Study On Detection Methods Of Pathogenic E.coli O157:H7 Based On Two Protein Probes And Nanoflowers

E.coli O157:H7 is commonly found in every corner of our lives.It can be transmitted to humans through undercooked meat,vegetables,and animal manure.E.coli O157:H7 is known as one of the most dangerous foodborne pathogens,and has been widely concerned by scientists at home and abroad.Scientists have studied a variety of detection methods for detecting E.coli O157:H7,such as traditional detection methods for bacterial culture counting.And modern molecular biology detection methods such as polymer chain reaction?PCR?.However,the traditional detection methods take a long time and the actual operation process is cumbersome.And modern molecular biology detection methods require expensive experimental equipment,high drug costs and strong professional operability.In order to meet the needs of popularization,this research based on organic-inorganic nanoflower technology,handheld blood glucose meter and colorimetric method,established two biosensors to detect E.coli O157:H7,1)based on sucrose invertase-calcium hydrogen phosphate detection of E.coli O157:H7 by nanoflower combined blood glucose meter.2)Visual detection of E.coli O157:H7 based on Hemin-antibacterial peptide-copper phosphate hybrid nanoflower.Recently,the nanoenzyme was successfully synthesized,the activity of the enzyme was amplified,and the detection efficiency was improved.In this study,sucrose invertase was used to catalyze sucrose to produce glucose.Sucrose invertase was mixed with calcium hydrogen phosphate buffer solution and concanavalin A?Con A?to synthesize sucrose invertase nanoflower as immunosensor.Fe₂O₃ magnetic beads attached with antibodies were used as capture probes to connect target pathogenic bacteria,which were further sandwiched with sucrose invertase nanoflower.Sucrose was added into the solution,and the content of pathogenic bacteria in it was detected by a blood glucose detector.The detection method has a detection linear range of 10⁴-10⁸ CFU/mL and a minimum detection limit of 10.2×10¹ CFU/mL.It achieves the goal of rapid detection and easy operation.Nano-enzymes has attracted great interest from researchers due to its high activity,stability and biocompatibility.In this study,a colorimetric immunosensor based on Hemin@MI was developed to detect the pathogen E.coli O157:H7 with visual,high sensitivity and selectivity.Hemin@MI nanoflowers with peroxidase mimic enzyme activity and Fe₃O₄@Ab nanoflowers with magnetic properties were synthesized by the"one-pot method".This method involved simply mixing antimicrobial peptide E.coli O157:H7?magainin I?and Hemin in a copper sulfate sodium phosphate saline buffer and adding antibodies?Antibody:Ab?against E.coli O157:H7 and Fe₃O₄ in the sodium copper sulfate buffer solution.Hemin@MI nanoflowers with target recognition and signal amplification functions were developed and integrated as signal probes for real-time colorimetric detection,combined with pathogenic E.coli O157:H7 and as a capture probe Fe₃O₄@Ab nanoflower.In the presence of H₂O₂,the peroxidase could catalyze the oxidation of2'-azidobis?3-ethylbenzothiazoline-6-sulfonic acid?ABTS??to ABTS^-,resulting in a color change that allowed immediate visual detection of E.coli O157:H7.The detection range was 10² to 10⁸ CFU/mL,and the detection limit was 8.5×10¹ CFU/mL.These data indicate that the two immunosensors can be used to quickly and conveniently evaluate the pathogenic E.coli O157:H7 in real samples.Compared with similar products,high sensitivity,low detection limit and all within the scope of international requirements.They provide new ways for the detection of foodborne pathogens.