Immunochromatographic fluorometric determination of clenbuterol with enhanced sensitivity

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−1. In addition, clenbuterol can be quantified in swine urine with a 0.03 μg·L−1 detection limit. This is 60-fold lower than current immunochromatography. Response is linear in the 0.06–0.59 μg·L−1 concentration range, and the recoveries from spiked swine urine range from 81 to 115%.”