Highly Sensitive Near-simultaneous Assay Of Multiple "Lean Meat Agent" Residues Using An Electrochemiluminescent Immunosensor Array

Nowadays,β2-agonists including clenbuterol (CLB), ractopamine (RAC), salbutamol (SAL) and so on, are abused illegally as "lean meat agents" for livestock breeding, in order to improve growth rate and reduce fat deposition. The residues of multiple β2-agonists, with some toxicological risks, have resulted in increasing serious food-safety accidents in some countries. In consideration of their potential hazard to human health, European Union, China, Japan and many other countries rigorously forbid using β2-agonists as growth promoter for food-producing animals. The routine monitoring of β2-agonists abuse also becomes exceedingly important. So far, a great variety of chromatographic methods, such as LC-MS, GC-MS, HPLC and capillary electrophoresis have been applied to detection of trace residues of various β2-agonists. These chromatographic methods all require time-consuming sample pre-treatments, and sophisticated and expensive apparatus. Thus, they are usually used as precise quantitative and confirmatory methods. In recent years, some immunoassay methods based on ELISA, electrochemistry, fluorescence and chemiluminescence, have shown great promise in assay of single β2-agonist. These immunoassay methods often require simple sample pre-treatments, and show high throughput. Therefore they are especially suitable for rapid screening. Unfortunately, we only find a few multianalyte immunoassay (MIA) methods reported for simultaneous detection of multiple "lean meat agents". Therefore, a highly sensitive MIA method for multiple "lean meat agents" would be very valuable.Recently, electrochemiluminescent (ECL) assay has already drawn growing attention. Due to its favorable spatial and time controllability, ECL assay is deemed to be well suited for construction of immunosensor. Furthermore, owing to its excellent sensitivity, it is assumed that ECL immunoassay can detect β2-agonist residues at very low level. In this paper, a highly sensitive ECL immunosensor was developed to detect CLB residue at first. Then, combined above experimental results, an ECL immunosensor array based on a screen-printed carbon electrodes array was designed to multianalyte detection of multiple β2-agonist residues, in which RAC and SAL were detected as the models. The work was proposed as follows:Part1Highly sensitive detection of trace amount of clenbuterol residue in swine urine utilizing an electrochemiluminescent immunosensorA highly sensitive ECL immunosensor is firstly applied for assay of trace amount of CLB residue in swine urine. In this investigation, gold nanoparticles doped chitosan composite film was utilized for antigen immobilization, and Ru(bpy)32+labeled antibody was synthesized as tracer antibody. With a competitive immunoassay format, good analytical performance was obtained. The proposed method shows a linear range of0.010-1.0ng/mL, with a limit of detection of0.0050ng/mL for CLB. Acceptable results for detection of CLB in swine urine were obtained using the proposed immunosensor. This method is suitable for screening of trace amount of CLB residue, due to its simple manipulation, short assay time, and high detection sensitivity.Part2Highly sensitive near-simultaneous assay of multiple "lean meat agent" residues in swine urine using a electrochemiluminescent immunosensor arrayCombined above experimental results, a screen-printed carbon electrodes array was assembled and acted as the substrate of the immunosensor array. Then the immunosensor array was constructed by site-selectively immobilizing the antigens of RAC and SAL on the working electrodes of array. After the competitive immuno-binding, with the aid of a homemade single-pore-four-throw switch, the ECL signals of the two β2-agonists were sequentially detected using a photomuitplier. The linear ranges for RAC and SAL were0.025-3.0and0.050-10ng/mL; the limits of detection for RAC and SAL were8.5and17pg/mL, respectively. This ECL immunosensor array successfully avoided the cross-talk between the adjacent reactive zones arising from photodiffusion or electroactive product diffusion. Compared with other routine methods based on chromatography and ELISA, this method is more suitable for screening of multiple "lean meat agent" in quantities of samples, owing to its merits of low cost, user-friendliness and high throughput, and shows great promise in food safety and agonist surveillance.