Study On Biomimetic Immunosorbent Assay Method Based On Nanozyme Label For Detection Of Histamine

Histamine(HA)is a biogenic amine present in foods and organisms,particularly in fermented foods.HA is produced through bacterial decarboxylation,and its accumulation leads to a decline in food quality.Allergic reactions are caused by excessive intake of HA.Therefore,monitoring the level of HA in foods is very important for human health.During recent years,HA has been determined in foods by several techniques.ELISA has attracted wide attention because of its high sensitivity and specificity.However,the biology antibody used in this assay is greatly limited due to its low stability during detection.To solve this problem,molecularly imprinted polymers(MIPs)with their excellent properties of mechanical/chemical stability have been selected as biomimetic antibodies.Currently,nanozymes,nanomaterials with enzyme-like properties for application in biosensing and immunoassay,are being developed which act as unique artificial enzymes.As these nanozymes possess the advantages of high stability with a simple and large-scale methods of preparation,they have emerged as alternatives to natural enzymes.In this study,the nanozyme take the place of natural enzymes,and MIPs were used as a biomimetic antibody.Two biomimetic immunoassay methods for the detection of HA were established.The main findings are as follows:1.Study on Pt@SiO₂nanoenzyme combined with PAF-45@MIP to detect HA.Using PAF-45@MIP as a biomimetic antibody,a novel direct competitive biomimetic immunoassay method for rapidly detecting HA was developed.Under optimal conditions,the limit of detection and sensitivity of the method were 0.128 and 8.306 mg/L,respectively.In order to verify the selectivity of the method,cross-reaction(CR)experiments were performed on tyramine and tryptamine,and the cross-reaction rates were 24.70%and 15.64%,respectively.In order to evaluate the accuracy of the method,the recovery experiments of vinegar samples were carried out,and the recoveries were 81.50%-90.38%.After that,shrimp and soy sauce samples were analyzed by both HPLC and the method.There were no significant difference between the two method.2.Study on biomimetic nanozyme-linked immunoassay method based on Au@Pt@Au nanozyme labeling for the detection of HA.With histamine dihydrochlorideused as template molecules,trimethylolpropane trimethacrylate as functional monomer,the imprinted film was prepared on the surface of a 96-well plate.Using Au@Pt@Au nanozyme as a marker,a novel direct competitive biomimetic immunoassay method for rapidly detecting HA was developed.Under optimal conditions,the limit of detection and sensitivity of the method were 0.069mg/L and 7.194 mg/L,respectively.In order to evaluate the accuracy of the method,the recovery experiments of yellow rice wine and liqueur samples were carried out,and the recoveries were 84.28%-92.36%.After that,white wine and red wine samples were analyzed by both HPLC and the method.There was no significant difference between the two method.