Surface-enhanced Raman Spectroscopy(SERS) Methods For Detection Of Ethyl Carbamate And Escherichia Coli

Food contaminants are substances that have not been intentionally added to food. But they may be present in food as a result of the various stages of its production (including cereal cultivation, animal grooming, and veterinary drug use), processing, treatment, packaging, transport, or holding. They also might result from environmental contamination. Such contamination, including chemical contaminant and microorganism contaminant, generally has a negative impact on the quality of food and may imply a risk to human health. Ethyl carbamate (EC) is one kind of chemical contaminant, which is produced in food processing. It is a by-product naturally formed in fermented foodstaff especially in alcoholic beverages, posing a potential threaten to human health, which can cause cancer. E.coli is one of the most important species in foodborne pathogens, some of which can lead people to illness even to death.Adopting surface-enhanced Raman spectroscopy (SERS), combined with latest research results of nanotechnology, quantum physics, optics, molecular biology, and chemometrics, this project, aimed to develop a series of new methods for rapid detection of ethyl carbamate and bacteria in beverages.The main contents and results are summarized as follows.(1) A rapid method for ethyl carbamate detection based on density functional theory (DFT) and surface-enhanced Raman spectroscopy (SERS) was established. Firstly, the optimized geometry structure of EC molecule was operated based on DFT calculation. Secondly, the combination of DFT, Raman spectroscopy (RS), and SERS was used to analyze the bands of EC molecule and assign the Raman vibrational modes of EC. In addition, the EC adsorption geometry on the SERS-substrate silver nanoparticle surface was discussed via molecular electrostatic potential (MEP). Based on theoretical and experimental results, the feasibility of SERS technique for qualitative and quantitative detection of EC in aqueous solution was investigated. The characteristic peak for quantitative detection was then chosen, and the limit of detection based on this method was found in the study. The results indicated that:①The calculated results of EC molecule based on DFT using B3LYP/6-31G (d) and aug-cc-pVTZ basis level were found to be in good agreement with experimental Raman data. The characteristic Raman bands observed for EC molecule are396,512,672,854,996,1076,1127,1150,1273,1346,1440,1457,1622,1688,2934,2965,2991, and3414cm-1, which are mostly assigned to carbonyl group, C-C, C-H and N-H stretching and deformation vibarations.②According to the surface selection rules of SERS, MEP3D contour map and SERS vibrational modes were used to discuss the EC molecule adsorption geometry on the silver nanoparticle surface, which indicated that the EC molecule may be absorbed on the silver nanoparticle surface via amide group, C-C and C-H vibrational bands. The bands assigned to the amide group which existed in the SERS spectrum were not strongly enhanced however. It is supposed that the EC molecule arranged in a way that the amide group stayed almost parallel to the surface. The C-C bond became oriented perpendicular to the surface and the vibrations specific to this bond were highly enhanced.③The Raman band at1006cm-1assigned to νs(CC)+ω(CH) was the highest and best reproducible peak in SERS spectrum of EC, which could be chosen as the characteristic peak for quantitative detection of EC in aqueous solution. It was found that Raman signal had a good linear relationship with EC concentration ranging from1×10-6M to1×10-3M with a corresponding correlation coefficient0.9253. The limit of detection (LOD) is2×10-7M (17.8μg/L). The method established here provided reference for Raman fingerprint bank, furthermore confirmed the feasibility of SERS technique for qualitative and quantitative detection of EC. The LOD obtained was lower than the standard value set up in every country, which opened a new possibility of EC rapid evaluation.(2) A quantitative method for ethyl carbamatc detection based on Au@Ag NPs-dependent SERS technique was contructed. Au@Ag NPs with different thicknesses of Ag shell were synthesized through seed-growing method. The performance of Au@Ag NPs with different Ag shell thicknesses at different laser was evaluated. The Au@Ag NPs with optimized condition was used as SERS amplifier to detect EC with different concentrations in aqueous solution. Based on the assignment result of SERS characteristic peaks of EC molecule, the optimized peak was chosen for quantitative analysis of EC in aqueous solution and alcoholic beverages. The LOD of EC in alcoholic beverages was investigated based on this method. The results indicated that:①The SERS performance of Au@Ag NPs with different thicknesses of Ag shell at different wavelength lasers was evaluated by a dye molecule rhodamine6G (R6G) and EC molecule, which indicated Au@Ag NPs with7-nm Ag shell thickness at633nm laser possessed the best SERS effect.