Detection Of Escherichia Coli O157:H7 By Gold Nanocluster Composite Based On Colorimetric Aptasensor In Milk

Escherichia coli O157:H7,as a foodborne pathogen that can be widely spread among people and animals,can cause various intestinal diseases and greatly threaten food safety all over the world.Dairy products are one of the main ways of transmission of E.coli O157:H7.A small amount of invasion of human body can cause food poisoning and abdominal pain,and death in severe cases.Timely and accurate detection of E.coli O157:H7 is the key to ensure the safety of dairy products.At present,the detection of pathogenic bacteria in dairy products mostly depends on culture method and amplification method,and the realization of rapid detection is still a challenge.Therefore,it is urgent to establish a safe,simple and efficient detection method to control the pollution of E.coli O157:H7 in dairy products.In this study,gold nanoclusters were prepared by papain and chloroauric acid which were characterized by optical properties,hydrodynamic diameter,surface charge,functional group composition,surface morphology,element composition and energy changes.Using gold nanoclusters and sulfhydryl modified aptamers covalently combined through Au-S bond,the peroxidase activity of the system changed significantly under the action of energy transfer.Based on this principle,a colorimetric aptamer sensor for detecting E.coli O157:H7 in different liquid milk was constructed.Using the unique optical properties of gold nanoparticles,gold nanoparticles can be adsorbed with bacteria after being induced by high concentration salt solution,and then dropped onto the surface of filter paper soaked by aptamer modified gold nanoclusters.The aptamer specifically captures the target bacteria to develop color in the reaction area.At the same time,the color change of paper-based color developing area is analyzed by using smartphone color gamut model.Based on this principle,a colorimetric paper-based aptamer sensor for detecting E.coli O157:H7 in different liquid milk is constructed.The specificity,sensitivity and practicability of the above two colorimetric aptasensors were evaluated.This provides a new theoretical support for the colorimetric detection of E.coli O157:H7 in different liquid milk.（1）Preparation and characterization of gold nanoclusters.The compound volume ratio of papain and chloroauric acid was 1:1（v/v）,the concentration of Na OH was 1 M,and the reaction time was 150 min.The complexes were characterized.The negative charge of gold nanoclusters was determined by Zeta potential;the disappearance of the stretching vibration peak at 1542 cm^-1 proved that the secondary structure of papain changed during the synthesis process in Fourier Transform Infrared Spectroscopy;Transmission Electron Microscopy showed that the gold nanoclusters were spherical and well dispersed;Energy Spectrum analysis shows that the gold nanoclusters have C,N,O,S and Au elements at the same time.The above results show that gold nanoclusters have been successfully prepared.（2）A colorimetric aptamer sensor was constructed and evaluated.The optimal condition of colorimetric aptamer sensor is that the concentration of gold nanoclusters is 5 mg/ml and incubated with aptamers for 2 hours.The optimum time of the system was determined to be 50 min by enzymatic reaction kinetics.At the same time,it was determined that the peroxidase activity of the system was the strongest when the substrate concentration was 3,3’,5,5’-tetramethylbenzidine（TMB）was 0.1 m M and hydrogen peroxide（H₂O₂）was 1 M.Under the pure culture of E.coli O157:H7,the colorimetric aptamer sensor has specific detection,and the detection limit is 39 CFU/m L（S/N=3）.In the detection of different contaminated liquid milk and raw milk,the colorimetric aptamer sensor has good practicability and sensitivity.After pre incubation for 2 hours,the detection limits in high-temperature sterilized milk,pasteurized milk and raw milk can reach 5.6×10²CFU/m L,5×10² CFU/m L and 4.9×10² CFU/m L,respectively.The standard rate of practical detection is 100%.（3）Preparation and characterization of gold nanoparticles.The synthetic gold nanoparticles were characterized,and there was a maximum absorption peak at 525 nm;Transmission Electron Microscopy showed that the gold nanoparticles were uniform,spherical and well dispersed,with an average diameter of 44 nm.The optimum salt concentration for aggregation and discoloration of gold nanoparticles was 4 M.（4）A colorimetric paper-based aptamer sensor was constructed and evaluated.The optimal condition of colorimetric paper-based aptamer sensor is that the adsorption time between induced gold nanoparticles and bacteria is 20 min.The colorimetric paper-based aptamer sensor has good specificity.Under the pure culture of E.coli O157:H7,the detection limit is 8.2×10² CFU/m L（S/N=3）.In the detection of liquid milk and raw milk with different pollution,the colorimetric paper-based aptamer sensor shows good practicability and sensitivity.The detection limits in high-temperature sterilized milk,pasteurized milk and raw milk can reach 1.3×10³ CFU/m L,1.2×10³CFU/m L and 1.45×10³ CFU/m L,respectively.The standard rate of practical detection is 100%.