Rapid Detection Of Salmonella In Food By A Compound Fluorescent Nanoprobe Based On Fluorescence Resonance Energy Transfer Effect

Salmonella is a common foodborne zoonotic intestinal pathogenic bacteria,a gram-negative short bacillus of enterobacteriaceae.It is reported to have more than 2,600serotypes.Diseases caused by Salmonella infection have become a major public health threat both in China and around the world.Because Salmonella is mainly spread through contaminated food,it can cause various diseases such as acute gastroenteritis,typhoid fever,and sepsis.Salmonella has always been an important test item in food safety risk prediction and hazard assessment.Therefore,development of a timely and efficient method for rapid detection of Salmonella has always been the direction of researchers'efforts,which has very important practical significance.The national standard for Salmonella inspection GB4789.4-2016"Food Microbiology Inspection Salmonella Inspection"requires the isolation and cultivation of pathogens,physiological and biochemical identification.Generally,it takes 4 to 7 days to determine the test results.The operation steps are cumbersome and the detection cycle is time-consuming.In this study,nanomaterials were combined with immunological methods to construct a switched-on composite fluorescent nanoprobe for Salmonella detection.according to the fluorescence resonance energy transfer(FRET)effect between the probe and gold nanoparticles,a new method for rapid detection of Salmonella in food was established.The main research results obtained are as follows:(1)First,functionalized immunomagnetic beads were prepared by coupling the functionalized magnetic beads modified by silanization and aminoization with Salmonella specific antibodies,which were used as capture probes to rapidly enrich Salmonella.The characterization and analysis of magnetic beads by near infrared spectroscopy confirmed that Fe3O4 beads had been successfully coated by Si O2,and the upper amino group was successfully modified by chemical modification.in order to explore the effect of different antibody concentrations on magnetic bead coupling,it was characterized by UV-Vis spectroscopy.The optimal dosage of Salmonella specific antibody in 1 m L(10 mg/m L)immunomagnetic beads was determined to be 200?L.At this time,the coupling rate was the highest,reaching 91%.Subsequently,different amounts of immunomagnetic beads were compared with Salmonella for magnetic bead capture performance experiments.it was determined that the capture efficiency of Salmonella was the highest when the amount of immunomagnetic beads was 10 mg,up to 98.52%.(2)In this study,quantum dots with good fluorescence performance were used as fluorescent signal elements to construct magnetic beads-antibody-quantum dots composite fluorescent nanoprobe integrating magnetic enrichment separation and detection by orderly assembly of functional magnetic beads,antibodies and quantum dots.The as-prepared QDs were characterized by uv-vis spectroscopy(UV-Vis)and fluorescence spectroscopy.It was proved that the qds coated Zn S shell had higher fluorescence intensity,their uv absorption peak was at 456 nm,and the fluorescence emission peak was at 516 nm.The charged condition of QDs was characterized by Zeta potentiometer.The QDs potential value prepared in this study was-35.7 m V,negatively charged.(3)This study used the FRET effect between quantum dots and gold nanometers to construct a fluorescence switch-on detection system.Transmission electron microscopy and uv spectroscopy characterization were carried out to determine that the prepared mercaptoethylamine modified gold nano-colloidal particles were basically the same size and good dispersion,with a particle size of 34 nm,concentration of about 0.83 nmol/L,and the uv absorption peak at 523 nm.The as-prepared gold nanoelectroelectricity was characterized by Zeta potentiometers,and the gold nanoelectroelectricity value of this study was determined to be+15.4 m V,positively charged.Satisfying the condition that electrostatic interaction can attract each other and thus electrostatic self-assembly occurs,and the fluorescence probe added with gold nanometers was characterized by fluorescence spectra.It was found that the fluorescence intensity of the fluorescent probe decreased obviously after the addition of gold nanometers,and at this time the fluorescence probe was quenched.(4)In this study,gold nanometers were used as fluorescent quenchers.after quenching the fluorescence of the composite fluorescent nanoprobe,the target bacteria were added to establish a rapid detection system according to the recovery of fluorescence,that is,the qualitative and quantitative detection of Salmonella was realized by the close-open of fluorescence.after optimizing the experimental conditions of the system for rapid detection of Salmonella constructed by composite fluorescent nanoprobes,it was determined that when the optimal p H of gold nanoparticles for quenching composite fluorescent nanoprobes is 8 and the optimal dilution of antibodies is 15 times,the quenching effect of gold nanoparticles on composite fluorescent nanoprobes is optimal.The fluorescence recovery of the composite fluorescent nanoprobe showed a good linear relationship with the logarithmic value of bacteria concentration.The linear regression equation was y=103.5x+121.4(R~2=0.9956),the detection limit was 10~2 CFU/m L(S/N=3),and the detection time was less than 2 h..the detection limit of this method is low,the interference of background matrix is low,the sensitivity is high,the detection of target bacteria is specific and time-consuming,which provides a new idea and platform for the rapid detection of Salmonella.