Establishment Of Rapid Detection Method For Escherichia Coli O157:H7 Based On Nanoflowers

Food safety issues have gradually become a global public concern.Food microbial contamination is one of the most important health problems in the world and the leading cause of foodborne diseases.Since 1993,the incidence of foodborne diseases has continued to rise.Billions of cases occur every year.Food-borne diarrhea diseases alone cause 2.2 million deaths every year and millions of people are infected every day.Food-borne pathogens are the main culprits.Therefore,the control of food contamination and detection of foodborne pathogens are given priority in China.Escherichia coli is one of the most common pathogenic pathogenic pathogens.It is widely found in nature and is a serious threat to human life and health in water,air,humans and animal faeces.However,the conventional detection methods have a series of problems,such as time-consuming and labor-intensive operation,cumbersome operation,high professional requirements for operators,large instruments,high cost,low sensitivity,and specific instability.Therefore,in order to improve the deficiencies of conventional detection methods,it is extremely important to establish a rapid,convenient,and visually detectable method for detecting foodborne pathogens.A method for visualizing E.coli O157:H7 was established based on organic-inorganic hybrid nanoflower technology combined with BCA reagent（Dioctanoic acid）.The 96-well plate coated with streptavidin was used as a reaction platform,and the biotin（Biotin）E.coli O157:H7 specific aptamer was used to capture Escherichia coli O157:H7,which was fixed in micro in the pore,the concanavalin（Con A）contained in the organic-inorganic hybrid nanoflower can non-specifically bind to the glycoprotein on the surface of the bacteria,so that the nanoflower is indirectly fixed in the micropores,and the nanoflower is used.The enzyme activity and stability characteristics of the enzyme,the alkaline phosphatase（ALP）embedded in the nano flower catalyzes the acid production of sodium ascorbate（AAP）,and the acid can make the BCA reagent change from light green to purple,visible to the naked eye,through the enzyme label.The instrument measures the absorbance of the solution to generate a standard curve,which in turn allows quantitative detection of the concentration of the bacteria based on the change in color.The optimal incubation time and reaction system temperature of the aptamer and nanoflower were optimized in the experiment.The optimal incubation time for the aptamer and nanoflower was 60 min and 30 min,respectively,and the reaction temperature was room temperature（25℃）,the minimum detection limit was up to 10² cfu/mL.Compared with the traditional detection method of E.coli O157:H7,the portable,efficient and visual detection of foodborne pathogenic bacteria is feasible.A method for visualizing E.coli O157:H7 was established based on organic-inorganic hybrid nanoflowers,click chemistry and G-quadruplex.The test uses a magnetic bead of streptavidin（SA）as a carrier,and biotin（Biotin）modified on E.coli O157:H7 antibody can specifically bind to streptavidin（SA）,and the antibody specifically captures Escherichia coli O157:H7,and immobilized E.coli O157:H7 on the magnetic beads,and then indirectly coupled to the magnetic beads by using the concanavalin contained in the nanoflowers,and adding ascorbic acid to promote the release of the nanoflowers.Copper ions(Cu ²⁺),and the released divalent copper ions are reduced to monovalent copper ions.Under the action of monovalent copper ions,the azide and alkyne modified on the short G-rich sequence can occur[3+2].The cycloaddition reaction forms a complete G-quadruplex structure,and the added heme（Hemin）is encapsulated in the intact G-quadruplex to form a horseradish peroxidase（HRP）-like property.The DNase（DNA zyme）,under the action of hydrogen peroxide,catalyzes the blue color of the substrate TMB to qualitatively detect the target bacteria.Optimization of reaction conditions in the experiment were carried out including optimization of ascorbic acid concentration,optimization of concentration of short G-rich fragments,time of click chemical reaction,and optimization of hemoglobin concentration.The result was an optimal concentration of ascorbic acid and a short G-rich fragment of 2mM and 5μM,respectively,and a click chemistry reaction time of 60 min.The minimum detection limit was up to 10¹ cfu/mL,and the results of other food-borne pathogens except the target bacteria were negative,which proved that the experiment was specific.The detection method is rapid,sensitive,and specific,and has good applicability in clinical rapid detection.