Development And Application Of Enhanced Clenbuterol Hydrochloride Immunoassay Technology Based On Nanozyme

Clenbuterol hydrochloride（CLL）is a typicalβ-adrenoceptor agonist,which is widely used as"leptin"and illegally added to animal feed.As a result,CLL residues in animal food can be ingested by consumers through the food chain,causing food poisoning and even malignant tumors.Therefore,it is important to achieve rapid and sensitive CLL detection to ensure food safety.Nanozymes are a class of nanomaterials with enzyme-like activity and better stability and environmental tolerance than natural enzymes,which can be used in immunoassay to enhance the analytical performance by acting as signal amplifiers.Therefore,this study is based on the hazards of CLL to food safety.Cu-histidine nanozyme（CHzyme）with catechol oxidase-like activity and Fe-Gallic acid nanozyme（FGN）with peroxidase-like activity were used for the development of enhanced ratiometric Enzyme-linked immunosorbent assay（ELISA）and immunochromatographic assay（ICA）strips to detect CLL,respectively.The main contents and results of the paper are as follows:1.Development of a ratiometric fluorescent ELISA based on CHzyme with catechol oxidase activity:CHzyme with catechol oxidase activity was prepared by a one-step hydrothermal synthesis method.Since CHzyme can catalyze the oxidation of catechol（CAT）to o-phenylenediamine,and the carbonyl group in o-phenylenediamine can react with the amino group in o-phenylenediamine（OPD）via Schiff-base to generate fluorescent substances,and thus a catalytic substrate sensing model was constructed for CAT detection.Ascorbic acid（AA）can also be oxidized by CHzyme to form a carbonyl compound,which in turn reacts with OPD to form another fluorescent substance,thus generating a new fluorescence emission peak and reducing the fluorescence emission peak generated by CAT,and a competitive substrate sensing model was designed for the ratiometric fluorescence sensing of AA.In addition,alkaline phosphatase（ALP）can catalyze the generation of AA by L-ascorbic acid-2-phosphate（AA2P）,which is used for the detection of ALP activity by competitive substrate sensing mode,and this sensing mode was named as the generated substrate sensing mode.In order to obtain a universal detection of analytes in food,a ratiometric fluorescence ELISA for CLL detection was constructed by introducing the generated substrate sensing mode into the ELISA system using ALP-labeled goat anti-mouse secondary antibody.The analytical performance was 0.0172–4 ng m L^-1.Compared with conventional ELISA,the sensitivity was improved by nearly 15 times.Finally,the recoveries of the assay ranged from 83.55%to 113.32%with the relative standard deviation（RSD）less than 16.34%in real samples such as ham and pork,demonstrating the good applicability of the assay in food samples.2.Development of dual colorimetric ICA test strips mediated by FGN signal amplification based on peroxidase-like activity:Firstly,FGN was prepared by sol-gel method.The potential of FGN as a nanozyme for signal amplification was demonstrated by investigating the peroxidase-like activity of FGN.Subsequently,the gallic acid in FGN was used as a recognition molecule to couple with monoclonal antibodies（m Abs）against CLL to prepare immunoprobes,and then ICA test strips were developed.In order to obtain the best detection performance,key parameters such as FGN concentration,BSA-CLL concentration,p H of running solution,detection time,anti-CLL m Abs concentration,and immunoprobe dosage in the ICA test strips were optimized.Under the optimal conditions,the developed dual colorimetric ICA test strips showed an LOD of 0.172 ng m L^-1for CLL and a detection range of 0.172–6 ng m L^-1.Finally,the standard addition recovery test was performed using pork and chicken as the actual samples,and the recoveries ranged from 93.93%–123.90%with RSD was less than 14.48%,which proved the good practical applicability of the developed dual colorimetric ICA test strips.