A Study On Dual Recognition And Highly Sensitive Detection Method Of Listeria Monocytogenes Based On Fe₃O₄@ZIF-8 Magnetic Separation

Listeria monocytogenes(L.monocytogenes),is a foodborne pathogen that can cause major foodborne diseases.It can grow in foods with low water content,high salt concentration and even cold storage temperature(4?),which seriously threatens human health and social and economic development.Therefore,the establishment of a highly sensitive and specific detection method of L.monocytogenes is the key to prevent the outbreak of Listeria disease.The traditional detection method of Listeria monocytogenes has the disadvantages of high labor intensity and time-consuming,which is difficult to meet the current detection needs.Nano materials combined with sensor method and visual colorimetry are widely used in the field of foodborne pathogenic bacteria detection because of their high detection accuracy.In this study,two highly sensitive methods for the detection of L.monocytogenes were established by combining functionalized nano materials with fluorescence enhancemen,colorimetric detection and UV spectroscopy.The main research results were as follows:(1)Superparamagnetic Fe₃O₄ with carboxyl group on the surface was prepared by solvothermal method,and then Fe₃O₄@ZIF-8 was synthesized in situ on its surface by layer-by-layer assembly strategy.The successful preparation of composite Fe₃O₄@ZIF-8 was confirmed by transmission electron microscopy and other characterization methods.By comparing its capture efficiency of L.monocytogenes with the other two magnetic materials,Fe₃O₄@ZIF-8 with the best capture efficiency(>90%)was selected as the basic material for the detection of L.monocytogenes.Gold particles(Au NPs)were synthesized by sodium citrate reduction method,and the characterization results of scanning electron microscope and fourier infrared spectroscopy all indicated the successful preparation of Au NPs.The synthesized Au NPs were wine red in color,with a particle size of 45.9±0.2 nm and zeta potential of-36.1±0.3 m V,exhibited a strong absorption peak at 528 nm.The addition of 16?L of Na Cl solution(1 mo L/L)to 200?L of Au NPs resulted in a discoloration of the Au NPs due to massive aggregation,which indicated the tolerance of the Au NPs to Na Cl.(2)A detection method for L.monocytogenes based on magnetic organic metal framework combined with fluorescence enhancement was established.The present method was based on the principle of fluorescence enhancement to achieve the purpose of improving sensitivity through the amplification of the detection signal,while the dual recognition function of“aptamer bacteria”and“antibody bacteria”was utilized to enhance the specificity of the detection results.The results showed that there was a good linear relationship between fluorescence intensity and the concentration of L.monocytogenes in the range of 1.4×10¹ to1.4×10⁷ CFU/m L.The linear equation was Y=1056.941+37.353X,and the correlation coefficient(R²)was 0.978,as the detection limit of L.monocytogenes was 0.88 CFU/m L,and the coefficient of variation(CV)of intra batch test was less than 2%.This method had good selectivity for L.monocytogenes.Meanwhile,the recoveries of pork and milk spiked samples ranged from 88.0%to 103.8%,and the coefficient of variation was less than 13%.It indicated that the detection system had a wide dynamic range,good repeatability and high detection sensitivity.(3)To meet the demand of on-site visual detection,a visual detection system for L.monocytogenes was further constructed by combining Au NPs.Detection of L.monocytogenes as well as artificially contaminated chicken breast meat samples was performed by colorimetry and UV spectroscopy using aptamer modified Fe₃O₄@ZIF-8 as capture probe and immunofunctionalized Au NPs as signal probe.The visual detection limit of L.monocytogenes was 1.2×10³ CFU/m L,the detection results of ultraviolet spectroscopy had a good linear relationship in the range of 1.2×10¹?1.2×10⁸ CFU/m L(Y=0.519-0.043X,R²=0.978),the detection limit was as low as 0.45 CFU/m L,and the intra batch coefficient of variation(CV)of each concentration gradient was 0.31%?1.30%.The detection results of actual samples showed that the visual detection limit of L.monocytogenes is 6.7×10⁴ CFU/m L,and the recovery was between 91.1%and 108.7%withthe coefficient of variation less than 7.4%.In addition,the experimental results were verified by plate counting method.The recovery was between 94.7%and 105.4%,and the coefficient of variation was less than 3.0%.The results showed that the method for the detection of L.monocytogenes established in this study had high sensitivity and good specificity,and had great application prospects in the detection of foodborne pathogens.