量子点荧光探针用于猪链球菌病快速检测新方法研究
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摘要
猪链球菌病是由猪链球菌(SS)引起的一种人畜共患疾病,在猪链球菌的35个血清型中以2型(SS2)流行最广,致病性最强,因此发展和建立基于SS2型重要毒力因子—溶菌酶释放蛋白(MRP)的猪链球菌病快速检测新方法对于该病的检测和诊断具有重要意义。量子点作为一种新型荧光探针,在光、电、化学方面具有很多独特的优势,已经成为化学、材料科学、生命医学和环境检测方面的重要研究工具。本论文结合荧光免疫分析技术,采用量子点-MRP抗体蛋白探针和量子点-兔抗猪IgG探针对猪链球菌病进行了检测,主要研究内容及结果如下:
1.合成了巯基乙酸修饰的CdSe/ZnS核壳量子点并制备了CdSe/ZnS量子点-MRP抗体蛋白探针,利用聚丙烯酰胺凝胶电泳和分子光谱法研究了MRP抗体蛋白与CdSe/ZnS量子点的结合机理,结果表明MRP抗体蛋白与CdSe/ZnS量子点可能通过表面配位作用结合。荧光光谱法优化了CdSe/ZnS量子点-MRP抗体蛋白探针制备的影响因素,建立了一种测定MRP抗原蛋白的新方法,其线性范围为5.0×10~(-8)～1.5×10~(-6) mol/L,线性相关系数为0.998。
2.合成了谷胱甘肽修饰的水溶性CdTe量子点,并以EDC·HCl/Sulfo-NHS为共价偶联剂制备了基于MRP抗体蛋白的量子点荧光探针。用分子光谱、毛细管电泳、透射电子显微镜和荧光显微镜的方法对探针进行了表征,结果表明量子点探针保持着MRP抗体蛋白的生物活性和生物特异性。荧光免疫分析法对MPR抗原蛋白进行了检测,荧光强度与抗原浓度具有很好的线性关系,线性浓度范围为5.0×10~(-8)～1.5×10~(-5) mol/L。与平板计数法相比,本试验中量子点探针对猪内脏血清的检测也获得了较满意的结果。同时,试验中采用96孔板的方法进行检测,为实现猪链球菌病的高通量检测提供了重要参考依据。
3.制备了基于兔抗猪IgG的CdTe量子点探针,并将其用于MRP抗原蛋白和猪内脏血清的检测,结果表明,该方法对浓度在2.5×10~(-7)～1.5×10~(-5) mol/L范围之间的抗原具有较好的线性检测关系。对猪内脏血清的检测,本试验的方法与平板检测法和基于MRP抗体蛋白的量子点探针的检测方法相比获得较好的一致性。同时考查了量子点探针的时间稳定性,结果表明制备的量子点探针具有较好的稳定性,为猪链球菌病检测试剂盒的制备提供了重要参考依据。
本研究将量子点探针用于动物重大疾病领域的研究,不仅提供了新颖的、简单快速的、高通量的猪链球菌病检测方法,还为猪链球菌病检测试剂盒的制备以及量子点荧光探针用于其他动物重大疾病研究提供了重要的参考依据。
Porcine streptococcal disease which caused by Streptococcus Suis (SS) was a kind of zoonotic diseases. In the 35 different serotypes of SS, SS2 is the most pathogenic and prevalent capsular type. So, to develop a novel detection method of porcine streptococcal disease based on muramidase-released protein (MRP) antibody (an important virulence factor of SS2) is with important significance for the detection and diagnoses of the disease. As a novel fluorescent probe, quantum dots (QDs) combine of unique optical, electrical and chemical traits, and has been widely used in chemistry, materials science, biology and environmentalology. In this study, QDs-MRPAb probe and QDs-rabitt anti pig IgG probe were used for the detection and diagnosis of porcine streptococcal disease by fluorescence—linked immunosorbent assay method. The main concents and results are listed as following:
1. Mercaptoacetic acid (MAA) capped CdSe/ZnS core/shell quantum dots and the CdSe/ZnS quantum dot-MRP antibody probe was prepared. The conjugation mechanism was studied according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular Spectrometry. The factors influencing the preparation of CdSe/ZnS quantum dot-MRP antibody probe were optimized by fluorescence Spectrometry. Under the optimum conditions, the probe showed good sensitivity to MRP antigen. The calibration graph was linear in the concentration range from 5.0×10~(-8) to 1.5×10~(-6) mol/L of MRP antigen concentration, with a correlation coefficient of 0.998.
