Highly Sensitive And Rapid Detection Of SEB Based On Immune Bio-barcode And Hybridization Chain Reaction In Milk

Staphylococcal enterotoxin B（SEB）,an exotoxin produced and secreted by Staphylococcus aureus,is a typical biotoxin that causes food poisoning due to its high temperature resistance,acid and alkali resistance,easy preparation of aerosol.It is also a potential biological warfare agent.Therefore,SEB poses a great threat to human health and has become one of the important biotoxins that many countries and regions concerned about.At present,the non-target screening methods for SEB mainly rely on omics and biomarkers technology for research,which require high-resolution mass spectrometry and high performance liquid chromatography（HPLC）equipment for analysis,and it is difficult to popularize in various departments.Targeted detection methods mainly rely on the antigen–antibody interactions,including immuno-agglutination test,agar diffusion,and enzyme-linked immunosorbent assay.However,the cost of monoclonal antibody preparation in conventional immunological assays is high.Batch-to-batch variations of antibody makes it difficult to meet the need for SEB high sensitivity detection.In this paper,based on the requiement for new high sensitivity and rapid detection technology of food safety research,the corresponding antibody and aptamer are used as SEB molecular recognition element,respectively.Hence,dual signal amplification method through bio-barcode and real-time Polymerase Chain Reaction and fluorescence hybridization chain reaction amplification method for SEB detection were established.On the basis of anti-SEB monoclonal antibody and the goat anti-SEB polyclonal antibody.The immue bio-barcode method was built through the modification of the goat anti-SEB polyclonal antibody and the DNA barcode on the surface of the Au nanoparticles,and antibody-Au NPs-DNA barcode probe was used to replace the traditional antibody-HRP label.In the meantime,anti-SEB monoclonal antibody was modified on the surface of magnetic nanomaterials for the rapid identification and enrichment of SEB in liquid food samples;The HCR amplification method provided a new strategy for aptamer-based molecular switching.This method offers the advantage of the design of an intelligent hairpin“switch”based on the fluorescent resonance energy transfer（FRET）.This signal“switch”mainly depends on the excellent signal transduction of molecular beacons（MBs）.In our design,the 6-FAM and BHQ-1 are linked covalently at the end of H1’s arm.The stem of H1 leads to the close proximity of the fluorescence and quencher which result in the lowest fluorescence intensity.This competitive aptasensor for sensitive detecting SEB is constructed combing with HCR amplification.The introduction of HCR amplification technique is expected to provide a rapid,low-cost,and sensitive aptasensor fluorescence strategy.（1）The traditional double-antibody"sandwich"ELISA assay was established,and the goat anti-SEB polyclonal antibody was used as the coating antibody and the anti-SEB monoclonal antibody as the second antibody.The result indicated that the detection range was 15.63 ng m L^-1 to 625 ng m L^-1.The detection limit was 0.39 ng m L^-1 and the SEB recovery test rate ranged from 77.62±5.72%to 113.64±4.45%（2）Antibody-AuNPs-DNA barcode probe was prepared using goat anti-SEB polyclonal antibody and DNA modification of 13 nm AuNPs.The results were characterized by UV-vis spectroscopy and transmission electron microscope respectively.The optimal amount of 1 m L colloidal gold labeled antibody was 200μg m L^-1,200μL by using Mey’s stabilization assay.Optimum conditions in the experiment include the concentration of AuNPs added,the amount of MMPs added,and the type and concentration of the optimal blocking solution.Under the optimal conditions,the CT values had a better linear relationship with SEB concentration ranged from 0.001ng m L^-1 to 100 ng m L^-1,and the detection limit of 0.269 pg m L^-1.The recoveries ranged from 89.17±5.07%to 110.29±2.52%（3）Based on the c DNA sequence of SEB aptamer,the hairpin H1 and H2 were designed which could trigger the hybridization chain reaction.H1 was designed as molecular beacon to construct a new intelligent enzyme-free fluorescence sensor.Under the optimal conditions,the fluorescence intensity and SEB standard concentration ranged from 3.13 ng m L^-1 to 100 ng m L^-1.Through the spiked recovery experiment of milk samples,the recoveries ranged from 87.83±2.91%to 112.68±2.94%.Based on the traditional double-antibody sandwich ELISA,immune bio-barcode combined with real-time PCR,and aptamer-hybridization chain reaction method for the analysis of SEB in milk samples.The bio-barcode assay enabled the ultrasensitive detection of SEB,while aptamer-hybridization chain reaction provided a simple and rapid method.These methods could make rapid and accurate SEB screening according to the degree of food contamination,and provide more ideas and platforms for on-site testing of SEB.