Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing Laboratory for Food Quality and Safety, China Agricultural University, 100193, Beijing, People's Republic of China.
Mikrochim Acta. 2019 Mar 8;186(4):225. doi: 10.1007/s00604-019-3326-8.
A method is described to enhance the sensitivity of an immunochromatographic assay for clenbuterol (CLE) by making use of dually-labeled gold nanoparticles (GNPs), background fluorescence blocking, and immunomagnetic separation. The GNPs were labeled with biotinylated antibody and streptavidin, respectively, and dually labeled GNPs were obtained via the biotin-streptavidin interaction to amplify the detection signal. The fluorescent signal was blocked by dually labeled GNPs and decreased as the dually labeled GNPs aggregation increases on nitrocellulose membrane, which derived from fluorescent polyvinylchloride card. However, fluorescence (measured at excitation/emission wavelengths of 518/580 nm) recovers when CLE reacts with dually labeled GNPs. Immunomagnetic separation was first applied for sample pretreatment. This can offset the matrix effect and improves the sensitivity and accuracy of the assay. Under the optimal conditions, the limits of detection of CLE visually were 0.25 μg·L. In addition, clenbuterol can be quantified in swine urine with a 0.03 μg·L detection limit. This is 60-fold lower than current immunochromatography. Response is linear in the 0.06-0.59 μg·L concentration range, and the recoveries from spiked swine urine range from 81 to 115%." Graphical abstract Schematic presentation of the strategies for improving sensitivity of immunochromatographic assay. It includes immunomagnetic separations, dually-labeled gold nanoparticles and background fluorescence blocking. The assay was applied to detect clenbuterol (CLE) in swine urine with an excellent performance.
一种利用双重标记金纳米粒子（GNPs）、背景荧光阻断和免疫磁分离来提高克伦特罗（CLE）免疫层析分析灵敏度的方法。GNPs 分别用生物素化抗体和链霉亲和素标记，通过生物素-链霉亲和素相互作用获得双重标记的 GNPs 以放大检测信号。荧光信号被双重标记的 GNPs 阻断，随着双重标记的 GNPs 在硝酸纤维素膜上聚集增加而减少，这是从荧光聚氯乙烯卡上获得的。然而，当 CLE 与双重标记的 GNPs 反应时，荧光（在 518/580nm 的激发/发射波长下测量）会恢复。免疫磁分离首先应用于样品预处理。这可以抵消基质效应，提高测定的灵敏度和准确性。在最佳条件下，CLE 的视觉检测限为 0.25μg·L。此外，克伦特罗可以在猪尿中定量检测，检测限为 0.03μg·L。这比目前的免疫层析法低 60 倍。在 0.06-0.59μg·L 的浓度范围内，响应呈线性，从添加的猪尿中回收的浓度范围为 81-115%。
