Construction Of Biosensors Mediated By Non-immunomagnetic Separation And Study On The Detection Of Listeria Monocytogenes

Listeria monocytogenes is a zoonotic pathogen that can survive in extreme environments such as low temperature and high osmotic pressure.Listeria monocytogenes can exist in every link of the food industry chain,which is an important cause of food safety problems and a serious threat to human health.Listeria monocytogenes has become the focus of attention in food-borne disease surveillance systems around the world.At present,the detection methods for Listeria monocytogenes mainly include traditional microbial cultures counting method,enzyme-linked immunosorbent method,nucleic acid amplification method,etc.Although to a certain extent,these methods enriched the detection system for Listeria monocytogenes,but most of the testing time,equipment,cost,sensitivity and other factors limited,which cannot meet the increasing food safety requirements.According to the above problem,based on magnetic separation technology and non-antibody biological recognition molecules,this study has built new fluorescence and magnetic relaxation biosensors that have been used to rapidly,sensitively detect Listeria monocytogenes,aiming to overcome the disadvantages of traditional detecting methods for Listeria monocytogenes and providing new ideas and new methods for rapid detection of foodborne pathogens.The main research contents include the following three parts:1.Development of fluorescence sensor based on two-site recognition strategy and fluorescence internal filtration effect for detection of Listeria monocytogenesIn this study,the upconversion nanoparticles（UCNPs）mediated fluorescence biosensor was constructed using vancomycin and aptamer as two-site recognition molecules,and was used for the rapid detection of Listeria monocytogenes.Vancomycin was used as the first recognition molecule to prepare MNPs-Van magnetic nano-probe;A biotinylated aptamer,as a second recognition molecule,could bind to the internalized protein-A on the cell wall of Listeria monocytogenes.Because the two recognition molecules target different sites on the target bacteria,they can specifically capture the target bacteria to form a sandwich type complex（MNPs-Van/Listeria monocytogenes/aptamer）.Through the horseradish peroxidase marker（HRP-SA）,the HRP-TMB enzyme catalysis system was introduced to produce blue substance.Combined with the fluorescence internal filtration effect of UCNPs,the fluorescence intensity of UCNPs was reduced,so as to realize the quantitative detection of Listeria monocytogenes.The results showed that the linear equation of the method was y=342.16x-532.25（R²=0.9969）with the linear range of 2×10³ to 2×10⁸ CFU/m L.The limit of detection（LOD）was 2.8×10² CFU/m L with good specificity,and the detection of target bacteria was realized in about 1.5 h.The recovery rate in the spiked ham samples was 88.0-108.5%.Compared with the traditional ELISA,the detection system not only avoids the use of antibodies,but also transforms the color signal into fluorescent signal,which can amplifie the signal and improve the accuracy of the method.The establishment of this sensor provides a useful supplement for fluorescence analysis of Gram-positive bacteria such as Listeria monocytogenes.2.Development of magnetic relaxation DNA sensor based on ALP-mediated Mn（VII）/Mn（II）conversion for detection of Listeria monocytogenesIn this study,the ALP-mediated Mn（VII）/Mn（II）magnetic relaxation sensing system was organically combined with DNA hybridization technology to construct a magnetic relaxation DNA sensor,which was used for the quantitative analysis of Listeria monocytogenes.Based on the hly A gene of Listeria monocytogenes,two oligonucleotide probes have been designed,and bound to magnetic nanoparticles surface（MNPs-probe1）and biotinylated（bio-probe2）,respectively,which triggered hybridization reaction by double probe identifying the target DNA.Through the streptavidin-biotin system,alkaline phosphatase（ALP）was introduced,forming"MNP-target DNA-ALP"structure;and ALP induced the conversion of Mn（VII）and Mn（II）,resulting in a significant change in transverse relaxation time（T₂）,thus achieving the quantitative detection of Listeria monocytogenes.The results showed that the linear equation of the method was y=62.45x+35.67（R²=0.9976）with the linear range from 2×10² to 2×10⁷ CFU/m L,and LOD of 10² CFU/m L.The method had good specificity and the recovery rate was 87.2-101.3%in the spiked ham samples.Compared with the traditional nucleic acid amplification methods,the new detection system does not require DNA amplification and pre-enrichment treatment,which effectively simplifies the analytical process and improves the analytical efficiency.In addition,this method can effectively improve the detection sensitivity by enzymatic reaction and Mn（VII）/Mn（II）-mediated magnetic relaxation signal opening system.Compared with the traditional magnetic immunosensor,it does not need the use of antibodies,effectively reducing the detection cost,and has a great advantage in the detection of food-borne pathogens.3.Development of magnetic relaxation DNA sensor based on differential magnetic separation strategylarge without enzyme labeling for detection of Listeria monocytogenesIn this study,a magnetic relaxation DNA sensor without enzyme labeling was constructed on the basis of two-probe hybridization system combined with differential magnetic separation strategy,and it was used for the highly sensitive detection of Listeria monocytogenes.Magnetic nano-probes(MNP₂₅₀-probe1 and MNP₃₀-probe2)with different magnetic separation speeds were prepared based on magnetic nanoparticles with different particle sizes.Then,combined with DNA hybridization reaction,unbound MNP₃₀-probe2 after magnetic separation was collected to establish the one-to-one correspondence between target DNA and MNP₃₀-probe2 in the supernatant,so as to realize the quantitative detection of Listeriosis monocytogenes.The results showed that the linear equation of the method was y=42.2x-32.36（R²=0.9930）with the linear range from 10² to 10⁷CFU/m L,and the limit of detection was50 CFU/m L.The recovery rate rate of target bacteria was 83.4-105.3%in the spiked ham samples.The system combines high sensitivity magnetic sensing and high efficiency hybridization reaction,which provides a good platform for pathogen detection.Compared with the traditional detection methods,this method has the advantages of rapid detection and high sensitivity.Compared with the two methods established above,this detection system does not need enzyme markers,DNA amplification and multiple washing steps,which can simplify the analytical process,and quantitatively detect Listeria monocytogenes only through one-step reaction.