基于磁性纳米线的免疫检测技术的研究
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摘要
磁性纳米线不仅具有普通一维纳米材料的特性,还拥有优异的磁性能,成为当前纳米材料中较为活跃的研究领域和前沿。目前对磁性纳米线的研究工作,以研究各种不同材料纳米线的制备及其各种特性的表征居多,而磁性纳米线在免疫传感器应用领域的深入研究工作还鲜见报道。
本论文首次将磁性纳米线引入到免疫检测传感器中,提出了一种免疫检测的新方法,即磁性纳米线作为生物载体的技术。这不仅对磁性纳米材料的应用进行了新的尝试,而且为生物活性体提供了新的固载技术,为免疫传感器的优化和发展开拓了更开阔的空间。以自行成功研制出的磁性纳米线/壳聚糖/α-HCG抗体生物探针为基础,组装了免疫层析试纸条,进行了HCG抗原的快速免疫层析测试;并创新性地首次搭建了基于磁性纳米线的小型免疫传感检测系统,利用此系统对不同浓度的磁性纳米线进行定量检测。为免疫传感器检测的集成化和微型化,以及生物样品在线分析和快速检测,提供了新的方法和探索途径。
利用两次阳极氧化法制备了多孔阳极氧化铝模板,对其形貌及化学组成进行了表征,同时研究了多孔阳极氧化铝膜的形成机理,并对实验条件进行了优化。所制备的多孔阳极氧化铝模板的纳米孔道分布均匀,彼此平行且垂直于模板表面,呈高度有序的正六边形排列。纳米孔平均直径:50 nm,纳米孔密度:10~6个/mm~2,纳米孔长度:大约10μm。
以经过阶梯降压法修饰后的多孔阳极氧化铝模板为基础,通过交流电化学沉积的方式制备Fe、Ni纳米线阵列,并对其形貌及化学组成进行了表征,结果显示,纳米线直径大约50 nm,长度大约750 nm;对纳米线的磁性分析结果表明,纳米线阵列的易磁化方向具有明显的单轴各向异性,其主导因素是纳米线的形状各向异性。
以壳聚糖包被Fe纳米线,以戊二醛为交联剂对壳聚糖表面进行醛基化,制备磁性纳米线/壳聚糖复合体,并对其形成机理进行了探讨。同时,在不同缓冲液pH值和不同BSA初始浓度的情况下,对BSA进行了吸附性能测试。结果表明:BSA质量浓度为2.0 mg/mL时,缓冲液pH值为5.0时,即BSA的等电点时,达到最大平衡吸附量,其值为148.4 m/g。
以壳聚糖包被Fe纳米线,以戊二醛为交联剂对壳聚糖表面进行醛基化,实现与α-HCG抗体的交联,制备磁性纳米线/壳聚糖/α-HCG抗体生物探针。以此生物探针为基础,与β-HCG、IgG多抗一起,组装HCG磁性纳米线标记免疫层析试纸条,分别对不同浓度的HCG抗原进行测试,测试结果表明:检测灵敏度为10 mIU/mL。与市场上销售的胶体金早孕试纸条的灵敏度25 mIU/mL相比,本实验制备的试纸条可以将早孕检测时间提前2～3天。
提出了一种操作简便、便于携带、响应迅速的磁性免疫检测的具体方案。通过对系统感应线圈的几何参数进行优化设计,研制了磁性纳米线免疫传感器检测系统,并对不同浓度的样品进行了检测,实验结果显示:该检测系统的灵敏度可达0.2 mg/mL。
The research of magnetic nanowires is hot spot in nanomaterial fields, because of both the normal characteristic of nanomaterial and the unique excellent magnetic properties. At present, many researches are the preparation and characterization of nanowires. However, the deep research of nanowires in immunosensors application is less reported.