②The SERS method based on Au@Ag NPs was used to detect EC with different concentrations (from1×10-9M to1×10-4M) in aqueous solution. It was found that three characteristic bands at1003cm-1,1083cm-1, and1117cm-1, assigned to vibrational modes of us(CC)+ω(CH),δ(NH)+νs(OC)+δ(CH2), and δ(NH2+CH3) respectively, were strongly enhanced. The linear relationship between SERS signal and EC concentration was found in the range of1×10-9M to1×10-7M, with a good value of R2(0.9682,0.9428, and0.9679). The band at1003cm-1represented the best reproducibility and linear relationship, could be chosen as the characteristic peak for quantitative analysis of EC in alcoholic beverages.③EC solution with different concentrations spiked into three kinds of alcoholic beverages (Vodka, White rum, and a fruit snack Obstler) were detected by SERS method. A linear relationship between SERS signal and EC concentration was found from1×10-9M to1×10-7M, with a good value of R2(0.8065,0.9106, and0.9283, respectively). LOD of9.0×10-9M (0.8μg/L),1.3×10-7M (11.6), and7.8×10-8M (6.9μg/L) for such three alcoholic beverages was obtained. The total assay time of the method was only15min, and only a small volume was required in a real-world sample without any complicated sample preparation. The result indicated that this sensitive Au@Ag NPs-dependent SERS method could successfully fulfill the detection of trace amount of EC in alcoholic beverages, which may offer an intersting alternative for simple, rapid assessment of EC in the alcoholic beverage industry.(3) A rapid label-free SERS detection method of E.coli in aqueous solution was established. In this case, E.coli DSM1116was chosen as model E.coli bacteria for the study. The bacteria were cultivated in silver nanoparticle colloids with different growing conditions including shaking speed, cultivated time, and cultivated temperature. An optimized cultivated condition was picked up to help construct a rapid SERS method. The feasibility of rapid detection of E.coli and classification of three E.coli strains (E. coli DSM498/1116/5695) based on SERS was investigated. Finally, a SERS mapping method for single bacterium and bacteria with different concentrations was construced. The results indicated that:①When E.coli DSM1116grew in Ag NP colloids for3h with100rpm shaking speed at37℃, the optimized cultivated condition was obtained. Raman signal at732cm-1of bacteria with1×108cells/mL concentration could be reached to the largest value of18000cps.②Compared to previous reported simply mixing method, the SERS reproducibility of bacteria was improved, and the Raman signal was raised to3.5fold.③The color of Ag NP colloids was obviously observed from yellow-green to grass-green. Meanwhile, a new broad absorbance band from500to700nm in UV-vis spectrum was shown. A close-linked structure seen in TEM image indicated that a new complex compound between Ag NPs and bacteria was supposed to form. According to the SERS assignment, the appearance of three bands at658,732and1330cm-1indicated a close adsorption between the Ag NPs and cell wall of bacteria.④Fifteen SERS spectra for three different E.coli strains (E. coli DSM498/1116/5695) were obtained, normalized and used for the establishment of the classified model using discriminant analysis (DA) method. The three E.coli strains could be successfully discriminated.⑤A single bacterium could be successfully spotted and detected by SERS method, with the same Raman shift of bacteria in aqueous solution, which provided experimental foundation for SERS mapping.⑥Different concentrations of bacteria were detected by SERS method with Raman mapping. The lowest concentration can be obtained at1×105cells/mL. The method can provide higher sensitity and reproduciblity of SERS spectra of E. coli compared to previous reported simply mixing method.(4) A rapid grasping bacteria chip based on label-free SERS was established. The normal glass slide was modified with diamino-PEG method to make the surface covered with diamino group. The diamino-PEG-glass slide was then protonized with HC1which could enable the glass slide with NH3+. As the bacteria possess negetive charge and the glass slide gives a positve one, the NH3+modified glass slide can trap and grasp bacteria due to the electrostatic adsorption principle. The modified glass slide was incubed with bacteria for half an hour followed by Ag NP colloids for another half an hour. Then bacteria were detected by SERS based on such chip. In addition, the feasibility of discriminating three different UT1bacteria was investigated using this method. The results indicated that:①The rapid grasping chip possessed positive charge can tightly adsorb negative-charged bacteria via electrostatic adsorption principle. When the chip was incubated with concentrated Ag NP colloid, good reproducible SERS spectra of bacteria can be obtained with recognizable bacteria characteristic peaks in5min.②Three kinds of bacteria (CFT, PSAE, and PRM1) extracted from urinary tract infection (UTI) can be discriminated using SERS and DA method. The rapid grasping bacteria chip based on SERS is easily prepared and disposed, which can provide a rapid portable detection method of bacteria.