2. CdTe quantum dots capped with glutathione (GSH) was prepared and the quantum dots probe based on MRP antibody were made through the conjugate material of EDC·HCl/Sulfo-NHS. Molecular Spectrometry, capillary electrophoresis (CE), transmission electron microscopy (TEM) and fluorescence microscope image results showed that the probe kept the bioactivity and bio-recognition of MRP antibody after the coupling process. In succession, the probe was carried out to detect MRP antigen by fluorescence-linked immunosorbent assay (FLISA), results depicted that the fluorescence intensity was linearly proportional to the MRP antigen concentration from5.0×10~(-8) to 1.5×10~(-5) mol/L. The determination of clinical serum samples from diseased pigs with the proposed method were satisfactory consistent with that of plate count method. Besides, 96 wells plate detection method was used in the research, which makes a promise of high-throughput diagnose.
3. CdTe quantum dots probe based on rabbit anti pig IgG were prepared and used in the detection of MRP antigen and the clinical serum samples from diseased pigs. Linearity concentrations range from 2.5×10~(-7) to 1.5×10~(-5) mol/L of MRP antigen was observed in the FLISA study. The sensitivity for the determination of pig viscera serum with the proposed method has good consistency with that of plate count method and the CdTe quantum dot-MRP antibody probe method. Future study indicated that the probe was time stable, which could offers references for its preparation, store for future use, and the development of the detection kit.
In conclusion, novel menthods for the rapid detection of porcine streptecoccal diseases based on quantum dots fluorescence probes were studied. The new methods hold promising potential for simple, fast and high-throughput detection of porcine streptococcal disease, also offers important references for the development of the detection kit and the application on the diagnostics and determination for animal desease of quantum dots florescent probe.
1.合成了巯基乙酸修饰的CdSe/ZnS核壳量子点并制备了CdSe/ZnS量子点-MRP抗体蛋白探针,利用聚丙烯酰胺凝胶电泳和分子光谱法研究了MRP抗体蛋白与CdSe/ZnS量子点的结合机理,结果表明MRP抗体蛋白与CdSe/ZnS量子点可能通过表面配位作用结合。荧光光谱法优化了CdSe/ZnS量子点-MRP抗体蛋白探针制备的影响因素,建立了一种测定MRP抗原蛋白的新方法,其线性范围为5.0×10~(-8)～1.5×10~(-6) mol/L,线性相关系数为0.998。
2.合成了谷胱甘肽修饰的水溶性CdTe量子点,并以EDC·HCl/Sulfo-NHS为共价偶联剂制备了基于MRP抗体蛋白的量子点荧光探针。用分子光谱、毛细管电泳、透射电子显微镜和荧光显微镜的方法对探针进行了表征,结果表明量子点探针保持着MRP抗体蛋白的生物活性和生物特异性。荧光免疫分析法对MPR抗原蛋白进行了检测,荧光强度与抗原浓度具有很好的线性关系,线性浓度范围为5.0×10~(-8)～1.5×10~(-5) mol/L。与平板计数法相比,本试验中量子点探针对猪内脏血清的检测也获得了较满意的结果。同时,试验中采用96孔板的方法进行检测,为实现猪链球菌病的高通量检测提供了重要参考依据。
3.制备了基于兔抗猪IgG的CdTe量子点探针,并将其用于MRP抗原蛋白和猪内脏血清的检测,结果表明,该方法对浓度在2.5×10~(-7)～1.5×10~(-5) mol/L范围之间的抗原具有较好的线性检测关系。对猪内脏血清的检测,本试验的方法与平板检测法和基于MRP抗体蛋白的量子点探针的检测方法相比获得较好的一致性。同时考查了量子点探针的时间稳定性,结果表明制备的量子点探针具有较好的稳定性,为猪链球菌病检测试剂盒的制备提供了重要参考依据。
本研究将量子点探针用于动物重大疾病领域的研究,不仅提供了新颖的、简单快速的、高通量的猪链球菌病检测方法,还为猪链球菌病检测试剂盒的制备以及量子点荧光探针用于其他动物重大疾病研究提供了重要的参考依据。
