用中引起了越来越多研究者的广泛关注。因为其具有细微的尺寸,低毒性和很好的生物相容性的优势,相比于现已存在的荧光金属纳米粒子,以DNA为模板合成的铜纳米颗粒是一种新型的功能化生物化学探针。末端脱氧核苷酸转移酶(TdT),是一种特别的DNA聚合酶,它通过在DNA引物的3′-OH端不断添加碱基,催化无模板的DNA发生聚合反应。TdT广泛用作分子生物工具,用来标记DNA末端。本文基于TdT的扩增反应,提出了一种合成DNA-CuNPs的新型方法,并应用于酶的活性检测和分析。具体如下:1、利用TdT催化单链DNA(ss DNA)聚合反应的性质,开发出了一种新型的DNA-CuNPs合成方法。在一条原本不能合成CuNPs的DNA 3′-OH端通过TdT催化发生聚合反应,从而在该DNA引物3′-OH端不断添加碱基,得到一段聚合胸腺嘧啶(polyT)的DNA序列,然后以这段poly T为模板合成能够发射荧光的DNA-CuNPs。此外,我们优化了TdT扩增反应的条件,以及可能会影响合成DNA-CuNPs的因素。并且比较分别以DNA-P-polyT和T40为DNA模板合成DNA-CuNPs,发现以DNA-P-poly T为模板合成的DNA-CuNPs发射出更高的荧光强度。同时,我们通过透射电子显微镜(TEM)来表征DNA-CuNPs。根据TEM图,我们分析以长链为模板合成的DNA-CuNPs荧光强度更大的原因是:1)长链DNA序列可以移除CuNPs表面极性溶剂,从而更好的保护CuNPs;2)荧光共振能量转移(FRET)的现象可能存在于CuNPs之间。2、基于新型DNA-CuNPs合成方法的构建,我们提出了一种TdT的活性分析检测方法。该方法实现了对TdT进行无标记、低背景、高灵敏地“turn-on”酶活分析,检测限为3.75 U/mL。此外,该方法具有较稳定的重复性,在复杂生物环境中的添加回收取得了较满意的结果。接着,我们考察了TdT抑制剂焦磷酸钠对TdT的抑制效果。3、基于TdT对随机DNA底物扩增及新型DNA-CuNPs的合成,我们构建了一个酶活检测的通用性平台。我们选取限制性内切酶(BamHI)和碱性磷酸酶(ALP)作为目标分析物。实验得到,BamHI的检测限为0.005 U/mL,信背比为31.4,跟相关文献报道的相仿;ALP的检测限为0.052×10-3 U/m L,信背比为44.6,比相关文献报道较低。由于酶对底物具有特异性,所以Bam HI和ALP的选择性都很好。此外,只需要换成不同的酶对应的DNA底物,就可以应用于核酸外切酶、切刻内切酶、DNA连接酶的活性分析。4、基于核酸内切酶V(Endonuclease V)可以识别并切割脱氨基损伤以及切刻内切酶Nt.BbvCI特异性识别并切割含有其识别序列的DNA,我们把前面提出的新型DNA-CuNPs合成方法用于Endonuclease V的“turn-on”放大检测。通过合理地设计三条DNA探针─一条含脱氧次黄嘌呤核苷的探针(Sub-DNA),一条与(Sub-DNA)互补的探针(Com-DNA),一条含有切刻内切酶Nt.BbvCI的酶切位点并与Sub-DNA部分互补的探针(Amp-DNA),我们成功实现了对Endonuclease V的高灵敏度放大检测。另外,由于Endo V特异性识别并切割脱氨基损伤,该方法的选择性很好。只要把脱氨基损伤位点换成其他的特殊位点,就可以分析检测更广泛的目标分析物。因此,我们提出的新方法在生物传感上有着非常广泛的应用前景。
[Abstract]:The nanotechnology opens up a new field for the development and application of nano materials science. Modern scientific research in nanotechnology, combined with physical, chemical, biological and medical imaging method and ultra sensitive detection is becoming more and more important in the analysis of chemistry and life science, is a promising field due to the particle size. Small (1-100 nm), metal nano materials in chemical, physical and electronic properties is very unique, can be used for construction of new sensing devices, especially in the biochemical and electrochemical sensing and so on. The use of metal nano materials can reduce the detection limit, improve the sensor sensitivity, develop some materials can complete the new job. In recent years, using DNA as template to synthesis of copper nanoparticles (DNA-CuNPs) attracted more and more attention of researchers in biological imaging and sensing applications. Because it has small size, The compatibility of the advantages of low toxicity and good biological fluorescence, metal nanoparticles compared to existing, with DNA as the template for the synthesis of copper nanoparticles probe is a novel bio chemical functionalization. Terminal deoxynucleotidyl transferase (TdT), is a kind of special DNA polymerase, DNA primer in it the 3 'end of -OH adding bases, catalytic polymerization template free DNA.TdT is widely used as molecular biological tools, used to mark the end of the DNA amplification reaction. In this paper, based on TdT, proposed a new method for the synthesis of DNA-CuNPs, and applied to the detection and analysis of activity of enzymes. The details are as follows: 1, catalyzed by TdT single stranded DNA (SS DNA) nature of the polymerization, developed a new method for the synthesis of DNA-CuNPs. In a unrescetable synthesis of CuNPs DNA 3' terminal -OH of the TdT catalytic