Foam Immunoassay Of Au@Pt Nanozyme And Fluorescence Enhanced Immunochromatography Of Immunomagnetic Beads Assay For E. Coli O157:H7

Escherichia coli O157:H7?E.coli O157:H7?is a very important foodborne pathogen.Standard and fast detection techniques are available,but there is still much room for improvement.The integration of new knowledge,materials and technologies in many fields has led to the development of many aspects of good performance,especially the ability to simultaneously achieve qualitative and quantitative analysis,which is of great significance for rapid detection.In recent years,gas immunosensors have attracted the attention of relevant researchers because of their environmental protection,sensitivity and simplicity.The new multifunctional nano-enzymes have also received much attention in the field of rapid detection.Based on this,this project has developed three kinds of rapid detection methods with simple operation,low detection cost,accurate detection results and innovation,and all of them have achieved qualitative and quantitative detection of E.coli O157:H7.The specific research contents are as follows:1.A quantitative foam immunoassay for detection of E.coli O157:H7 based on bimetallic nanocatalyst-gold platinumIn this chapter,a foam immunoassay technology was developed to quantificationally detect E.coli O157:H7 by combining the foam generation reaction indicating system with the pathogen recognition component and using a low-cost measuring tool-straight ruler to directly measure the change in foam height.Fe₃O₄ magnetic nanoparticles?MNP?modified with mouse anti-E.coli O157:H7 monocolonol antibody for capture?mAb?were used to capture,enrich and separate target bacteria in positive samples.Gold-platinum nanoparticles?Au@Pt NP?were loaded on the surface of silica nanoparticles?SiO₂ NP?to form Au@Pt/SiO₂ NP complex,and mouse anti-E.coli O157:H7 monocolonol antibody for labeling?mAb2/mAb?was functionalized to form a mAb2-Au@Pt/SiO₂ NP signal probe.In the resulting sandwich immune complex,Au@Pt NP catalyzes the decomposition of hydrogen peroxide?H₂O₂?to produce a large amount of oxygen?O₂?,which is captured by sodium dodecyl sulfate?SDS?to produce dense foam.Rising foam in the acrylic tube produces a highly sensitive and directly visually amplified signal-foam height that is linear with the logarithm of the E.coli O157:H7 concentration.After selective enrichment and under the optimal conditions,the linear range was 1.19×10¹¹.19×10⁵CFU·mL^-1,and the detection limit was 8.64 CFU·mL^-1?3??.The Au@Pt nanozyme-catalyzed foam immunoassay achieves accurate detection of E.coli O157:H7 in milk samples and has good practical value.The catalysis,generation and measurement of this new signal technology opens up new avenues for simple,portable,and highly sensitive biological assays in a variety of environments.2.An unplugged and quantitative foam based immunochromatographic assay for E.coli O157:H7 using Au@Pt nanozymes to catalyze hydrogen peroxide decomposition reactionThis chapter is further optimized based on the research in the previous chapter.The immunochromatographic assay?ICA?is introduced as a qualitative detection method,which simplifies the washing step,shortens the detection time,and adopts the same foam quantification method as in the previous chapter.Here we developed a cost-effective immunochromatographic foam assay for E.coli O157:H7 in foods without the use of any instrument for on-site quantitative detection.First,the target bacteria in the selective enrichment liquid were captured,enriched and separated by immunomagnetic beads?MNP-mAb1 conjugates,MIc?,and the Au@Pt NP was labeled with mAb,and horseradish peroxidase?HRP?was used to block the active site on the surface of Au@Pt NP in place of bovine serum albumin?BSA?.The synergistic catalysis of Au@Pt NP and HRP further enhances the final signal.In the ICA process,mouse anti-E.coli O157:H7 polycolonol antibody for capture?pAb?on the detection line?T line?,the E.coli O157:H7 and mAb-Au@Pt-HRP NP complexes form a sandwich structure forming a gray T-line by antigen-antibody recognition.The T-line was cut out in a 96-well plate containing H₂O₂ and reacted at 50°C for 8 min to convert the E.coli O157:H7 concentration to a foam height.The linear range of the method is 1.93×10²¹.93×10⁶ CFU·mL^-1,and the detection limit is1.77×10² CFU·mL^-1?3??.Therefore,the immunochromatographic sandwich foam method can perform low-cost,effective,quantitative determination of the target object,and does not require complicated operation steps or high skill requirements of the tester,and is easy to popularize and apply.3.Quantitative detection of E.coli O157:H7 by immunochromatography with fluorescence enhancement of terephthalic acid based on magnetic beads catalyzed hydrogen peroxideIn this chapter,a quantitative rapid detection of E.coli O157:H7 by fluorescence enhanced immunochromatography was developed using MNP highly efficient peroxidase properties and terephthalic acid?TA?sensitive fluorescence enhancement properties.Firstly,the target bacteria in the selective enrichment liquid were captured,enriched and separated by MIc to improve the sensitivity of detection,and then as a labeled probe formed a brown T-line during the ICA qualitative detection process.The T line was then cut into the MNP/H₂O₂/TA detection system to accelerate the decomposition of H₂O₂ to produce·OH which oxidizes the typical non-fluorescent substrate TA of peroxidase to strong fluorescent o-hydroxyterephthalic acid?OHTA?.Under the optimal conditions,the linear range of E.coli O157:H7 was 2.40×10²².40×10⁶ CFU·mL^-1,and the detection limit was 1.75×10² CFU·mL^-1?3??.This method has been successfully used for the detection of E.coli O157:H7 in spiked milk samples,and has good potential practical application value.In summary,the three detection methods developed in this paper can achieve fast and accurate quantitative detection of E.coli O157:H7,with high sensitivity,good specificity and stability.The new method developed can realize the research and application of different detection targets by replacing antigen and antibody,and provides a basic model for the field of rapid detection.Because it is a quantitative test for initial research and development,there are still many areas that can be improved and optimized.