基于纳米探针技术的DNA杂交分析与免疫分析方法研究

发布时间：2018-07-07 14:02

本文选题：纳米探针 + DNA杂交　；参考：《江南大学》2008年硕士论文

【摘要】： DNA杂交分析及免疫分析在生命科学研究中具有重要的意义,广泛应用于生物学、医学诊断等方面。已有分析方法的局限性促使研究者们积极的研究普遍适用性好、操作简便、灵敏度高的DNA杂交检测及免疫检测新方法。本文将纳米探针技术与高灵敏化学发光技术结合应用于DNA杂交检测及免疫检测,对特定序列的寡聚核苷酸,人IgG及黄曲霉毒素B1进行了超微量检测,取得了令人满意的结果。以CoFe_2O_4/Au核壳复合纳米颗粒标记巯基化沙门氏菌特异寡核苷酸序列,用纳米金标记沙门氏菌另一特异寡核苷酸序列,通过DNA杂交反应与沙门氏菌特异目标DNA序列互补形成夹心结构。经过简单的磁分离,去除其他没有杂交的部分,最后将磁分离得到部分的纳米金溶出成为Au~(3+),结合luminol化学发光体系实现对目标DNA的高灵敏检测。结果表明,发光强度和目标DNA浓度在1-100 pmol·L~(-1)范围内相关性良好,对目标DNA的检测限为0.3 pmol·L~(-1)(3S/N),相对标准偏差为3.6%(10 pmol·L~(-1), n=7)。同时以CoFe2O4/Au核壳复合纳米颗粒为载体与羊抗人IgG构建捕获探针复合结构,首先与目标分析物人IgG发生免疫反应,捕获的人IgG再与纳米金标记的二抗(金标羊抗人IgG)发生免疫反应,形成三明治夹心结构;通过磁分离去除未结合物质干扰,将分离得到的标记纳米金溶出成为Au~(3+),结合Au~(3+)催化luminol化学发光分析方法实现对目标分析物人IgG的高灵敏检测。在实验优化条件下,化学发光强度与人IgG的浓度在2-100 ng·mL~(-1)范围内呈良好的线性关系,检测限为0.5 ng·mL~(-1)。黄曲霉毒素B1的快速灵敏检测在食品安全检测工作中具有重要意义。本文建立了两种基于银增强纳米金标记探针的高灵敏度免疫分析方法。第一种方法用黄曲霉毒素B1(AFB1)抗体与金标抗原、待测抗原进行竞争免疫反应,然后加入银增强溶液,以金为核沉积生长银,通过检测吸光度来确定待测物中AFB_1的含量,该方法的检出限可达到0.01 ng·mL~(-1)。第二种方法在前一种方法的基础上,将银化学溶出,通过化学发光法检测沉积的银的量来确定待测物中AFB1的含量,该方法的检出限可达到0.002 ng·mL~(-1)。论文还建立了纳米金标记-银增强-化学发光联用检测沙门氏菌的新方法。通过沙门氏菌捕获探针、金标沙门氏菌显示探针与沙门氏菌目标核酸序列之间的DNA杂交,形成三明治复合体,然后通过银增强在标记的纳米金表面选择性沉积银,实现第一次信号放大;随后结合溶出化学发光检测技术,实现信号第二次放大。结果表明,在优化条件下,化学发光强度和目标DNA浓度在1-1000 fmol·L~(-1)范围内相关性良好,对目标DNA的检测限为0.3 fmol·L~(-1)(3S/N),相对标准偏差为2.2%(10 fmol·L~(-1), n=7)。
[Abstract]:DNA hybridization analysis and immunoassay are of great significance in life science research, and are widely used in biology, medical diagnosis and so on. The limitations of existing analytical methods have prompted researchers to actively study new methods of DNA hybridization and immunoassay with good applicability, simple operation and high sensitivity. In this paper, the nanoprobe technique combined with highly sensitive chemiluminescence technique was applied to the detection of DNA hybridization and immunoassay. The specific sequences of oligonucleotides, human IgG and aflatoxin B1 were detected in ultramicro amounts, and satisfactory results were obtained. CoFe2O4 / au core-shell composite nanoparticles were used to label the specific oligonucleotide sequence of Salmonella mercaptobacter, and another specific oligonucleotide sequence was labeled with gold nanoparticles. The sandwich structure was formed by DNA hybridization with the specific target DNA sequence of Salmonella. After simple magnetic separation, the other parts without hybridization were removed. Finally, the partial gold nanocrystalline from magnetic separation was dissolved into Au3, and the highly sensitive detection of target DNA was realized by combining with the chemiluminescence system of luminol. The results showed that there was a good correlation between the luminescence intensity and the concentration of target DNA in the range of 1-100 pmol L ~ (-1). The detection limit of target DNA was 0.3 pmol L ~ (-1) (3s / N), and the relative standard deviation was 3.6% (10 pmol L ~ (-1), 7). At the same time, CoFe2O4 / au core-shell composite nanoparticles were used as carrier and goat anti-human IgG as carrier to construct the structure of trap probe. Firstly, the captured human IgG reacted with the target analyte human IgG, and then the captured human IgG reacted with gold labeled second antibody (gold-labeled goat anti-human IgG). The sandwich structure was formed, and the labeled gold nanoparticles were dissolved into Au3 by magnetic separation, and the high sensitive detection of human IgG was realized by using Au3 catalytic luminol chemiluminescence analysis method. Under the optimized conditions, there was a good linear relationship between the intensity of chemiluminescence and the concentration of human IgG in the range of 2-100 ng mL ~ (-1). The detection limit was 0.5 ng mL ~ (-1). The rapid and sensitive detection of aflatoxin B1 is of great significance in food safety detection. In this paper, two high sensitivity immunoassay methods based on silver-enhanced gold nanoparticles labeling probe were established. The first method is to use aflatoxin B1 (AFB1) antibody and gold-labeled antigen to carry on competitive immune reaction with the antigen to be tested, then to add the silver enhancement solution, then deposit the growing silver with gold as the nucleus, and determine the content of AFB1 in the object to be tested by measuring absorbance. The detection limit of this method is 0.01 ng mL ~ (-1). The second method is based on the former method, the silver is chemically dissolved, and the content of AFB1 in the sample is determined by chemiluminescence method. The detection limit of the method can reach 0.002 ng mL ~ (-1). A new method for the detection of Salmonella by gold-silver-enhanced chemiluminescence assay was established. The DNA hybridization between the probe and the target nucleic acid sequence of Salmonella was demonstrated by the capture probe of Salmonella aureus to form a sandwich complex, and then the selective deposition of silver on the labeled gold surface was enhanced by silver. The first signal amplification is realized, and then the second signal amplification is realized with the dissolution chemiluminescence detection technology. The results showed that the chemiluminescence intensity was well correlated with the concentration of target DNA in the range of 1-1000 fmol L ~ (-1). The detection limit of target DNA was 0.3 fmol L ~ (-1) (3s / N), and the relative standard deviation was 2.2% (10 fmol L ~ (-1), n ~ (7).
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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