本文關(guān)鍵詞： 銅納米顆粒 末端脫氧核苷酸轉(zhuǎn)移酶 限制性內(nèi)切酶 堿性磷酸酶 核酸內(nèi)切酶V 放大檢測(cè)　出處：《湖南大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：納米技術(shù)的提出為納米材料科學(xué)的發(fā)展及應(yīng)用開(kāi)辟了新的領(lǐng)域。現(xiàn)代科學(xué)研究中,納米技術(shù)結(jié)合物理、化學(xué)、生物和醫(yī)學(xué)成像和超靈敏檢測(cè)的方法在分析化學(xué)和生命科學(xué)中的地位越來(lái)越重要,是一個(gè)極具前景的領(lǐng)域。由于粒徑微小(1-100 nm),金屬納米材料在化學(xué)、物理和電子性質(zhì)非常獨(dú)特,可用作構(gòu)建新型的傳感設(shè)備,特別在生化傳感和電化學(xué)傳感等方面。金屬納米材料的運(yùn)用可以降低檢測(cè)限,提高傳感器的靈敏性,開(kāi)拓一些已有材料不能完成的新的工作。近年來(lái),以DNA為模板來(lái)合成銅納米顆粒(DNA-CuNPs)在生物成像及傳感應(yīng)用中引起了越來(lái)越多研究者的廣泛關(guān)注。因?yàn)槠渚哂屑?xì)微的尺寸,低毒性和很好的生物相容性的優(yōu)勢(shì),相比于現(xiàn)已存在的熒光金屬納米粒子,以DNA為模板合成的銅納米顆粒是一種新型的功能化生物化學(xué)探針。末端脫氧核苷酸轉(zhuǎn)移酶(TdT),是一種特別的DNA聚合酶,它通過(guò)在DNA引物的3′-OH端不斷添加堿基,催化無(wú)模板的DNA發(fā)生聚合反應(yīng)。TdT廣泛用作分子生物工具,用來(lái)標(biāo)記DNA末端。本文基于TdT的擴(kuò)增反應(yīng),提出了一種合成DNA-CuNPs的新型方法,并應(yīng)用于酶的活性檢測(cè)和分析。具體如下:1、利用TdT催化單鏈DNA(ss DNA)聚合反應(yīng)的性質(zhì),開(kāi)發(fā)出了一種新型的DNA-CuNPs合成方法。在一條原本不能合成CuNPs的DNA 3′-OH端通過(guò)TdT催化發(fā)生聚合反應(yīng),從而在該DNA引物3′-OH端不斷添加堿基,得到一段聚合胸腺嘧啶(polyT)的DNA序列,然后以這段poly T為模板合成能夠發(fā)射熒光的DNA-CuNPs。此外,我們優(yōu)化了TdT擴(kuò)增反應(yīng)的條件,以及可能會(huì)影響合成DNA-CuNPs的因素。并且比較分別以DNA-P-polyT和T40為DNA模板合成DNA-CuNPs,發(fā)現(xiàn)以DNA-P-poly T為模板合成的DNA-CuNPs發(fā)射出更高的熒光強(qiáng)度。同時(shí),我們通過(guò)透射電子顯微鏡(TEM)來(lái)表征DNA-CuNPs。根據(jù)TEM圖,我們分析以長(zhǎng)鏈為模板合成的DNA-CuNPs熒光強(qiáng)度更大的原因是:1)長(zhǎng)鏈DNA序列可以移除CuNPs表面極性溶劑,從而更好的保護(hù)CuNPs;2)熒光共振能量轉(zhuǎn)移(FRET)的現(xiàn)象可能存在于CuNPs之間。2、基于新型DNA-CuNPs合成方法的構(gòu)建,我們提出了一種TdT的活性分析檢測(cè)方法。該方法實(shí)現(xiàn)了對(duì)TdT進(jìn)行無(wú)標(biāo)記、低背景、高靈敏地“turn-on”酶活分析,檢測(cè)限為3.75 U/mL。此外,該方法具有較穩(wěn)定的重復(fù)性,在復(fù)雜生物環(huán)境中的添加回收取得了較滿意的結(jié)果。接著,我們考察了TdT抑制劑焦磷酸鈉對(duì)TdT的抑制效果。3、基于TdT對(duì)隨機(jī)DNA底物擴(kuò)增及新型DNA-CuNPs的合成,我們構(gòu)建了一個(gè)酶活檢測(cè)的通用性平臺(tái)。我們選取限制性內(nèi)切酶(BamHI)和堿性磷酸酶(ALP)作為目標(biāo)分析物。實(shí)驗(yàn)得到,BamHI的檢測(cè)限為0.005 U/mL,信背比為31.4,跟相關(guān)文獻(xiàn)報(bào)道的相仿;ALP的檢測(cè)限為0.052×10-3 U/m L,信背比為44.6,比相關(guān)文獻(xiàn)報(bào)道較低。由于酶對(duì)底物具有特異性,所以Bam HI和ALP的選擇性都很好。此外,只需要換成不同的酶對(duì)應(yīng)的DNA底物,就可以應(yīng)用于核酸外切酶、切刻內(nèi)切酶、DNA連接酶的活性分析。4、基于核酸內(nèi)切酶V(Endonuclease V)可以識(shí)別并切割脫氨基損傷以及切刻內(nèi)切酶Nt.BbvCI特異性識(shí)別并切割含有其識(shí)別序列的DNA,我們把前面提出的新型DNA-CuNPs合成方法用于Endonuclease V的“turn-on”放大檢測(cè)。通過(guò)合理地設(shè)計(jì)三條DNA探針─一條含脫氧次黃嘌呤核苷的探針(Sub-DNA),一條與(Sub-DNA)互補(bǔ)的探針(Com-DNA),一條含有切刻內(nèi)切酶Nt.BbvCI的酶切位點(diǎn)并與Sub-DNA部分互補(bǔ)的探針(Amp-DNA),我們成功實(shí)現(xiàn)了對(duì)Endonuclease V的高靈敏度放大檢測(cè)。另外,由于Endo V特異性識(shí)別并切割脫氨基損傷,該方法的選擇性很好。只要把脫氨基損傷位點(diǎn)換成其他的特殊位點(diǎn),就可以分析檢測(cè)更廣泛的目標(biāo)分析物。因此,我們提出的新方法在生物傳感上有著非常廣泛的應(yīng)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.

【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：TB383.1;TP212.3

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本文編號(hào)：1555737