發(fā)布時(shí)間：2018-05-31 02:33

  本文選題：Au + Pt　； 參考：《天津醫(yī)科大學(xué)》2016年碩士論文

【摘要】：生物硫醇參與人體的蛋白合成、代謝、解毒等生化過(guò)程,其水平的失衡極易誘發(fā)心腦血管疾病、阿爾茨海默癥等多種疾病,因此對(duì)生物體內(nèi)生物硫醇含量的監(jiān)測(cè)有著重要意義。根據(jù)巰基官能團(tuán)與金屬離子、納米團(tuán)簇、納米結(jié)晶的相互作用,目前發(fā)現(xiàn)了許多檢測(cè)方法:如電化學(xué)法、化學(xué)發(fā)光法、熒光分析法和比色法等。其中比色法,以生成有色產(chǎn)物的顯色反應(yīng)為基礎(chǔ),通過(guò)比較或測(cè)量有色產(chǎn)物溶液的顏色深淺確定待測(cè)組分的含量,此方法操作簡(jiǎn)單、顏色可見(jiàn),在定量分析中常被選用,但是實(shí)現(xiàn)超高靈敏度的比色檢測(cè),仍然是一個(gè)挑戰(zhàn)。本文以富胞嘧啶(Cytosine)寡核苷酸為模板合成雙金屬納米顆粒Aux Pty,并采用比色法將其應(yīng)用于生物硫醇的超靈敏檢測(cè)。首先,實(shí)驗(yàn)以富胞嘧啶C序列RET2為模板合成金屬納米顆粒Au、Pt、Au_xPt_y,分別采用高倍-透射電子顯微鏡(HR-TEM),圓二色光譜儀(CD),紫外可見(jiàn)分光光度計(jì)(UV-vis),熒光分光光度計(jì)(FL)和X射線表面光電子能譜儀(XPS)對(duì)金屬納米顆粒進(jìn)行性質(zhì)表征。結(jié)果顯示4.8 nm-Au、4.2 nm-Pt、2.6 nm-Au_1Pt_1、3.6 nm-Au_2Pt_1納米顆粒中,Au~(3+)幾乎全部被還原成Au0,Pt~(2+)部分被還原成Pt0,Au_2Pt_1中Pt0/Pt~(2+)的百分比比Pt、Au_1Pt_1中的高,說(shuō)明Au~(3+)比Pt~(2+)易于還原,Au~(3+)與核苷酸的作用影響了DNA與Pt~(2+)結(jié)合的比例。此外UV-vis、CD光譜掃描表明Au_xPt_y雙金屬納米顆粒中Au、Pt共同存在于富C的核酸模板上。其次,評(píng)價(jià)了金屬納米顆粒催化TMB-H_2O_2反應(yīng)的酶活性,其催化初始反應(yīng)速率規(guī)律為:PtAu_1Pt_1Au_2Pt_1,其中700 nM Au_2Pt_1催化TMB氧化反應(yīng)10 min時(shí)已達(dá)到平衡,可見(jiàn)雙金屬納米顆粒Au_2Pt_1具有較高的過(guò)氧化物酶活性。采用米氏方程-雙倒數(shù)作圖法獲得各自對(duì)底物的動(dòng)力學(xué)參數(shù),如Km,Vmax。雖然Au_2Pt_1對(duì)3,3',5,5'-四甲基聯(lián)苯胺(TMB)的親和性比天然辣根過(guò)氧化物酶HRP差,但是催化TMB-H_2O_2反應(yīng)的最大反應(yīng)速率比HRP快。最后,通過(guò)對(duì)比Pt、Au_xPt_y檢測(cè)生物硫醇的檢測(cè)限,獲知雙金屬納米顆粒Au_2Pt_1的檢測(cè)靈敏度最好,對(duì)L-半胱氨酸(L-Cys)、L-同型半胱氨酸(L-Hcy)的檢測(cè)限分別為3.5 nM、1.6 nM,比表2.2列舉的其他比色法檢測(cè)Cys、Hcy的檢測(cè)限低;采用等溫滴定量熱法(ITC)、HR-TEM、XPS分析了金屬納米顆粒Au_xPt_y的巰基檢測(cè)性能,發(fā)現(xiàn)其與Au:Pt濃度比(x:y)有關(guān),另外,選擇性實(shí)驗(yàn)證明Au_2Pt_1對(duì)生物硫醇檢測(cè)的特異性。雙金屬納米顆粒Au_2Pt_1由于與含巰基化合物發(fā)生作用形成Au-S鍵,造成納米顆粒Au_2Pt_1發(fā)生聚集,進(jìn)而使其在催化TMB-H_2O_2反應(yīng)時(shí)金屬納米模擬酶的活性降低�；贑ys對(duì)Au_2Pt_1催化TMB-H_2O_2反應(yīng)表現(xiàn)出抑制效應(yīng),可知雙金屬納米顆粒Au_2Pt_1檢測(cè)生物硫醇具有高度的特異性。因此,將Au_2Pt_1應(yīng)用到人血清中生物硫醇的檢測(cè),最終計(jì)算出人血清中生物硫醇濃度為312μM,此值與有關(guān)文獻(xiàn)報(bào)道一致�；謴�(fù)補(bǔ)加實(shí)驗(yàn)中,合適的恢復(fù)率證明了Au_2Pt_1檢測(cè)方法的可靠性。未來(lái)會(huì)展開(kāi)對(duì)雙金屬納米顆粒的進(jìn)一步研究,有望將其應(yīng)用于含巰基藥物的檢測(cè)中。
[Abstract]:Biothiol participates in the biochemical processes of human protein synthesis, metabolism, detoxification and other biochemical processes. The imbalance of its level is very easy to induce cardiovascular and cerebrovascular diseases, Alzheimer's disease and many other diseases. Therefore, it is of great significance to monitor the content of biological thiol in the organism. At present, many detection methods have been found, such as electrochemical method, chemiluminescence method, fluorescence analysis method and colorimetric method, in which colorimetric method is based on the color reaction of colored products, and determines the content of the components by comparing or measuring the color depth of the colored product solution. This method is easy to operate, color is visible, and is often used in quantitative analysis. Selection, but the ultra high sensitivity colorimetric detection is still a challenge. In this paper, Cytosine oligonucleotides are used as a template to synthesize bimetallic nanoparticles Aux Pty, and it is applied to the ultra sensitive detection of biologic thiol by colorimetric method. First, the synthesis