A Novel Lateral Flow Immunoassay For Clenbuterol Hydrochloride Residues In Animal Products

Clenbuterol hydrochloride（CLE）is a syntheticβ-adrenoceptor agonist,which is widely used in livestock farming because its presence in animals can promote lipolysis and protein synthesis,thus improving the lean meat rate of animals.However,CLE is stable and has a long half-life,and its accumulation in the human body through the food chain can easily cause food poisoning and even death.Therefore,it is important to establish an immediate detection method for CLE in livestock products to achieve effective food safety control.Lateral flow immunoassay（LFIA）is one of the most important methods for immediate detection because of its simplicity,rapid detection and high specificity.However,the traditional immunochromatographic method based on colloidal gold（Au NPs-LFIA）,on the one hand,has a single signal output and cannot avoid visual error signal;on the other hand,the signal label preparation is costly and unstable.Based on the above problems,two immunochromatographic rapid detection methods were constructed in this study to achieve highly sensitive and rapid detection of CLE in livestock products,using CLE in livestock products as the target.The main research contents and results are as follows:1.LFIA construction and performance characterization based on tannic acid-loaded tungsten disulfide nanosheets（TA-WS₂）:TA-WS₂ was firstly prepared by TA-assisted aqueous phase exfoliation method,which effectively improved the exfoliation efficiency and stability of WS₂.TA-WS₂ exhibited excellent colorimetric signal and also good photothermal effect.TA-WS₂ labeled with anti-CLE monoclonal antibody(Ab _CLE)was used as the detection probe to construct a colorimetric/photothermal dual-signal immunochromatographic rapid detection method.Under the optimal conditions,the visual limit of detection（v LOD）and cut-off value（COV）of TA-WS₂-LFIA for CLE were 0.7 ng m L^-1 and 7 ng m L^-1,respectively,and the v LOD and COV of the photothermal signal were 0.3 ng m L^-1 and 11 ng m L^-1,respectively.Meanwhile,immunochromatographic test strips were constructed with WS₂ without TA stripping as the signal label.The photothermal mode sensitivity of TA-WS₂-LFIA was enhanced 16-fold than Au NPs-LFIA.In addition,TA-WS₂-LFIA showed no positive reaction to other interferents,demonstrating good specificity.The method was applied to the residue detection of CLE in chicken meat and skim milk with the v LOD of 4.2μg kg^-1 and 1.8μg L^-1,respectively,and the recoveries ranged from 90%to 106.67%,indicating its good application in the rapid detection of livestock products.2.Construction and characterization of ultrafast LFIA with indirect probe-assisted one-step dyeing:Using a negatively charged gallocyanine dyestuff solution（GC）as a signal label,goat anti-mouse immunoglobulin（Ab₂）binds Ab _CLE by non-covalent forces to form a positively charged Ab₂-Ab _CLE biohybrid.Among them,commercial GC does not require artificial synthesis.Compared with synthetic nano signal labels,GC has the advantages of short preparation time and small difference between batches.Compared with synthetic nano signal labels,GC has the advantages of short preparation time and smalldifferencese between batches.On this basis,Ab₂-Ab _CLE was labeled with GC to form a probe,and a rapid detection method for LFIA（GC-AA-LFIA）was constructed.Under optimal conditions,the v LOD and COV of the method for CLE were 2 ng m L^-1,12 ng m L^-1,and the calculated detection limit（c LOD）was 0.411 ng m L^-1.Compared with Au NPs-LFIA,the sensitivity of GC-AA-LFIA was enhanced at least 4 fold.In addition,the method had no positive reaction to other interferinterferonsproved good specificity.GC-AA-LFIA was applied to the detection of CLE residue in skim milk,beef and pork ham.The v LOD and COV were 2,2 and 1 ng m L^-1,and the COV were 18,20 and 20 ng m L^-1,respectively.The recoveries ranged from 80%to 113.1%.It is proved that this method has good application value in food substrate.