In this paper, a new immunoassay, magnetic nanowires as biological carrier, is given out. Magnetic nanowires are use in immunosensors first time. And new attempts for immobilization technology of biological active material and application of magnetic nanowires are tried. These widen thought of immunosensors' optimization and development. Based on magnetic nanowires/chitosan/α-HCG, the test strips of immunochromatography were prepared and tested. Mini immunosensor detection system based on magnetic nanowires was developed. Magnetic nanowires solution with different concentration was detected by it. This provides a new way in integration and miniaturization of immunosensors, as well as on-line analysis and rapid detection of biological sample.
Pour anodic aluminum oxide (AAO) template was prepared by two-step oxide method and characterized. Forming mechanism was researched and experiment condition was optimized. The nano-holes equably distribute and parallel each other in AAO template. They are equilateral hexagon with 50 ran diameter, 10~6/ mm~2 density and 10 urn length.
Based on AAO template after modified with step-reduce-voltage method, Fe and Ni nanowire arrays were prepared by AC electrochemical deposition method and characterized. Their diameter is 50 nm. And their length is 750 nm. Magnetism of nanowires was analyzed and the result shown that the magnetization direction was obvious uniaxial magnetic anisotropy because of figure anisotropy of nanowires.
Fe nanowires were coated by chitosan. Magnetic nanowires/chitosan was prepared by glutaraldehyde as cross-linking reagent. Forming mechanism was researched. Adsorbing bovine serum albumin (BAS) capacity was tested with different pH buffer solution and different concentration BAS. The result shown that when the concentration of BSA is 2.0 mg/mL and pH of buffer solution is 5.0, the maximum of adsorption is 148.4 mg/g.
The biological probes of magnetic nanowires/chitosan/α-HCG were prepared by cross-linking withα-HCG. The immunochromatographic test strips of were prepared by magnetic nanowires/chitosan/α-HCG,β-HCG and IgG. And different concentration HCG antigen was tested. The result shown that sensitivity of test strips was 10 mlU/mL. Compared with 25 mlU/mL sensitivity of colloidal gold test strips in market, the detection time of pregnancy can be forward 2～3 days.
An easy operating, easy taking and rapid reacting magnetic immunosensor detection system was developed. After optimized the geometry parameter of detect coil, different concentration sample were detecting. The result shown that the sensitivity of system was 0.2 mg/mL.
本论文首次将磁性纳米线引入到免疫检测传感器中,提出了一种免疫检测的新方法,即磁性纳米线作为生物载体的技术。这不仅对磁性纳米材料的应用进行了新的尝试,而且为生物活性体提供了新的固载技术,为免疫传感器的优化和发展开拓了更开阔的空间。以自行成功研制出的磁性纳米线/壳聚糖/α-HCG抗体生物探针为基础,组装了免疫层析试纸条,进行了HCG抗原的快速免疫层析测试;并创新性地首次搭建了基于磁性纳米线的小型免疫传感检测系统,利用此系统对不同浓度的磁性纳米线进行定量检测。为免疫传感器检测的集成化和微型化,以及生物样品在线分析和快速检测,提供了新的方法和探索途径。
利用两次阳极氧化法制备了多孔阳极氧化铝模板,对其形貌及化学组成进行了表征,同时研究了多孔阳极氧化铝膜的形成机理,并对实验条件进行了优化。所制备的多孔阳极氧化铝模板的纳米孔道分布均匀,彼此平行且垂直于模板表面,呈高度有序的正六边形排列。纳米孔平均直径:50 nm,纳米孔密度:10~6个/mm~2,纳米孔长度:大约10μm。
以经过阶梯降压法修饰后的多孔阳极氧化铝模板为基础,通过交流电化学沉积的方式制备Fe、Ni纳米线阵列,并对其形貌及化学组成进行了表征,结果显示,纳米线直径大约50 nm,长度大约750 nm;对纳米线的磁性分析结果表明,纳米线阵列的易磁化方向具有明显的单轴各向异性,其主导因素是纳米线的形状各向异性。
以壳聚糖包被Fe纳米线,以戊二醛为交联剂对壳聚糖表面进行醛基化,制备磁性纳米线/壳聚糖复合体,并对其形成机理进行了探讨。同时,在不同缓冲液pH值和不同BSA初始浓度的情况下,对BSA进行了吸附性能测试。结果表明:BSA质量浓度为2.0 mg/mL时,缓冲液pH值为5.0时,即BSA的等电点时,达到最大平衡吸附量,其值为148.4 m/g。
