寡核苷酸模板法制备银团簇及其作为荧光探针的可行性研究

发布时间：2019-06-22 11:30

【摘要】：银团簇(Silver Nanoclusters, Ag NCs)是指Ag元素的几个至几十个原子组成核心,有机单分子如硫醇类化合物或生物分子如DNA、蛋白质等作为保护基团组装而成的核／壳型分子级聚集体。Ag NCs具有良好的生物相容性和光稳定性,在实验相关时间尺度上无闪烁,为生物分析及医学诊断研究提供了新的标记手段,开辟了新的应用领域。本文利用茎环结构的寡核苷酸作为模板合成了Ag NCs,对其物理化学性质进行了表征,探讨了几种因素对Ag NCs稳定性的影响：并进一步利用该团簇制备了银团簇探针(Ag NCs-probe),通过芯片杂交技术对其作为荧光探针在应用中的可行性进行了研究。得到了以下研究结果： 1以一段20bp的特定寡核苷酸序列为模板合成了水溶性Ag NCs。该溶液在可见光下呈淡红色,365 nm紫外光下呈现明亮的红色荧光。其最大吸收波长为262 nm,最大荧光激发及发射波长分别295 nm和660 nm,其量子产率(quantum yield, QY)为0.105。该团簇粒径约0.9 nm,其核心由约6-8个Ag原子组成。荧光显微镜下观察Ag NCs分散性良好。聚丙烯酰胺凝胶电泳结果显示该团簇为由至少两种组分组成的混合物,迁移最快和最慢的组分的迁移率分别为1.74和1.37,寡核苷酸的迁移率为1.67。 2 Ag NCs制备及应用的最适条件。pH 6.5-7,5适宜Ag NCs的制备,并且在温度小于等于60℃时Ag NCs都能保持较明亮的荧光,Ag+、Na+、K+、Zn2+、Ba2+、Ca2+、Mg2+、Co2+、Bi3+、Sr2+、Al3+、Pb2+、Fe3+、F-、Br-、Cl-、CH3COO-、B4O72-、SO32-、SO42-、C2O42-、IO3-对Ag NCs的荧光影响较小或几乎无影响,而Cu2+、Hg2+、IO4-、S2O32-对Ag NCs有较为严重的猝灭作用。 3利用Cu2+、Hg2+对Ag NCs荧光的猝灭作用,成功地将Ag NCs作为光学传感器对Cu2+、Hg2+进行高灵敏度检测,并分别绘制了其工作曲线,根据IUPAC标准计算得到检出限分别为5.6 nmol/L、5.5 nmol/L。并讨论了Cu2+、Hg2+对Ag NCs荧光的猝灭机理,EDTA溶液可以有效地使Ag NCs溶液的荧光部分恢复,其荧光恢复率分别为70%和40%。 4.利用IO4-、S2O32-对Ag NCs荧光的猝灭作用,成功地将Ag NCs作为光学传感器对IO4-、S2O32进行高灵敏度检测,并分别绘制了其工作曲线,根据IUPAC标准计算得到检出限分别为12.6nmol/L、3.8nmol/L。讨论了IO4-、S2O32-对Ag NCs荧光的猝灭机理,对比研究了IO3-、BrO3与I04-氧化性强弱对Ag NCs荧光猝灭的不同影响。 5.利用连接了探针序列的寡核苷酸模板制备了Ag NCs-probe。该探针在365 nm紫外光下呈现出橙红色荧光,在630 nm和325 nm处均有较强的发射峰,而且其发射光谱较宽,在300-400 nm和500-800 nm之间均有荧光发射。30%-70%甲酰胺使Ag NCs-probe最大发射峰红移到645 nm处,发射光谱变窄,且荧光强度明显升高,最大提高了16.5%,而325 nm处的荧光强度有所降低；80%和90%的甲酰胺溶液则使Ag NCs-probe的荧光强度减弱。 6.利用芯片杂交技术检测了0157：H7的特异序列。制备了寡核苷酸芯片,利用制备的Ag NCs-probe与芯片杂交,并在芯片上检测到0157：H7的特异序列。证明其作为荧光探针在生物检测中是可行的。
[Abstract]:Silver cluster (Ag NCs) refers to the core of several to several tens of atoms of the Ag element, and the organic single molecule such as a thiol compound or a biological molecule such as DNA, a protein, or the like is assembled as a protecting group to form a core/ shell type molecular level aggregate. The Ag-NCs have good biocompatibility and light stability, and no flicker on the time scale of the experiment, and provides a new marking method for the biological analysis and the medical diagnosis and research, and opens up a new application field. The chemical properties of Ag-NCs were characterized by using the oligonucleotide of the stem-ring structure as the die plate, and the effect of several factors on the stability of the Ag-NCs was discussed. The silver cluster probe (Ag NCs-probe) was further prepared by using the cluster cluster. The feasibility of using the chip hybridization technique as a fluorescent probe in the application was studied. The following results were obtained: 1. The water-soluble Ag NC was synthesized by a 20-bp specific oligodeoxynucleotide sequence. S. The solution is light red under visible light and bright red color under the ultraviolet light of 365 nm. The maximum absorption wavelength of the light is 262 nm, the maximum fluorescence excitation and the emission wavelength are 295 nm and 660 nm, respectively, and the quantum yield (QY) is 0.10. 