羧基磁性微球检测急性心肌梗塞早期标志蛋白的研究
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摘要
随着纳米技术的迅速发展,新型纳米材料被逐渐应用于生物科学领域,为其研究和发展提供了新的技术和手段。免疫磁性微球(Immunomagnetic Microspheres, IMMS)技术,就是将纳米磁性微球作为固相载体应用于免疫分析试验中,由于纳米磁性微球体积小、比表面积大以及分散稳定性好,使得其表面包被的抗原或抗体较多,整个反应体系类似在均相中进行,加大了抗原抗体接触机率,加速了抗原抗体复合物的形成,从而提高了检测的灵敏度。本研究以羧基磁性微球为固相载体,采用双抗体夹心荧光免疫分析方法,对急性心肌梗塞(Acute Myocardial Infarction, AMI)早期标志蛋白——脂肪酸结合蛋白(H-FABP)和肌红蛋白(Myo)进行了半定量检测,从而建立了一种基于羧基磁性微球的新型荧光免疫分析方法。该方法特异性强、灵敏度高、耗时短,为今后疾病标志物检测和临床诊断提供了一种新的模型。本研究的具体内容和结果包括:
1.首先采用化学共沉淀法制备了油酸钠改性的纳米Fe_3O_4磁性粒子,然后以纳米Fe_3O_4磁性粒子为磁核,以苯乙烯、丙烯酸和二乙烯基苯为聚合单体,采用分散聚合法制备了羧基磁性高分子微球。利用透射电子显微镜(TEM)、扫描电子显微镜(SEM)、X-射线衍射仪(XRD)、振动样品磁强计(VSM)傅里叶变换红外光谱仪(FITC)、原子吸收分光光度计等对其进行了表征。结果表明纳米Fe_3O_4磁性粒子粒径在8～15 nm,分散性好,具有超顺磁性,饱和磁强度为48 emu/g。羧基磁性高分子微球平均粒径为1.7μm,粒径范围在0.19~3.50μm,具有超顺磁性,比饱和磁化强度为13.0 emu/g,磁含量为17.85%,羧基含量达0.386 mmol/g。
2.成功地进行了捕获抗体Anti-FABP10E1/Anti-Myo7C3与羧基微球连接,2.5 h后抗体吸附量分别达到最大值42.5和41.7 mg antibody/g;按照FITC试剂盒说明对检测抗体Anti-Myo-4E2/ Anti-FABP-9F3进行荧光标记;最后采用新型双抗体夹心免疫荧光分析法对AMI早期标志蛋白——Myo和H-FABP进行了检测,通过激光共聚焦显微镜对试验结果进行分析,并利用荧光分光光度计测定该方法的特异性。
Nowadays, with the development of nanotechnology, the study of new nano-material offers a new technology for the bioscience research. Immunomagnetic microspheres (Immunomagnetic Microspheres, IMMS) technology, is used the nano-magnetic microspheres as solid-phase in immunoassay. For the magnetic nano-microspheres are with small size, high specific surface area and good dispersion stability, their surfaces can coated more antigen or antibody, and the whole reaction system carried out in homogeneous solution, thereby increasing the contact probability of the antibody with the antigen, accelerating the formation of antigen-antibody complex, improving the detection sensitivity. In the present study, carboxylated magnetic microbead was used as solid phase carrier, and a carboxylated superparamagnetic microbead-assisted sandwich fluoroimmunoassay was successfully demonstrated for the semi-quantitative analysis of the early protein markers, myoglobin and human heart-type fatty acidbinding protein (H-FABP), associated with acute myocardial infarction (AMI). This assay approach was specificity, sensitively, rapidly. In addition, the protocol devised here can be used as a model for establishing general methods for other protein marker assays in the fields of clinical diagnostics and molecular biology. The results of each part are listed as follows:
One) Firstly iron oxide nanoparticles were synthesized via a modified chemical coprecipitation method, and the surface properties were changed from hydrophobic to hydrophilic by coating with sodium oleate. Then carboxylated magnetic polymer microspheres were prepared via a dispersion polymerization method using iron oxide nanoparticles as cores and styrene, acrylic acid and DVB as polymeric monomers. They were characterized using transmission electron microscopy (TEM), scanning electron microscopy(SEM), X-ray diffractometer (XRD), vibrating sample magnetometer (VSM), fourier transform infrared spectrophotometer (FT-IR) and atomic absorption spectrophotometry. The results of iron oxide nanoparticles indicated that their diameters are in the range of 8-15 nm, they have good dispersion stability, superparamagnetism and a saturation magnetization of 48 emu/g. The results of carboxylated magnetic polymer microspheres indicated that their diameters are in the range of 0.19-3.50μm and average diameter is 1.7μm. They have superparamagnetism, a saturation magnetization of 13.0 emu/g and a represented magnetic content of about 17.85%. The content of carboxyl group on the surface of the microsphere was 0.386 mmol/g.