of metal nanoparticles A with rich cytosine C sequence RET2 as template U, Pt, Au_xPt_y, high power transmission electron microscopy (HR-TEM), circular two color spectrometer (CD), ultraviolet visible spectrophotometer (UV-vis), fluorescence spectrophotometer (FL) and X ray surface photoelectron spectroscopy (XPS) are used to characterize the properties of metal nanoparticles. The results show that 4.8 nm-Au, 4.2 nm-Pt, 2.6 nm-Au_1Pt_1,3.6 nanoparticles. In grain, Au~ (3+) is almost completely reduced to Au0, and the part of Pt~ (2+) is reduced to Pt0, and the percentage of Pt0/Pt~ (2+) in Au_2Pt_1 is higher than that in Pt and Au_1Pt_1, indicating that Au~ is easier to restore than that of nucleotides. Pt co exists on the rich C nucleic acid template. Secondly, it evaluates the enzyme activity of TMB-H_2O_2 reaction catalyzed by metal nanoparticles, and its initial reaction rate is PtAu_1Pt_1Au_2Pt_1, in which 700 nM Au_2Pt_1 catalyzes the equilibrium of the reaction of TMB to 10 min, and it can be seen that the double gold nanoparticles Au_2Pt_1 has a higher peroxidase. Activity. Using the Michaelis equation and double reciprocal mapping method, the kinetic parameters of their respective substrates were obtained, such as Km, Vmax., although the affinity of Au_2Pt_1 to 3,3', 5,5'- four methyl diphenyl amine (TMB) was worse than the natural horseradish peroxidase HRP, but the maximum reaction rate of the TMB-H_2O_2 reaction was faster than HRP. Finally, the biological sulfur was detected by contrasting Pt and Au_xPt_y. The detection limit of alcohol was found to be the best detection sensitivity of bimetallic nanoparticles Au_2Pt_1. The detection limits for L- cysteine (L-Cys) and L- homocysteine (L-Hcy) were 3.5 nM and 1.6 nM respectively. The detection limit of Cys and Hcy was lower than that of other colorimetric methods listed in table 2.2, and the isothermal titration calorimetry (ITC), HR-TEM, XPS analyzed the metal nanoparticles. The detection performance of the sulfhydryl group of Pt_y was found to be related to the concentration ratio of Au:Pt (x:y). In addition, the selectivity experiment showed that Au_2Pt_1 was specific to the detection of bio-mercaptan. The bimetallic nanoparticle Au_2Pt_1 was formed by the formation of Au-S bonds with the sulfhydryl compounds, resulting in the aggregation of nanoparticle Au_2Pt_1, and then made it to catalyze the reaction of TMB-H_2O_2 in the reaction. The activity of the nanoscale enzyme is reduced. Based on the inhibitory effect of Cys on the Au_2Pt_1 catalytic reaction to TMB-H_2O_2, it is known that bimetallic nanoparticles Au_2Pt_1 has a high specificity for the detection of biothiol. Therefore, Au_2Pt_1 is applied to the detection of bio-mercaptan in human serum, and the concentration of bio-mercaptan in human serum is 312 u M, and this value is calculated. The appropriate recovery rate has proved the reliability of the Au_2Pt_1 detection method. The further study of bimetallic nanoparticles will be carried out in the future, and it is expected to be applied to the detection of sulfhydryl drugs.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：TB383.1;R914

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本文編號(hào)：1957962