5. The cluster particle size is about 0.9 nm, the core of which is from about 6 to about 8 Ag atom groups, The Study on the Dispersibility of Ag-NCs under the Fluorescence Microscope The results show that the cluster is a mixture of at least two components, the mobility of the fastest and slowest components is 1.74 and 1.37, and the mobility of the oligonucleic acid is 1.6. 7. Preparation and Application of 2Ag NCs Suitable conditions. The pH 6.5 -7,5 is suitable for the preparation of Ag NCs, and when the temperature is less than or equal to 60 DEG C, the Ag NCs can maintain bright fluorescence, Ag +, Na +, K +, Zn2 +, Ba2 +, Ca2 +, Mg2 +, Co2 +, Bi3 +, Sr2 +, Al3 +, Pb2 +, Fe3 +, F-, Br-, Cl-, CH3COO-, B4O 72-, SO32-, SO42-, C2O42-, IO3-have little or no effect on the fluorescence of Ag NCs, while Cu2 +, Hg2 +, IO4-, and S2O32-have a more serious influence on Ag NCs. The quenching effect of Cu 2 + and Hg2 + on the fluorescence of Ag NCs was used, and the Ag-NCs were successfully used as the optical sensor to detect the Cu 2 + and Hg2 +, and their working curves were plotted. The detection limit was 5.6 nmol/ L, 5.5n, respectively, according to the IUPAC standard. The quenching mechanism of Cu 2 + and Hg2 + on the fluorescence of Ag-NCs was also discussed. The fluorescence recovery of the Ag-NCs solution can be effectively recovered by the EDTA solution, and the fluorescence recovery rate of the solution is 70%. According to IUPAC standard, the detection limit was 12.6 nmol/ L, respectively. In this paper, the quenching mechanism of IO4-, S2O32-on the fluorescence of Ag-NCs was discussed. The fluorescence of IO3-, BrO3 and I04-oxidation on the fluorescence of Ag-NCs was studied. different effects of quenching.5. Ag is prepared using an oligonucleic acid template that is linked to the probe sequence NCs-probe. The probe exhibits orange-red fluorescence at 365 nm ultraviolet light, has a strong emission peak at 630 nm and 325 nm, and has a wider emission spectrum, and has a fluorescence emission between 300-400 nm and 500-800 nm. m, the emission spectrum is narrow, and the fluorescence intensity is obviously increased, the maximum increase is 16.5%, and the fluorescence intensity at the 325 nm is reduced; and 80% and 90% of the mealamine solution make the Ag NCs-pro The fluorescence intensity of be decreased.6. The hybridization technique of the chip was used to detect 0. 157: H7 specific sequence. The oligonucleotide chip is prepared, and the prepared Ag NCs-probe is used for hybridization with the chip, and 0 is detected on the chip. The specific sequence of 157: H7. It is proved to be a fluorescent probe.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：TB383.1

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