polymerization, resulting in the 3 DNA primers' -OH terminal Add a broken base, polymerization of thymine (polyT) DNA sequence, and then to the poly T as the template synthesis can emit fluorescence DNA-CuNPs. in addition, we optimized the TdT reaction conditions, and the factors which may influence the synthesis of DNA-CuNPs and DNA-P-polyT respectively. Compared with DNA and T40 as the template for the synthesis of DNA-CuNPs. DNA-P-poly T DNA-CuNPs is found in the template for the synthesis of fluorescence intensity is higher. At the same time, we by transmission electron microscopy (TEM) to characterize the DNA-CuNPs. according to the TEM diagram, we analyze the reasons for the long chain with the fluorescence intensity of DNA-CuNPs template is greater: 1) long chain DNA sequence can remove CuNPs surface polarity in order to better protect the solvent, CuNPs; 2) fluorescence resonance energy transfer (FRET) phenomenon may exist in the CuNPs.2, to build a new synthesis method based on DNA-CuNPs, we propose a TdT Activity analysis of detection method. This method realizes the TdT label free, low background, high sensitive turn-on enzyme activity analysis, the detection limit was 3.75 U/mL.. In addition, this method has more stable repeatability, recovery in complex biological environments and achieved satisfactory results. Then, we investigated the inhibitory effect of TdT inhibitor sodium pyrophosphate on TdT.3 on Synthesis of TdT random DNA model based on DNA-CuNPs amplification and substrate, we construct a universal platform for enzyme activity detection. We selected restriction endonuclease (BamHI) and alkaline phosphatase (ALP) as the target analyte. The experiment results show that the detection limit of BamHI for 0.005 U/mL, the signal to background ratio of 31.4, with the related literature reported similar; detection limit of 0.052 ALP * 10-3 U/m L, the signal to background ratio was 44.6, lower than reported in the literature. Because the enzyme is specific for the substrate, so the selective Bam and ALP HI Very good. In addition, only need to replace the enzyme corresponding to different DNA substrate, can be applied to exonuclease and endonuclease digestion, DNA ligase activity of.4 endonuclease V (Endonuclease V) based on can recognize and cleave the deamination damage and cutting endonuclease Nt.BbvCI specific recognition and identification of its containing cutting the sequence of DNA, we put the new DNA-CuNPs synthesis method proposed above for Endonuclease V's "turn-on" amplification detection. Through reasonable design of three DNA probes, a DNA probe containing hypoxanthine riboside (Sub-DNA), and (Sub-DNA) a complementary probe (Com-DNA), containing a cutting endonuclease Nt.BbvCI the restriction sites and Sub-DNA complementary probe (Amp-DNA), we successfully achieved a high sensitivity amplification detection of Endonuclease V. In addition, because the Endo V specific recognition and cutting deamination damage The selectivity of the method is very good. As long as the deamination site is changed to other special sites, we can analyze and detect a wider range of target analytes. Therefore, our new method has a very wide application prospect in biosensor.

【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1;TP212.3

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