A novel fluorescence internal filtration immunoassay for the detection of clenbuterol

Clenbuterol is a β-adrenergic receptor agonist that can be used as a bronchodilator for the treatment of respiratory diseases or as an additive feed in livestock to improve conversion rate of lean meat. However, the residue of clenbuterol in food can be a threat to human health. Therefore, it is important to monitor clenbuterol in foods. In this paper, a fluorescence inner filtration immunoassay (FIFI) method for the detection of clenbuterol was developed based on the principle of immunoreactive competitive method and the theory of fluorescence quenching, which was constructed from a fluorescence-quenching detection system and a separation system of magnetic immunization. In the magnetic immunization separation system, the detection antigen competes with the biotin-labeled antigen for binding to colloidal gold-labeled clenbuterol antibody and the immunocomplex is enriched by streptavidin- and biotin-specific reactions on the surface of magnetic beads. The remaining complex as a fluorescence quencher in the detection system after centrifugation, and rhodamine 6G acts as a fluorescence agent. The quantitative detection of clenbuterol was accomplished by achieving fluorescence quenching under excitation light irradiation. The standard solutions of clenbuterol in the range of 0.03–5.0 ng/mL exhibited good correlation (r = 0.9996) with the fluorescence intensity, with limit of detection 0.01 ng/mL and recoveries ranging from 98.1 to 101.2%. Moreover, there was no significant difference between the results obtained after testing valid samples by FIFI and ELISA. Therefore, our developed method is sensitive and efficient, simple to operate, and has great potential for on-site detection of clenbuterol.