Porcine streptococcal disease which caused by Streptococcus Suis (SS) was a kind of zoonotic diseases. In the 35 different serotypes of SS, SS2 is the most pathogenic and prevalent capsular type. So, to develop a novel detection method of porcine streptococcal disease based on muramidase-released protein (MRP) antibody (an important virulence factor of SS2) is with important significance for the detection and diagnoses of the disease. As a novel fluorescent probe, quantum dots (QDs) combine of unique optical, electrical and chemical traits, and has been widely used in chemistry, materials science, biology and environmentalology. In this study, QDs-MRPAb probe and QDs-rabitt anti pig IgG probe were used for the detection and diagnosis of porcine streptococcal disease by fluorescence—linked immunosorbent assay method. The main concents and results are listed as following:
1. Mercaptoacetic acid (MAA) capped CdSe/ZnS core/shell quantum dots and the CdSe/ZnS quantum dot-MRP antibody probe was prepared. The conjugation mechanism was studied according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular Spectrometry. The factors influencing the preparation of CdSe/ZnS quantum dot-MRP antibody probe were optimized by fluorescence Spectrometry. Under the optimum conditions, the probe showed good sensitivity to MRP antigen. The calibration graph was linear in the concentration range from 5.0×10~(-8) to 1.5×10~(-6) mol/L of MRP antigen concentration, with a correlation coefficient of 0.998.
2. CdTe quantum dots capped with glutathione (GSH) was prepared and the quantum dots probe based on MRP antibody were made through the conjugate material of EDC·HCl/Sulfo-NHS. Molecular Spectrometry, capillary electrophoresis (CE), transmission electron microscopy (TEM) and fluorescence microscope image results showed that the probe kept the bioactivity and bio-recognition of MRP antibody after the coupling process. In succession, the probe was carried out to detect MRP antigen by fluorescence-linked immunosorbent assay (FLISA), results depicted that the fluorescence intensity was linearly proportional to the MRP antigen concentration from5.0×10~(-8) to 1.5×10~(-5) mol/L. The determination of clinical serum samples from diseased pigs with the proposed method were satisfactory consistent with that of plate count method. Besides, 96 wells plate detection method was used in the research, which makes a promise of high-throughput diagnose.
3. CdTe quantum dots probe based on rabbit anti pig IgG were prepared and used in the detection of MRP antigen and the clinical serum samples from diseased pigs. Linearity concentrations range from 2.5×10~(-7) to 1.5×10~(-5) mol/L of MRP antigen was observed in the FLISA study. The sensitivity for the determination of pig viscera serum with the proposed method has good consistency with that of plate count method and the CdTe quantum dot-MRP antibody probe method. Future study indicated that the probe was time stable, which could offers references for its preparation, store for future use, and the development of the detection kit.
In conclusion, novel menthods for the rapid detection of porcine streptecoccal diseases based on quantum dots fluorescence probes were studied. The new methods hold promising potential for simple, fast and high-throughput detection of porcine streptococcal disease, also offers important references for the development of the detection kit and the application on the diagnostics and determination for animal desease of quantum dots florescent probe.
引文
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