Department of Analytical Chemistry, Institute of Fine Chemistry and Nanochemistry (IQFN), Campus of Rabanales, Annex to Marie Curie (C-3) Building, University of Cordoba, 14071 Cordoba, Spain.
Talanta. 2012 May 30;94:195-200. doi: 10.1016/j.talanta.2012.03.019. Epub 2012 Mar 12.
A heterogeneous immunoassay for monensin determination in milk samples using a tracer formed by anti-monensin antibodies bound to nile blue (NB)-doped silica nanoparticles (NPs), 96-well microplates as solid supports and long-wavelength fluorescence measurements is described for the first time. The assay relies on the competition of the monensin present in the samples with a monensin-bovine serum albumin conjugate, which was immobilized onto the well surface, for the active sites of anti-monensin antibodies. After subsequent incubation and washing steps, the fluorescence of the bound tracer fraction is measured onto the dry surface of the well. An antigen capture format was also assayed by immobilizing anti-sheep IgG previously to the incubation of sheep anti-monensin antibodies and using a tracer formed by monensin bound to nile blue-doped silica NPs, which competes with the analyte for binding the immobilized antibody. Although the fluorescence signal obtained in both formats can be correlated to the analyte concentration, better results were obtained using the antibody capture format. After the optimization of the system using this format, the method features a detection limit of 0.015 μg L(-1) and a dynamic range from 0.05 to 5 μg L(-1). The precision, assayed at two different analyte concentrations, 0.2 and 1 μg L(-1), and expressed as relative standard deviation, gave values of 5.9% and 4.0%, respectively. The method was satisfactorily applied to the analysis of milk samples, which only required a simple extraction step in order to remove the proteins from samples, giving recoveries in the range 83.3-107.5%.
首次描述了一种使用尼罗蓝（NB）掺杂硅纳米粒子（NPs）标记的抗莫能菌素抗体形成的示踪剂，通过 96 孔微板作为固体支持物和长波长荧光测量，用于牛奶样品中莫能菌素的异质免疫测定。该测定依赖于样品中莫能菌素与固定在孔表面的莫能菌素-牛血清白蛋白轭合物竞争抗莫能菌素抗体的活性位点。随后进行孵育和洗涤步骤后，测量干燥孔表面结合示踪剂部分的荧光。还通过在孵育绵羊抗莫能菌素抗体之前固定抗绵羊 IgG 来检测抗原捕获形式，并使用与尼罗蓝掺杂硅 NPs 结合的莫能菌素形成的示踪剂，该示踪剂与分析物竞争结合固定的抗体。尽管两种形式都可以将荧光信号与分析物浓度相关联，但使用抗体捕获形式可以获得更好的结果。在使用这种格式优化系统后，该方法的检测限为 0.015 μg L(-1)，动态范围为 0.05 至 5 μg L(-1)。在两个不同分析物浓度（0.2 和 1 μg L(-1)）下测定的精密度，以相对标准偏差表示，分别为 5.9%和 4.0%。该方法令人满意地应用于牛奶样品的分析，仅需要简单的提取步骤即可从样品中去除蛋白质，回收率在 83.3-107.5%范围内。