以壳聚糖包被Fe纳米线,以戊二醛为交联剂对壳聚糖表面进行醛基化,实现与α-HCG抗体的交联,制备磁性纳米线/壳聚糖/α-HCG抗体生物探针。以此生物探针为基础,与β-HCG、IgG多抗一起,组装HCG磁性纳米线标记免疫层析试纸条,分别对不同浓度的HCG抗原进行测试,测试结果表明:检测灵敏度为10 mIU/mL。与市场上销售的胶体金早孕试纸条的灵敏度25 mIU/mL相比,本实验制备的试纸条可以将早孕检测时间提前2～3天。
提出了一种操作简便、便于携带、响应迅速的磁性免疫检测的具体方案。通过对系统感应线圈的几何参数进行优化设计,研制了磁性纳米线免疫传感器检测系统,并对不同浓度的样品进行了检测,实验结果显示:该检测系统的灵敏度可达0.2 mg/mL。
The research of magnetic nanowires is hot spot in nanomaterial fields, because of both the normal characteristic of nanomaterial and the unique excellent magnetic properties. At present, many researches are the preparation and characterization of nanowires. However, the deep research of nanowires in immunosensors application is less reported.
In this paper, a new immunoassay, magnetic nanowires as biological carrier, is given out. Magnetic nanowires are use in immunosensors first time. And new attempts for immobilization technology of biological active material and application of magnetic nanowires are tried. These widen thought of immunosensors' optimization and development. Based on magnetic nanowires/chitosan/α-HCG, the test strips of immunochromatography were prepared and tested. Mini immunosensor detection system based on magnetic nanowires was developed. Magnetic nanowires solution with different concentration was detected by it. This provides a new way in integration and miniaturization of immunosensors, as well as on-line analysis and rapid detection of biological sample.
Pour anodic aluminum oxide (AAO) template was prepared by two-step oxide method and characterized. Forming mechanism was researched and experiment condition was optimized. The nano-holes equably distribute and parallel each other in AAO template. They are equilateral hexagon with 50 ran diameter, 10~6/ mm~2 density and 10 urn length.
Based on AAO template after modified with step-reduce-voltage method, Fe and Ni nanowire arrays were prepared by AC electrochemical deposition method and characterized. Their diameter is 50 nm. And their length is 750 nm. Magnetism of nanowires was analyzed and the result shown that the magnetization direction was obvious uniaxial magnetic anisotropy because of figure anisotropy of nanowires.
Fe nanowires were coated by chitosan. Magnetic nanowires/chitosan was prepared by glutaraldehyde as cross-linking reagent. Forming mechanism was researched. Adsorbing bovine serum albumin (BAS) capacity was tested with different pH buffer solution and different concentration BAS. The result shown that when the concentration of BSA is 2.0 mg/mL and pH of buffer solution is 5.0, the maximum of adsorption is 148.4 mg/g.
The biological probes of magnetic nanowires/chitosan/α-HCG were prepared by cross-linking withα-HCG. The immunochromatographic test strips of were prepared by magnetic nanowires/chitosan/α-HCG,β-HCG and IgG. And different concentration HCG antigen was tested. The result shown that sensitivity of test strips was 10 mlU/mL. Compared with 25 mlU/mL sensitivity of colloidal gold test strips in market, the detection time of pregnancy can be forward 2～3 days.
An easy operating, easy taking and rapid reacting magnetic immunosensor detection system was developed. After optimized the geometry parameter of detect coil, different concentration sample were detecting. The result shown that the sensitivity of system was 0.2 mg/mL.
引文
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