Optimization Of NMR Polysaccharide Macromolecular Gadolinium Probe Synthesis Process And Its Application In Rapid Detection Of Salmonella

Food-borne disease is the core problem of global food safety,which has attracted global attention.Therefore,rapid screening and early warning of pathogenic bacteria in food is the key to prevent and control the spread of food-borne diseases.Salmonella has been recognized as the leading cause of food-borne illness outbreaks,posing a serious threat to public health.It can enter the food supply chain at all stages of production,processing,transportation and marketing.High outbreak rates of Salmonella require efficient and rapid methods for early identification and detection.Due to the time-consuming and laborious traditional laboratory methods such as microbial culture,polymerase chain reaction（PCR）and enzyme-linked immunosorbent assay（ELISA）,new and rapid detection methods are urgently needed for field and online applications.Biosensors have many advantages over traditional laboratory analysis methods in terms of specificity,sensitivity and accuracy,as well as in terms of rapid response and portability.They are now considered a promising alternative tool and one of the most applicable detection methods in the field.In this study,a nuclear magnetic resonance（NMR）biosensor based on O-carboxymethyl chitosan target gadolinium（Gd）probe was developed to quickly detect Salmonella in milk by combining nuclear magnetic resonance（NMR）technology,bioimmunoassay and membrane filtration technology.The polysaccharide macromolecule O-carboxymethyl chitosan（O-CMC）with high biocompatibility,rich multifunctional groups and chelating adsorption to metal ions was used as the probe probe skeleton,and the paramagnetic metal nanoparticle Gd³⁺was used as the magnetic signal material,combining streptavidin-biotin system,the antibody and magnetic signal substance Gd were coupled by polysaccharide macromolecule O-CMC to prepare the target probe,Salmonella was captured by the interaction between the antibody on the target probe and the surface antigen of Salmonella,then,Salmonella and free probe were separated by membrane filtration method,finally,the longitudinal relaxation time（T₁）of the probe in the collected filtrate was measured by NMR.The main research contents are as follows:1.Research on the design and preparation of polysaccharide macromolecular NMR biosensor:The polysaccharide macromolecule O-carboxymethyl chitosan（O-CMC）was used as the probe skeleton,and the paramagnetic metal nanoparticle Gd was used as the magnetic signal substance,combined with the streptavidin（SA）-biotin（Biotin）system,the target probe was prepared by coupling Biotin-O-CMC-Gd with biotin-antibody using SA as an intermediate bridge.The synthesis conditions of Biotin-O-CMC-Gd were optimized.The optimized results showed that the molar ratio of Biotin to O-CMC was 10:1,the reaction time was 3h,the p H of the buffer system for chelating Gd by O-CMC was 4.5,and the reaction time and temperature were 3 h and 25℃,respectively.The successful synthesis of Biotin-O-CMC-Gd was characterized by Zeta potential,Fourier transform infrared spectroscopy（FT-IR）,X-ray photoelectron spectroscopy（XPS）and scanning electron microscope images（SEM）.The gadolinium loading of Biotin-O-CMC-Gd was determined by inductively coupled plasma emission spectrometry（ICP-OES）,and the longitudinal relaxation time（T₁）of Biotin-O-CMC-Gd was measured under different Gd concentrations to determine the longitudinal relaxation rate r₁of Biotin-O-CMC-Gd.In addition,the biotin activity on the Biotin-antibody was experimentally analyzed by colorimetry to verify the successful synthesis of the Biotin-antibody.2.Construction of Salmonella detection system:In order to improve the specificity of the target probe and achieve rapid detection of Salmonella,the study optimized the main conditions such as the amounts of SA in the Salmonella detection system,the mole ratio of Biotin-antibody to SA,the amounts of Biotin-O-CMC-Gd and the capture time of Salmonella by the target probe.The results showed that the optimal amounts of SA was 6μg,the optimal molar ratio of Biotin-antibody to SA was 4:1,the optimal amounts of Biotin-O-CMC-Gd was 60μg,and the optimal capture time of Salmonella by the target probe was 45 minutes.Therefore,under the optimal conditions,the output of NMR signal was relatively stable,and the target probe prepared had the best capture effect on Salmonella.3.Performance evaluation of NMR biosensor and application in the detection of Salmonella in milk samples:In order to ensure the detection effect of the probe on real samples,it is necessary to evaluate the performance of the NMR biosensor.Other non-target bacteria were introduced to test the specificity of NMR biosensor,and other non-target bacteria were added into the sample solution containing Salmonella to test the anti-interference capability.In addition,Salmonella was diluted to different concentrations for sensitivity detection,and the sensitivity detection effect in milk and the reproducibility of the Salmonella detection in milk were analyzed in detail.The experimental results show that the sensor had good specificity for the detection of Salmonella,and it can also recognize Salmonella in the presence of non-target bacteria.The sensitivity of detection in PBS buffer and milk can reach 1.8×10³CFU/m L.In addition,the sensor has a good reproducibility for the detection of Salmonella in milk.Therefore,the NMR biosensor designed in this study has great application potential in the detection of Salmonella,can be used for the direct detection of complex biological samples,and has important guiding significance for the detection of other pathogenic bacteria in food.