| Surface-enhanced Raman Scattering (SERS) is a highly-efficient spectrum detection technology. SERS can reflect abundantly structure information of molecular, therefore, it often referred to be the fingerprint spectrum of molecular. SERS not only inherits all the advantages of traditional Raman scattering, such as nondestructive detection, simple sample preparation and rapid detection, but also overcomes the disadvantages such as low sensitivity and susceptible to fluorescence interference. The enhancement of SERS is as high as 105-106, and it can even realize the detection of single molecule. Noble metals such as gold (Au), silver (Ag) and copper (Cu) are widely used as SERS-active substrates. Ag is extremely efficient in enhancing the Raman scattering, but it is easily oxidized. Compared with Ag, Au possesses weaker enhancement but has excellent resistance against oxidation.In this paper, the periodical three dimensional nanostructure of cicada wing was used as Au/cicada wing substrate with high sensitivity, good stability, good reproducibility and recyclability by the electron beam evaporation (EBE) of Au NPs onto cicada wings. Systematic characterization and structure analysis had been done for morphology structure and SERS performance of the substrate. Finite different time domain (FDTD) simulation had also been done for the Au/cicada wing substrate to explore the distribution regularity of "hot spots" and the enhancement mechanism. The detection of unsaturated fatty acids in vegetable oils and the quantitative and detection of trace reduced nicotinamide-adenine dinucleotid (NADH) had been done based on the Au/cicada wing substrate. These two application studies possess the certain innovation, and build novel detection systems for detecting biological moleculars. Finally, the identification of vegetable oils was implemented successfully and the trace NADH was detected quantitatively. The main content of the this paper is divided into the following four aspects:The first is preparation and structure characterization of the Au/cicada wing substrate. All the complete cicada wings were first ultrasonically cleaned in acetone, ethanol and deionized water in turn for 5 min each to remove impurities and keep the nanopillar array structure of surface. Then the wings were modified by Au NPs with 50nm thickness in the EBE system, the Au/cicada wing substrate was prepared. Film emission-scanning electron microscope (FE-SEM) shows that The average diameters of Au NPs covered on the tops and the sides of the surface nanopillar were ?65 nm and 35 nm, respectively. The regular nanopillar array ensure reproducibility of the substrate. Contact angle of the substrate is 144.4°, indicating that the substrate is close to super hydrophobic which is very beneficial for improving SERS sensitivity. X-ray diffraction (XRD) analysis show that cicada wing covered with Au NPs successfully and ultraviolet-visible absorption spectrometry (Uv-vis) analysis show that the absorbing ability for visible light of cicada wing covered with Au NPs increase.The second is the SERS performance test and FDTD simulations of the Au/cicada wing substrate. Series concentration of Rhodamine 6G (R6G) were detected on the Au/cicada wing substrate, the limit of detection as low as 10"7M. The linear relationship curve between the SERS logarithmic integrated intensity of the peaks centered at 1361 cm-(S1361)and the concentration of R6G was established, and trace R6G can be detected quantitatively. Saturated adsorption was studied to calculate the accurate enhancement factor (EF), and the EF is ?3.4×105. Two kinds of SERS reproducibility, substrate-to-substrate and spot-to-spot reproducibility, were measured using R6G solution. SERS signals were measured at 100 randomly chosen spots, and the Relative Standard Deviation (RSD) were less than 15%. R6G was used to measure the three-month stability test of the SERS substrate, the intensity of the Raman peaks change slightly, verifying the long-time stability of the Au/cicada wing substrate. This may owes to gold's resistance against oxidation. Two kinds of analyte with (p-aminothiophenol) and without (R6G, methyl orange, Nile blue A and Crystal violet) mercapto group (-SH) were used to measure the recyclability of the substrate. It shows that NaBH4 can remove the molecular physical or chemical absorbing on the substrate in the four cycles, and the SERS enhancement was undiminished. FDTD was utilized to simulate the electric field distribution of the substrate surface and the theoretical EF can be calculated which. EF calculated by SERS measurement was in the same order magnitude with theoretical EF, indicating the main enhancement mechanism of the Au/cicada wing is electromagnetic field enhancement mechanism.The third is the establishment of converse relation between common unsaturated fatty acids, and the accurate identification of vegetable oils sold in market. Vegetable oils are important to our daily life, which are rich in essential fatty acids. The main fatty acids in vegetable oils include oleic acid (OA), leinoleic acid (LA), a-linolenic acid (ALA) and palmitic acid (PA). Among these, the former three are unsaturated fatty acids and the latter is saturated fatty acid. In this paper, the four kinds of fatty acids and their mixtures were analyzed by SERS on the substrate, and quantitative detection of typical and the most abundant unsaturated fatty acids in the PA environment was achieved. More importantly, the S1656 values of LA and ALA are about 1.5 and 2.2 times respectively compared to that of OA. Therefore, the contents of LA and ALA can be converted to that of virtual OA, together with the actual OA content as the equivalent total content of OA to denote the unsaturated fatty acids content. Taking peanut oil, sesame oil and soybean oil as examples, the ranges of S1656 corresponding to the national standard of China were established and can authenticate whether the unsaturated fatty acids content of vegetable oil conforms to the national standard of China. Gas chromatograph-mass spectrometer analyses were also done to analyze the fatty acids content of the three vegetable oils, and the results show that the relative errors between calculated and measured S1656 values were less than 5%, indicating the accuracy of our method. Moreover, the conversion relationship obtained in this paper for fatty acids can also be applied in other fields. For example, by analyzing the unsaturated fatty acids content in the cell membrane and plasma, several diseases can be detected in advance.The forth is the quantitative detection of trace NADH. According to our previous experiments, it is not so satisfactory to detect NADH on the Au/Ag-based SERS substrates. Fortunately, the enzyme-based method can solve this problem, and NADH can be indirectly detected quantitatively. NADH generates nicotinamide adenine dinucleotide (NAD+) and hydrogen peroxide (H2O2) with the action of NADH oxidase, and chromogen can be oxidated into pigment by H2O2 with the catalysis of horseradish peroxidase. Pigments is usually easy to be detected by SERS for their strong Raman peaks, and NADH can be detected indirectly through quantifying the pigment. In our experiment, eight kinds of chromogens were screened by SERS and OT (o-tolidine) was selected as the most appropriate one. Experiments were done to determine the optimal concentration of OT, and 2 × 10-3M was finally selected through the SERS data analysis. Peak centered at 1448cm-1 of the corresponding pigment tolidine blue (TB) is different form OT, and the integrated intensity (S1448) was used to denote TB concentration. Liner relationship between the logarithmic concentration of H2O2 and the logarithmic S1448 was established, and 2×10-4 and 2×10-5 M NADH solutions were used to validate the linear relationship, the logarithmic relative errors were less than 5%. This method can overcome the instability and small Raman scattering cross section of NADH, realizing the quantitative detection of trace NADH effectively. |
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