Zhang Ruiqi, Nakajima Hizuru, Soh Nobuaki, Nakano Koji, Masadome Takashi, Nagata Kazumi, Sakamoto Kazuhira, Imato Toshihiko
Department of Applied Chemistry, Graduated School of Engineering, Kyushu University Motooka, Fukuoka 819-0395, Japan.
Anal Chim Acta. 2007 Sep 26;600(1-2):105-13. doi: 10.1016/j.aca.2007.02.052. Epub 2007 Feb 24.
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 100 ms using standard APnEOs sample solutions at various concentrations (0-1000 ppb) under optimum conditions. The lower detection limit defined as IC(80) is ca 10 ppb. The time required for analysis is less than 15 min per a sample. The present method was successfully applied to the determination of APnEOs in river water.
描述了一种基于顺序注射分析（SIA）并使用磁性微珠的快速灵敏免疫分析法，用于测定烷基酚聚乙氧基化物（APnEOs）。SIA系统由注射泵、切换阀、配备光子计数单元和钕磁铁的流通式免疫反应池构成。固定有抗APnEOs单克隆抗体的磁性微珠用作免疫分析中的固相载体。磁性微珠在免疫反应池中的引入、捕获和释放通过钕磁铁并调节载液流速来控制。该免疫分析基于固定在磁性微珠上的抗APnEOs单克隆抗体与样品中的APnEOs以及辣根过氧化物酶（HRP）标记的APnEOs之间的间接竞争免疫反应，并基于随后磁性微珠上的HRP与含有过氧化氢和对碘苯酚的鲁米诺溶液的化学发光反应。通过在已涂覆聚乳酸膜的磁性微珠表面的羧基部分活化后将抗体与磁性微珠偶联，将抗APnEOs抗体固定在磁性微珠上。固定有抗体的磁性微珠被引入免疫反应池，并被位于免疫反应池下方的钕磁铁捕获。根据SIA编程序列，将含有恒定浓度HRP标记的APnEOs的APnEOs样品溶液以及含有过氧化氢和对碘苯酚的鲁米诺溶液依次引入免疫反应池。通过位于免疫反应池上方的光子计数单元借助透镜收集发射光来监测化学发光发射。在最佳条件下，使用各种浓度（0 - 1000 ppb）的标准APnEOs样品溶液，当以100 ms内的光子数表示的化学发光强度对APnEOs浓度的对数作图时，获得了典型的S形校准曲线。定义为IC(80)的最低检测限约为10 ppb。每个样品的分析时间少于15分钟。该方法成功应用于河水样品中APnEOs的测定。
