Sequential injection chemiluminescence immunoassay for nonionic surfactants by using magnetic microbeads.
Sequential injection chemiluminescence immunoassay for nonionic surfactants by using magnetic microbeads.
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DOI：
10.1016/j.aca.2007.02.052
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发表时间：
2007-09
影响因子：
6.2
通讯作者：
Ruiqi Zhang;Hizuru Nakajima;N. Soh;K. Nakano;T. Masadome;Kazumi Nagata;Kazuhira Sakamoto;T. Imato
中科院分区：
文献类型：
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作者：
Ruiqi Zhang;Hizuru Nakajima;N. Soh;K. Nakano;T. Masadome;Kazumi Nagata;Kazuhira Sakamoto;T. Imato
建立了一种基于磁性微球顺序注射分析（SIA）的烷基酚聚氧乙烯醚（APnEOs）快速、灵敏的免疫分析方法。SIA系统由注射泵、开关阀、配备有光子计数单元和钕磁体的流通型免疫反应池构成。将固定有抗APnEOs单克隆抗体的磁珠用作免疫测定中的固体支持物。通过钕磁体和调节载体溶液的流量来控制磁珠在免疫反应细胞中的引入、捕获和释放。该免疫测定基于固定在磁珠上的抗APnEOs单克隆抗体与样品APnEOs和辣根过氧化物酶（HRP）标记的APnEOs在同一样品溶液中的间接竞争免疫反应，并且基于随后在磁性微珠上的HRP与含有过氧化氢和对碘苯酚的鲁米诺溶液的荧光反应。在已经用聚乳酸膜包被的磁珠表面上的羧酸酯部分活化后，通过将抗体与磁珠偶联，将抗APnEOs抗体固定在磁性微珠上。将抗体固定化磁珠引入免疫反应池中，并通过安装在免疫反应池下方的钕磁体将抗体固定化磁珠捕获在免疫反应池中。将含有恒定浓度的HRP标记的APnEOs的APnEOs样品溶液和含有过氧化氢和对碘苯酚的鲁米诺溶液按照SIA程序化顺序依次引入免疫反应池中。通过位于免疫反应室上侧的光子计数单元，通过用透镜收集发射的光，监测化学发光发射。在最佳条件下，用不同浓度（0- 1000 ppb）的APnEOs标准样品溶液，将APnEOs浓度的对数与化学发光强度（100 ms内的光子数）作图，得到典型的S形校准曲线。IC_（80）的检测下限约为10 ppb。每个样品分析所需时间小于15分钟。该方法已成功应用于河水中APnEOs的测定。
A rapid and sensitive immunoassay based on a sequential injection analysis (SIA) using magnetic microbeads for the determination of alkylphenol polyethoxylates (APnEOs) is described. An SIA system was constructed from a syringe pump, a switching valve, a flow-through type immunoreaction cell equipped with a photon counting unit and a neodymium magnet. Magnetic beads, to which an anti-APnEOs monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in and from the immunoreaction cell were controlled by means of a neodymium magnet and adjusting the flow of a carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-APnEOs monoclonal antibody immobilized on the magnetic beads with a sample APnEOs and a horseradish peroxidase (HRP)-labeled APnEOs in the same sample solution, and was based on the subsequent chemiluminscence reaction of HRP on the magnetic microbeads with a luminol solution containing hydrogen peroxide and p-iodophenol. The anti-APnEOs antibody was immobilized on the magnetic microbeads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of the magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced in the immunoreaction cell and trapped in it by the neodymium magnet, which was equipped beneath the immunoreaction cell. An APnEOs sample solution containing the HRP-labeled APnEOs at a constant concentration, and a luminol solution containing hydrogen peroxide and p-iodophenol were sequentially introduced into the immunoreaction cell, according to an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the immunoreaction cell by collecting the emitted light with a lens. A typical sigmoidal calibration curve was obtained, when the logarithm of the concentration of APnEOs was plotted against the chemiluminescence intensity as the number of photons in 100ms using standard APnEOs sample solutions at various concentrations (0–1000ppb) under optimum conditions. The lower detection limit defined as IC80is ca 10ppb. The time required for analysis is less than 15min per a sample. The present method was successfully applied to the determination of APnEOs in river water.