| Salmonella is a common kind of harmful pathogen that constantly threatens human health.At present,there are many methods for detecting microorganisms,but their advantages and disadvantages are different and cannot fully meet the needs of production testing.Therefore,in order to achieve rapid and accurate detection of pathogenic bacteria,this study aims to a new biosensor for rapid detection of Salmonella in food via low-field nuclear magnetic resonance(NMR)technology.In the study,we used paramagnetic gadolinium ions(Gd3+)as the magnetic signal source,which can have a great influence on the water longitudinal relaxation time(T1).To ensure the magnetic particles have good specificity to the target bacteria,gadolinium ions and polyamine polycarboxylated compounds(DTPA)were modified to various functional macromolecular ligands to synthesize macromolecular gadolinium probes with high relaxation efficiency and specificity by direct coupling,streptavidin-biotin mediated coupling and poly-L-lysine signal amplification methods,respectively.We regarded this probe as Salmonella specific marker and relaxation signal conversion medium.After the probe bound to the Salmonella in the sample,the mixture was filtered by microfiltration membrane filtration techniques to remove the excess probe.Finally,the filtrate was collected and measured by NMR technology.Because the longitudinal relaxation time(T1)between the negative group and the positive group had a significant change,this method was successfully used for the detection of Salmonella.The main results are as follows:1.The effects of different paramagnetic metal ions Gd3+,Fe3+,Mn2+ and Cu2+ on longitudinal relaxation time(T1)in solutions with concentration gradients ranging from 0.001 to 0.1 mmol/L were studied.The results showed that the relaxation efficiency of Gd3+was significantly higher than that of Fe3+,Mn2+ and Cu2+,and it had the condition of being a magnetic signal source for NMR biosensors.2.The effects of different synthesis methods on the relaxation efficiency and specificity of the probe were studied.Firstly,different kinds of probes were synthesized by direct coupling method,streptavidin-biotin mediated coupling method and poly-L-lysine signal amplification method,and the optimum process conditions for the synthesis of probes were determined by single factor experiments.Then the relaxation efficiency of various probes was measured by NMR technology.Finally,the sensitivity of three probes was evaluated by comparing the detection results of target bacteria at the same concentration.The results indicated that since a lot of negative charges on the surface of DTPA,it reduced the activity of the antibody when directly covalently bound to the antibody in the direct coupling method,and the specificity of the probe also decreased.However,when the streptavidin-biotin system was introduced into the preparation of the probe,the activity of the antibody could be retained because of the small molecular weight of biotin,which made sentivity of the probe to the detection of Salmonella higher than the direct coupling method.When poly-L-lysine was introduced as the backbone of the probe in the poly-L-lysine signal amplification method,because several amino groups were present on the surface of PLL,it achieved high-Gd loading by linking with multiple Gd-DTPA molecules.Furthermore,poly-L-lysine can increase the molecular weight of the probe and prolong its rotation correlation time,so that the relaxation efficiency of Gd-DTPA-PLL-SA-biotin-antibody(8.25mM-1 s-1)was significantly higher than Gd-DTPA-antibody and Gd-DTPA-SA-biotin-antibody,and the sensitivity to the detection of salmonella was also higher.3.The probe synthesized by the poly-L-lysine signal amplification method has a higher relaxation rate and specificity,so it was used for the construction of the NMR detection system.In the study,the optimum incubation conditions of Salmonella and probe were firstly determined by single factor experiment.The results showed that the optimal incubation time was 60 min,and the optimal amounts of streptavidin and biotinylated antibody were 20 ?g and 10 ?g,respectively.Then,the experiment analyzed the effects of different membrane filtration methods and membrane filtration materials on the stability and reliability of the experimental results.The results showed that the magnetic signal was not stable because the components above the membrane were difficult to be fully eluted and collected in the high pressure filtration method,which made the error of each experiment too large(P<0.05).However,the filter filtration method was simple and fast,and the filtrate was also easier to collect than the components above the membrane,so the magnetic resonance signal of the filtrate in the filter filtration method was taken as the output signal of the NMR sensor in this study.The experiment also found that the non-specific adsorption capacity of the four commonly water-based membranes(MCE,PES,PTFE,Nylon66)on the probe was negligible(P>0.05)due to the probe's good solubility.Finally,the specificity,anti-interference ability and sensitivity of the probe in the detection process of Salmonella were also studied.The results showed that this NMR biosensor based on streptavidin-biotin system and poly-L-lysine signal amplification system had good specificity and anti-interference ability for the detection of Salmonella.This method was successfully used in the simultaneous detection of Salmonella in pure culture and milk.The detection sensitivity was 2.4 × 104 CFU/mL,and the overall detection process could be finished in 2 h.Compared with other pathogen detection methods,the NMR biosensor is a simpler and faster system,and has great potential to become a very promising biological molecular detection method. |
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