| In recent years, foodborne pathogens are important factors that affect food safety. Foodborne pathogens often need to be seperated and purified from samples before idenfication in conventional detection method. It often takes long experimental period and much more efforts, limiting its application in food hygiene. Thus, it is particularly important to establish a simple, sensitive and specific method for rapid detection of pathogens. Surface enhanced Raman scattering(SERS) is a fingerprint recognition technique, which has been used in various biological appication, for instance, in the detection of molecule, protein and cancer. SERS technique was successfully used to achieve the quantitative detection of salmonella typhimurium in our work., Magnetic nanoparticles(MNPs) with good magnetic characteristics was used as substrate in this method, which make it easier for the separation process. SERS detection based on the aptamer functionalized nanopaiticles is of better specificity and sensitivity compared to SERS detection of pathogens using only nanoparticles. Work which had been done was as follow:First, A SERS-based sandwich structure detection method for S. typhimurium was investigated, using aptamers modified MNPs as capture probes, aptamers and 4-MBA functionalized GNPs as signal probes. The capture probes and S. typhimurium were firstly incubated, washed three times with phosphate buffer solution(10 mol/L PBS, p H 7.4). Then, the signal probes was added and incubated under the same conditions, washed three times with PBS for Raman test. 4-MBA exhibits a special peak at 1582 cm-1,which was used for the calculation in further experiment. Under the optimal conditions, the correlation between the logarithm of the colonies got from the plate count method and the intensity of Raman intensity at 1582 cm-1 was observed to be linear within the range of 102 cfu/m L~107 cfu/m L(y=129.3045+175.7883 x, R2=0.9922), and the limit of detection(LOD) for the proposed method was 25 cfu/m L. E. coli, Vibrio parahemolyticus, Bacucilius cereus, shigella and S. aureus were selected in the specificity experiment. Pork samples was used in the recovery experiment. The results of the two experiment performed well, indicating that the developed method could be used in real sample.Then, to improve the sensibility of the method, Aptamer-functionalized MGNPs were used as capture probes instead of MNPs. MGNPs have good dispersibility and biocompatibility, which can improve the uniformity of the reaction system. S. typhimurium signal probes and capture probes were connected through S. typhimurium, thus, generating a “hot spot” effect between MGNPs and GNPs. It can substantially enhance the SERS effect. Under the optimal conditions, the SERS intensity of 4-MBA at 1582 cm-1 and the logarithm of the colonies got from the plate count method exhibit a good linear relationship(y=494.3184+793.4222 x, R2=0.9961) in the range of 10 cfu/m L~107 cfu/m L. The LOD is 5 cfu/m L. The specificity experiment and detection of the pork samples proved that this developed method could be used for detection of the real sample.At last, S. aureus aptamers and S. typhimurium aptamers simultaneously modified MGNPs were used as capture probes. Aptamers and 4-MBA functionalized GNPs were used as signal probes of S. typhimurium; aptamers and DTNB functionalized GNPs were used as signal probes of S. aureus. Under optimal conditions, the SERS intensity of DTNB at 1333 cm-1 and 1582 cm-1 was observed in a good linear relationships with the logarithm of S. aureus(1333 cm-1, R2=0.9885; 1582 cm-1, R2=0.9774). Raman intensity of 4-MBA at 1582 cm-1 and the logarithm of S. typhimurium also performed a good linear relationships(1584 cm-1, R2=0.9820). In the real sample experiment, the number of S. aureus was was obtaioned depend on the Raman intensity at 1333 cm-1. Then, the Raman intensity generated by S. aureus at 1582 cm-1 was calculated. Thus, indirect detection of S. typhimurium was achieved. there was no significant difference between the counting method and the developed method for the detection of the pork samples, which confirms that this developed method could be used for the detection of the real samples. |
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