寡核苷酸引导金属纳米颗粒的合成及其检测生物硫醇的应用

发布时间：2018-05-31 02:33

本文选题：Au + Pt　；参考：《天津医科大学》2016年硕士论文

【摘要】：生物硫醇参与人体的蛋白合成、代谢、解毒等生化过程,其水平的失衡极易诱发心脑血管疾病、阿尔茨海默症等多种疾病,因此对生物体内生物硫醇含量的监测有着重要意义。根据巯基官能团与金属离子、纳米团簇、纳米结晶的相互作用,目前发现了许多检测方法:如电化学法、化学发光法、荧光分析法和比色法等。其中比色法,以生成有色产物的显色反应为基础,通过比较或测量有色产物溶液的颜色深浅确定待测组分的含量,此方法操作简单、颜色可见,在定量分析中常被选用,但是实现超高灵敏度的比色检测,仍然是一个挑战。本文以富胞嘧啶(Cytosine)寡核苷酸为模板合成双金属纳米颗粒Aux Pty,并采用比色法将其应用于生物硫醇的超灵敏检测。首先,实验以富胞嘧啶C序列RET2为模板合成金属纳米颗粒Au、Pt、Au_xPt_y,分别采用高倍-透射电子显微镜(HR-TEM),圆二色光谱仪(CD),紫外可见分光光度计(UV-vis),荧光分光光度计(FL)和X射线表面光电子能谱仪(XPS)对金属纳米颗粒进行性质表征。结果显示4.8 nm-Au、4.2 nm-Pt、2.6 nm-Au_1Pt_1、3.6 nm-Au_2Pt_1纳米颗粒中,Au~(3+)几乎全部被还原成Au0,Pt~(2+)部分被还原成Pt0,Au_2Pt_1中Pt0/Pt~(2+)的百分比比Pt、Au_1Pt_1中的高,说明Au~(3+)比Pt~(2+)易于还原,Au~(3+)与核苷酸的作用影响了DNA与Pt~(2+)结合的比例。此外UV-vis、CD光谱扫描表明Au_xPt_y双金属纳米颗粒中Au、Pt共同存在于富C的核酸模板上。其次,评价了金属纳米颗粒催化TMB-H_2O_2反应的酶活性,其催化初始反应速率规律为:PtAu_1Pt_1Au_2Pt_1,其中700 nM Au_2Pt_1催化TMB氧化反应10 min时已达到平衡,可见双金属纳米颗粒Au_2Pt_1具有较高的过氧化物酶活性。采用米氏方程-双倒数作图法获得各自对底物的动力学参数,如Km,Vmax。虽然Au_2Pt_1对3,3',5,5'-四甲基联苯胺(TMB)的亲和性比天然辣根过氧化物酶HRP差,但是催化TMB-H_2O_2反应的最大反应速率比HRP快。最后,通过对比Pt、Au_xPt_y检测生物硫醇的检测限,获知双金属纳米颗粒Au_2Pt_1的检测灵敏度最好,对L-半胱氨酸(L-Cys)、L-同型半胱氨酸(L-Hcy)的检测限分别为3.5 nM、1.6 nM,比表2.2列举的其他比色法检测Cys、Hcy的检测限低;采用等温滴定量热法(ITC)、HR-TEM、XPS分析了金属纳米颗粒Au_xPt_y的巯基检测性能,发现其与Au:Pt浓度比(x:y)有关,另外,选择性实验证明Au_2Pt_1对生物硫醇检测的特异性。双金属纳米颗粒Au_2Pt_1由于与含巯基化合物发生作用形成Au-S键,造成纳米颗粒Au_2Pt_1发生聚集,进而使其在催化TMB-H_2O_2反应时金属纳米模拟酶的活性降低。基于Cys对Au_2Pt_1催化TMB-H_2O_2反应表现出抑制效应,可知双金属纳米颗粒Au_2Pt_1检测生物硫醇具有高度的特异性。因此,将Au_2Pt_1应用到人血清中生物硫醇的检测,最终计算出人血清中生物硫醇浓度为312μM,此值与有关文献报道一致。恢复补加实验中,合适的恢复率证明了Au_2Pt_1检测方法的可靠性。未来会展开对双金属纳米颗粒的进一步研究,有望将其应用于含巯基药物的检测中。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TB383.1;R914

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