Two) The capture antibodies (Anti-FABP10E/Anti-Myo7C3) were successfully conjugated onto carboxylated microbeads. The maximum anti-H-FABP 10E1 antibody loading of 42.5 mg antibody/g microbeads and 41.7 mg anti-myoglobin 7C3 antibody/g microbeads were observed after 2.5 h reaction coupling time. The detection antibodies (anti-myoglobin 4E2 and anti-H-FABP 9F3) were labeled with FITC following the instructions for the EZ-Label TM FITC Protein Labeling Kit. Superparamagnetic polymer microsphere-assisted sandwich fluoroimmunoassay was used to detect two early biomarkers of acute myocardial infarction, myoglobin and heart-type fatty acid binding protein (H-FABP). The fluorescent reporter signals of the post-immunoassay microspheres were recorded using a confocal laser-scanning microscope. In addition, spectrofluorophotometer was also used to analyze the specificity of the immunoassay and photostability of the post-immunoassay microspheres.
1.首先采用化学共沉淀法制备了油酸钠改性的纳米Fe_3O_4磁性粒子,然后以纳米Fe_3O_4磁性粒子为磁核,以苯乙烯、丙烯酸和二乙烯基苯为聚合单体,采用分散聚合法制备了羧基磁性高分子微球。利用透射电子显微镜(TEM)、扫描电子显微镜(SEM)、X-射线衍射仪(XRD)、振动样品磁强计(VSM)傅里叶变换红外光谱仪(FITC)、原子吸收分光光度计等对其进行了表征。结果表明纳米Fe_3O_4磁性粒子粒径在8～15 nm,分散性好,具有超顺磁性,饱和磁强度为48 emu/g。羧基磁性高分子微球平均粒径为1.7μm,粒径范围在0.19~3.50μm,具有超顺磁性,比饱和磁化强度为13.0 emu/g,磁含量为17.85%,羧基含量达0.386 mmol/g。
2.成功地进行了捕获抗体Anti-FABP10E1/Anti-Myo7C3与羧基微球连接,2.5 h后抗体吸附量分别达到最大值42.5和41.7 mg antibody/g;按照FITC试剂盒说明对检测抗体Anti-Myo-4E2/ Anti-FABP-9F3进行荧光标记;最后采用新型双抗体夹心免疫荧光分析法对AMI早期标志蛋白——Myo和H-FABP进行了检测,通过激光共聚焦显微镜对试验结果进行分析,并利用荧光分光光度计测定该方法的特异性。
Nowadays, with the development of nanotechnology, the study of new nano-material offers a new technology for the bioscience research. Immunomagnetic microspheres (Immunomagnetic Microspheres, IMMS) technology, is used the nano-magnetic microspheres as solid-phase in immunoassay. For the magnetic nano-microspheres are with small size, high specific surface area and good dispersion stability, their surfaces can coated more antigen or antibody, and the whole reaction system carried out in homogeneous solution, thereby increasing the contact probability of the antibody with the antigen, accelerating the formation of antigen-antibody complex, improving the detection sensitivity. In the present study, carboxylated magnetic microbead was used as solid phase carrier, and a carboxylated superparamagnetic microbead-assisted sandwich fluoroimmunoassay was successfully demonstrated for the semi-quantitative analysis of the early protein markers, myoglobin and human heart-type fatty acidbinding protein (H-FABP), associated with acute myocardial infarction (AMI). This assay approach was specificity, sensitively, rapidly. In addition, the protocol devised here can be used as a model for establishing general methods for other protein marker assays in the fields of clinical diagnostics and molecular biology. The results of each part are listed as follows:
One) Firstly iron oxide nanoparticles were synthesized via a modified chemical coprecipitation method, and the surface properties were changed from hydrophobic to hydrophilic by coating with sodium oleate. Then carboxylated magnetic polymer microspheres were prepared via a dispersion polymerization method using iron oxide nanoparticles as cores and styrene, acrylic acid and DVB as polymeric monomers. They were characterized using transmission electron microscopy (TEM), scanning electron microscopy(SEM), X-ray diffractometer (XRD), vibrating sample magnetometer (VSM), fourier transform infrared spectrophotometer (FT-IR) and atomic absorption spectrophotometry. The results of iron oxide nanoparticles indicated that their diameters are in the range of 8-15 nm, they have good dispersion stability, superparamagnetism and a saturation magnetization of 48 emu/g. The results of carboxylated magnetic polymer microspheres indicated that their diameters are in the range of 0.19-3.50μm and average diameter is 1.7μm. They have superparamagnetism, a saturation magnetization of 13.0 emu/g and a represented magnetic content of about 17.85%. The content of carboxyl group on the surface of the microsphere was 0.386 mmol/g.
Two) The capture antibodies (Anti-FABP10E/Anti-Myo7C3) were successfully conjugated onto carboxylated microbeads. The maximum anti-H-FABP 10E1 antibody loading of 42.5 mg antibody/g microbeads and 41.7 mg anti-myoglobin 7C3 antibody/g microbeads were observed after 2.5 h reaction coupling time. The detection antibodies (anti-myoglobin 4E2 and anti-H-FABP 9F3) were labeled with FITC following the instructions for the EZ-Label TM FITC Protein Labeling Kit. Superparamagnetic polymer microsphere-assisted sandwich fluoroimmunoassay was used to detect two early biomarkers of acute myocardial infarction, myoglobin and heart-type fatty acid binding protein (H-FABP). The fluorescent reporter signals of the post-immunoassay microspheres were recorded using a confocal laser-scanning microscope. In addition, spectrofluorophotometer was also used to analyze the specificity of the immunoassay and photostability of the post-immunoassay microspheres.
引文
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