Research On Detection Method Of Foodborne Pathogens In Milk Based On Upconversion Fluorescence Technology

Milk is referred to as"white blood"because of its rich nutrition and easy absorption.As a perfect natural food,consumers'expectations for milk quality are continually rising as milk consumption rises.Therefore,the contamination of milk by harmful substances has attracted great attention.Among them is the problem of microbial contamination,particularly the presence of foodborne pathogens,which is difficult to detect due to its wide channels to produce toxins,endangering people's health and should be given more consideration.Traditional foodborne pathogens detection methods have problems such as time-consuming detection and cumbersome operation.Although some of the current rapid detection methods have the characteristics of being rapid requiring lower volumes of reagents and chemicals,however,requires professional operation and expensive equipment.Therefore,it is necessary to find a fast,accurate,economical,and efficient method to realize the detection of milk related pathogens for ensuring the healthy consumption of milk by consumers.In recent years,upconversion fluorescent nanomaterials doped with rare earth elements have been continuously applied in the field of food detection with the advantages of strong stability,high sensitivity,and low background interference due to their unique luminescence characteristics.In this context,this research looks into a new method for detecting foodborne pathogens contamination in milk.Utilizing the unique optical advantages of upconversion nanomaterials,combined with aptamer specific recognition,magnetic nanomaterial separation,antibody specific capture,immunochromatography and other technologies,a new method for upconversion fluorescence detection of food-borne pathogens in milk was established.The rapid detection and discrimination of foodborne pathogens in milk was successfully executed with the specific research contents as follows:1.Research on the rapid detection method of foodborne pathogens in milk based on upconversion fluorescence and magnetic separation.To achieve rapid and sensitive detection of foodborne pathogens in milk,this chapter establishes a rapid detection method for foodborne pathogens with aptamers as a"bridge"based on upconversion fluorescence and magnetic separation technology.Taking Bacillus cereus in milk as an example,first,aptamers(Apt)are modified on the surface of magnetic nanoparticles(MNPs)to form Apt-MNPs capture probes;then the aptamer complementary strands(c DNA)are connected to upconverting nanoparticles(UCNPs)to form UCNPs-c DNA signal probes.When the analyte is not present,the UCNPs-c DNA-Apt-MNPs complex will be formed due to the specific binding between the aptamer and the complementary strand.In the presence of analyte,due to the aptamer specific recognition,part of the signal probe is separated from the complex.Under the excitation of 980nm near-infrared light,the fluorescence intensity of the complex decreases after magnetic separation.Therefore,based on this principle,the calibration curve is constructed between the concentration of the analyte(Bacillus cereus)and the fluorescence intensity.The results show that the method has a good quantitative ability for Bacillus cereus in the range of 49-49×10~6cfu/m L under the optimal conditions with a detection limit(LOD)of 22 cfu/m L.Simultaneously,the detection method also exhibits a certain degree of specificity.The spiked recovery rate of the actual sample was in the range of 90.54%-111.28%,and the relative standard deviation(RSD)values in the range of 2.12%-3.13%,indicating that the method has good reproducibility and stability.This study demonstrates that the constructed method can be used successfully for the rapid detection of foodborne pathogens in milk.2.Research on rapid detection method of foodborne pathogens in milk based on upconversion fluorescence and multifunctional microspheres.In order to further improve the sensitivity of capture probes and signal probes in upconversion fluorescence detection,and realize the rapid capture and detection of foodborne pathogens in milk,this chapter establishes a method based on upconversion fluorescence and multifunctional microsphere capture technology.Based on dual fluorescence signals,a rapid and sensitive detection method for foodborne pathogens has been developed.Taking Staphylococcus aureus in milk as an example,first,the UCNPs(Tm-doped)and MNPs are filled in the polystyrene microspheres,and the Staphylococcus aureus antibody is connected to the surface of the microspheres to form multifunctional microspheres as fluorescent capture probes;to make a UCNPs-Apt signal probe,the aptamer is connected to UCNPs(Er-doped).The fluorescent capture probe and the signal probe exist independently in the absence of the target pathogen.When the target is present,its specific binding to the aptamer and antibody causes the formation of a signal probe-analyte-fluorescence capture probe complex.Under the excitation of 980 nm near-infrared light,the complex generates a fluorescent signal after magnetic separation.Therefore,based on this principle,the calibration curve is established between the concentration of the analyte(Staphylococcus aureus)and the fluorescence intensity.The results show,that the method has a good quantitative ability for Staphylococcus aureus in the range of 27-27×10~7 cfu/m L and LOD of 6 cfu/m L under optimal conditions.Moreover,the method has a good specificity for the detection of the target in real samples.The spiked recovery rate of the actual sample is in the range of 92.14%-116.26%,and the RSD value is in the range of 1.41%-4.11%,indicating that the method has good reproducibility and stability.This study shows that the constructed method can be successfully used for the rapid and highly sensitive detection of foodborne pathogens in milk.3.Research on rapid identification method of foodborne pathogens in milk based on upconversion fluorescence and immunochromatography.In order to further improve the application of upconversion fluorescence technology in actual detection and realize the rapid identification of foodborne pathogens in milk,this chapter aims to develop a new type of fluorescence immunochromatography test strip based on upconversion fluorescence and immunochromatography technology for rapid and sensitive detection of foodborne pathogen.Taking the Shigella flexneri in milk as an example,first,Shigella flexneri antibody A is connected with UCNPs to form a signal probe and spray it on a specific glass fiber membrane;then,the corresponding antibody B and the secondary antibody respond to antibody A are used as a capture probe to scribble on the nitrocellulose membrane to form a test line(T line)and quality control line(C line);the treated glass fiber membrane,nitrocellulose membrane and PVC base plate are assembled to develop this new type of fluorescence chromatography test strip.When a non-analyte is dropped into the sample pad of the test strip,the signal probe in the binding pad will not be combined with it and is not activated.The signal probe will only bind to the corresponding secondary antibody on the C line during the lateral flow process,and only the C line will generate a fluorescent signal when excited with 980 nm near-infrared light.The signal probe in the binding pad binds to the analyte when it is dropped into the test strip sample pad.In the process of lateral flow,it will bind to the corresponding antibodies and secondary antibodies on the T line and C line again.Therefore,the T line and C line both generate fluorescence signals under the excitation of 980 nm near-infrared light to realize the rapid identification of the analyte.The results show that under the optimal conditions,the constructed fluorescent test strip's lowest detection concentration is 10~2 cfu/m L with a detection time of only10 minutes.At the same time,the fluorescent test strip has good specificity and it was found that both the T-line and the C-line produced fluorescence in the actual sample of 10~2-10~7 cfu/m L under the imaging device,indicating that this method has a good identification effect.This study shows that the constructed fluorescence test strip can be used for rapid identification of foodborne pathogens in milk.