基于纳米探针技术的DNA杂交分析与免疫分析方法研究
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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)。
DNA hybridization analysis and immunoassay play important role in life sciences. They have been widely used in biochemistry, molecular biology and medical diagnostics. The disadvantage of the routine methods urged the researchers to find the new analytical methods with high sensitivity, simple operation and widely used in molecular biology, biochemistry and clinical diagnosis.
In the present work, we studied the applications of nano-robe in the detection of DNA hybridization and immunoassay by coulping with high sensitive chemiluminescence (CL) technique. The results of determination of specific sequence oligonucleotides, human IgG and aflatoxin B_1 (AFB_1) were satisfactory.
At first, CoFe_2O_4/Au nanoparticles were functionalized with the SH-tagged salmonella-specific oligonucleotide. Another salmonella-specific oligonucleotide was also labeled with gold nanoparticles. Through DNA hybridization, above oligonucleotide probes captured the target salmonella-specific oligonucleotide to form sandwich-type complex. After magnetic separation to remove the unbound section, the obtained section by magnetic separation was treated with gold dissolving reagents. The tagged gold then were dissolve to form Au3+, which can be sensitively measured by luminol CL system, and the target DNA can be determined by the light intensity. The result shows that the CL intensity is proportional to the concentration of target oligonucleotides over the range of 1-100 pmol·L~(-1) and the detection limit is 0.3 pmol·L~(-1) (3S/N), and the relative standard deviation is 3.6%(10 pmol·L~(-1), n=7).
Secondly, uing CoFe_2O_4/Au nanoparticles as the carrier, a probe complex were constructed by combining the gold-capped magnetic nanoparticles with sheep anti-human IgG (anti-IgG). The nanoparticles tagged anti-IgG firstly immune affinity to bind with IgG, which then captured by the gold nanoparticles labeled second anti-IgG and form a sandwich complex. After removal of free sections interference by magnetic separation, the labeled gold nanoparticles was dissolved to become Au3+, which can be used to catalyze luminol CL to achieve the objective of the highly sensitive detection of IgG. Under the selected conditions, it was found that the CL intensity is proportional with the concentration of IgG in 2-100 ng·mL~(-1) with a detection limit comes to 0.5 ng·mL~(-1) (3S/N).
Thirdly, two methods for the competitive heterogeneous immunoassay of aflatoxin B1 (AFB1) was developed. The rapid, sensitive measurement of AFB_1 is of great importance in food safty detection. The first one is based on the competitively immune bind to AFB1 antibody between the tested antigen and nanogold-labeled antigen. A silver enhancement process then was conducted to deposit silver around bond nanogold. Measuring the absorbance of silver at 630 nm can identify the content of AFB_1. The detection limit for the proposed method is 0.01 ng·mL~(-1). Based upon the first method, the deposited silver was then dissolved to form Ag+, which was measured with a sensitive CL system. And the CL intensity was found to be proportional with the content of AFB1 with a detection limit of 0.002 ng·mL~(-1).
Finally, a novel method based on nanogold labeling, silver enhancement and stripping CL analysis for ultrasensitive salmonella detection was proposed. It relies on a sandwich-type DNA hybridization analysis, in which the DNA targets were first hybridized to the captured oligonucleotide probes immobilized on polystyrene microwells, and then the gold nanoparticles modified with alkylthiol-capped oligonucleotides were used as probes to monitor the presence of the specific target DNA. After being anchored on the hybrids, the signals of gold nanoparticles were subsequently amplified by silver enhancement. Stripping CL was then applied for the second signal amplification. Under the selected conditions, it was found that the CL intensity is proportional to the concentration of target oligonucleotides over the range of 1-1000 fmol·L~(-1) with a detection limit of 0.3 fmol·L~(-1) (3S/N), and the relative standard deviation is 2.2%(10 fmol·L~(-1), n=7).
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)。
DNA hybridization analysis and immunoassay play important role in life sciences. They have been widely used in biochemistry, molecular biology and medical diagnostics. The disadvantage of the routine methods urged the researchers to find the new analytical methods with high sensitivity, simple operation and widely used in molecular biology, biochemistry and clinical diagnosis.
In the present work, we studied the applications of nano-robe in the detection of DNA hybridization and immunoassay by coulping with high sensitive chemiluminescence (CL) technique. The results of determination of specific sequence oligonucleotides, human IgG and aflatoxin B_1 (AFB_1) were satisfactory.
At first, CoFe_2O_4/Au nanoparticles were functionalized with the SH-tagged salmonella-specific oligonucleotide. Another salmonella-specific oligonucleotide was also labeled with gold nanoparticles. Through DNA hybridization, above oligonucleotide probes captured the target salmonella-specific oligonucleotide to form sandwich-type complex. After magnetic separation to remove the unbound section, the obtained section by magnetic separation was treated with gold dissolving reagents. The tagged gold then were dissolve to form Au3+, which can be sensitively measured by luminol CL system, and the target DNA can be determined by the light intensity. The result shows that the CL intensity is proportional to the concentration of target oligonucleotides over the range of 1-100 pmol·L~(-1) and the detection limit is 0.3 pmol·L~(-1) (3S/N), and the relative standard deviation is 3.6%(10 pmol·L~(-1), n=7).
Secondly, uing CoFe_2O_4/Au nanoparticles as the carrier, a probe complex were constructed by combining the gold-capped magnetic nanoparticles with sheep anti-human IgG (anti-IgG). The nanoparticles tagged anti-IgG firstly immune affinity to bind with IgG, which then captured by the gold nanoparticles labeled second anti-IgG and form a sandwich complex. After removal of free sections interference by magnetic separation, the labeled gold nanoparticles was dissolved to become Au3+, which can be used to catalyze luminol CL to achieve the objective of the highly sensitive detection of IgG. Under the selected conditions, it was found that the CL intensity is proportional with the concentration of IgG in 2-100 ng·mL~(-1) with a detection limit comes to 0.5 ng·mL~(-1) (3S/N).
Thirdly, two methods for the competitive heterogeneous immunoassay of aflatoxin B1 (AFB1) was developed. The rapid, sensitive measurement of AFB_1 is of great importance in food safty detection. The first one is based on the competitively immune bind to AFB1 antibody between the tested antigen and nanogold-labeled antigen. A silver enhancement process then was conducted to deposit silver around bond nanogold. Measuring the absorbance of silver at 630 nm can identify the content of AFB_1. The detection limit for the proposed method is 0.01 ng·mL~(-1). Based upon the first method, the deposited silver was then dissolved to form Ag+, which was measured with a sensitive CL system. And the CL intensity was found to be proportional with the content of AFB1 with a detection limit of 0.002 ng·mL~(-1).
Finally, a novel method based on nanogold labeling, silver enhancement and stripping CL analysis for ultrasensitive salmonella detection was proposed. It relies on a sandwich-type DNA hybridization analysis, in which the DNA targets were first hybridized to the captured oligonucleotide probes immobilized on polystyrene microwells, and then the gold nanoparticles modified with alkylthiol-capped oligonucleotides were used as probes to monitor the presence of the specific target DNA. After being anchored on the hybrids, the signals of gold nanoparticles were subsequently amplified by silver enhancement. Stripping CL was then applied for the second signal amplification. Under the selected conditions, it was found that the CL intensity is proportional to the concentration of target oligonucleotides over the range of 1-1000 fmol·L~(-1) with a detection limit of 0.3 fmol·L~(-1) (3S/N), and the relative standard deviation is 2.2%(10 fmol·L~(-1), n=7).
引文
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