| Invasive infections caused by Listeria monocytogenes(L.monocytogenes)in food are frequent and often lead to sepsis and meningoencephalitis in immunocompromised individuals such as infants and pregnant women.Compared to other foodborne pathogens,L.monocytogenes is more severe,with a fatality rate approaching 30%during some outbreaks.Therefore,sensitive,specific,and rapid detection of L.monocytogenes is essential to ensure food safety and public health.The gold standard for bacterial detection is the culture and colony-counting method,but this method is time-consuming and labor-intensive.The limitations of traditional methods have promoted the development of various emerging methods and techniques for the detection of pathogens,among which,fluorescent biosensors show great potential for detection of L.monocytogenes due to their quantitative properties and rapid response.In this thesis,the first step was to culture the targeted pathogen,prepared its immunogen,and immunized guinea pigs and New Zealand white rabbits respectively to obtain anti-L.monocytogenes antibodies.Then,the serum was purified by the gradient salting out method of n-octanoic acid and saturated ammonium sulfate to obtain specific antibodies,and the purity and relative molecular mass of the antibodies were analyzed and identified by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis and BCA protein detection kit.The titer and specificity of the antibody were analyzed by enzyme-linked immunosorbent assay and FITC immunofluorescence staining assay.In the second step,ultrasmall paramagnetic Fe3O4 magnetic nanoparticles(Fe3O4 NPs)with good dispersion in aqueous solution and CdZnTe quantum dots(CdZnTe QDs)with long-wavelength fluorescence emission were synthesized.The guinea pig antibody was bound to the magnetic nanoparticles by electrostatic force to form the guinea pig antibody-magnetic nanoparticle biocomplex(Fe3O4 NPs/pAb1),and then the bioconjugates of the rabbit antibody-CdZnTe quantum dot(CdZnTe QDs/pAb2)were formed under the action of amide bonds between rabbit antibodies and CdZnTe quantum dots.In the third step,a fluorescence immunosensing for detection of trace L.monocytogenes was designed based on magnetic nanoparticles functionalized by guinea pig antibodies and CdZnTe quantum dots anchored by rabbit antibodies.Fe3O4 NPs/pAb1 was used as an immunomagnetic separator to capture the targeted bacteria,and CdZnTe QDs/pAb2 was used as a specific signal indicator.The magnetic separator,signal indicator and target bacteria formed a stable sandwich mode under the interaction between antigen and antibody.Finally,quantitative analysis of L.monocytogenes was carried out by the detection of fluorescent signal.The results of this thesis are as follows:the purity of the obtained antibody by analyzing the SDS-PAGE electrophoresis had been significantly improved compared with that before purification,and the antibody titers of rabbit and guinea pig arrived 256 000 and 64 000,respectively.The prepared rabbit antibody and guinea pig antibody have the characteristics of high titer,good specificity and high sensitivity.After optimizing the conditions,the proposed fluorescent immunosensor has a wide detection range of 1 to 109 CFU mL-1 and a low limit of detection(LOD)of 1 CFU mL-1,realizing an exceptionally sensitive detection of L.monocytogenes.Spike recoveries of L.monocytogenes in tap water and pasteurized milk were within the acceptable range of 91-111%(RSD<4.39%).At the same time,the immunosensor took a total of 60 min,and achieve a fast and accurate determination in complex samples.Furthermore,the constructed immunosensing strategy exhibits excellent anti-interference performance against biological matrices due to the CdZnTe QDs/pAb2 with long-wavelength fluorescence and the ultrasmall paramagnetic Fe3O4 NPs/pAb1.In summary,immunosensors with high sensitivity,specificity,and anti-interference show great potential for trace L.monocytogenes.Interestingly,the immunosensing strategy proposed in this work is universal and can be applied to other foodborne pathogen by simply substituting specific antibodies.Finally,the full text is summarized as follows:1.The immunoassay strategy was constructed based on immunomagnetic separation and antibody-modified CdZnTe QDs indication with long-wave fluorescent emission.2.The immunosensor has the advantages of simple operation,less time-consuming,high sensitivity,and excellent anti-interference performance for biological matrices.3.The immunoassay strategy proposed in this work shows universal applicability,which can be applied for other foodborne pathogens detection via simply replacing specific antibodies.4.Showing great potential for detection of L.monocytogenes in practical applications and significance for the control of outbreaks of foodborne illnesses. |
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