A surface-enhanced Raman scattering substrate, a preparation method thereof and uses thereof

Abstract

The present invention provides a surface-enhanced Raman scattering substrate, which includes an anti-sea urchin array structure and is modified with a metal thin film on the anti-sea urchin array structure; the substrate has a periodically ordered structure and has high uniformity during actual application; and it has hydrophobicity, high sensitivity, a detection limit for rhodamine of 10-13 M, and can realize rapid detection of trace methanol in blended wine; the present invention also provides a preparation method and uses of the surface-enhanced Raman scattering substrate, and at the same time provides a method for detecting the methanol content in blended wine by using the surface-enhanced Raman scattering substrate. This method solves the huge influence of the strong background peak of ethanol on methanol analysis by adopting an analysis method of relative intensity ratio, and this method is rapid, simple and reliable.

Description

Technical Field

[0001] The present invention relates to the technical field of Raman spectroscopy, and particularly to a surface-enhanced Raman scattering substrate, a preparation method thereof, and uses thereof. Background Art

[0002] Chinese liquor is one of the most popular alcoholic beverages in China. Water and alcohol are the main components of Chinese liquor. In addition, there are various organic components. Chinese liquor brewed from raw materials with a large amount of pectin often contains a large amount of methanol. Methanol has a greater toxic effect on the nervous system and blood system of the human body. 4 grams can cause poisoning reactions, 10 grams can cause double blindness, and 30 milliliters can be fatal. Therefore, the detection of trace components such as methanol in Chinese liquor is of great significance for public health and food safety. There are mainly two methods for detecting the components of Chinese liquor in China: One is chromatographic analysis using special instruments, such as gas chromatographs and high-performance liquid chromatographs. Sorghum, food additives, etc. in Chinese liquor will show different colors under chromatographic analysis, so as to analyze the components in Chinese liquor. However, the detection process of this method is cumbersome and complex, and the instruments are expensive, which is greatly limited in practical applications. The other is that professional personnel conduct detections through the senses. This detection method is difficult to unify the standards of each professional detector and lacks scientificity.

[0003] As a highly sensitive detection technology, surface-enhanced Raman scattering has important application potential in chemical analysis and biological detection, and has been widely used in the field of food detection. A substrate with higher enhancement effect, lower detection limit, and better consistency is the basis for realizing its application. It has been reported that gold nanoparticles coated with silver shells are used as surface-enhanced Raman scattering substrates to detect the residue of the harmful component "carbofuran" in Chinese liquor. It is by adding 2,6-dichloroquinone-4-chloroimide and sodium hydroxide to Chinese liquor to form a complex with "carbofuran", and then mixing it with nanoparticles, so that the complex is coupled with the nanoparticles, and then an enhanced Raman signal is obtained. However, there is no report on using surface-enhanced Raman scattering substrates to quickly detect the harmful component methanol in Chinese liquor. Therefore, obtaining a new surface-enhanced Raman scattering substrate, combining Raman scattering technology and an accurate and fast method to detect the content of methanol in blended liquor is of great significance for the development of food safety. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a surface-enhanced Raman scattering substrate and a preparation method thereof, which are used to solve the problems of cumbersome detection and lack of scientificity in detecting the methanol content in blended liquor in the prior art.

[0005] To achieve the above purpose and other related purposes, the present invention provides a surface-enhanced Raman scattering substrate, including an inverse sea urchin array structure, and a metal thin film is modified on the inverse sea urchin array structure.

[0006] Preferably, the anti-urchin array is composed of a number of accommodating cavities in a two-dimensional hexagonal close-packed structure, and a number of conical spines are provided on the inner cavity wall of the accommodating cavity, and a sphere is provided at the top of the spine.

[0007] Preferably, the metal thin film is a silver thin film, a gold thin film or a copper thin film.

[0008] The present invention also provides a method for preparing a surface-enhanced Raman scattering substrate, which at least includes the following steps:

[0009] 1), forming a monolayer film of polystyrene microspheres on a substrate;

[0010] 2), preparing an inverse opal array structure on the basis of the monolayer film of polystyrene microspheres prepared in step 1);

[0011] 3), preparing an etching layer on the inverse opal array structure prepared in step 2);

[0012] 4), etching to obtain an anti-urchin nanoarray structure;

[0013] 5), coating to obtain a metal-anti-urchin array structure, that is, a surface-enhanced Raman scattering substrate.

[0014] Preferably, the monolayer film of polystyrene microspheres formed on the substrate in step 1) is prepared by an interfacial self-assembly method.

[0015] Preferably, the inverse opal array structure in step 2) is prepared by a template-assisted spin coating method.

[0016] Preferably, the etching layer in step 3) includes a polystyrene layer and a polystyrene microsphere layer, and is sequentially a polystyrene layer and a polystyrene microsphere layer starting from the inverse opal array structure.

[0017] Preferably, the etching in step 4) is carried out by oxygen plasma etching.

[0018] Preferably, the coating in step 5) is carried out by thermal evaporation coating.

[0019] Preferably, the metal in step 5) is gold, silver or copper.

[0020] The present invention also provides a use of the surface-enhanced Raman scattering substrate for detecting the methanol content in blended wine.

[0021] The present invention further provides a method for detecting the methanol content in blended wine, which at least includes the following steps: collecting the Raman spectrum of a blended wine sample by using the surface-enhanced Raman scattering substrate according to any one of claims 1 to 6, and obtaining the relative intensity ratio I of the methanol analysis peak and the ethanol analysis peak of the blended wine sample 甲醇 / I 乙醇, analyze the volume ratio V of methanol to ethanol in the blended wine sample using the standard curve 甲醇 / V 乙醇 , to quantitatively analyze the methanol content in the blended wine.

[0022] Preferably, the standard curve is obtained by the following steps:

[0023] S1. Collect the Raman spectrum of the blended wine standard sample using the surface-enhanced Raman scattering substrate described in any one of claims 1 to 6;

[0024] S2. Perform linear fitting on the relative intensity ratio I of the methanol analysis peak to the ethanol analysis peak in the blended wine standard sample 甲醇 / I 乙醇 and the volume ratio V of methanol to ethanol 甲醇 / V 乙醇 to obtain a linear graph.

[0025] Preferably, the blended wine standard samples in steps S1 and S2 are blended wines with different methanol / ethanol volume ratios V 甲醇 / V 乙醇 .

[0026] Preferably, the methanol analysis peak is the characteristic peak at 2837 cm of methanol; the ethanol analysis peak is the characteristic peak at 2882 cm of ethanol. -1 ; ethanol analysis peak is the characteristic peak at 2882 cm of ethanol -1 .

[0027] As described above, the surface-enhanced Raman scattering substrate of the present invention, its preparation method and uses have the following beneficial effects:

[0028] The surface-enhanced Raman scattering substrate of the present invention has a periodically ordered structure and has high uniformity during actual application.

[0029] The surface-enhanced Raman scattering substrate of the present invention has hydrophobicity, can achieve rapid detection; and has high sensitivity, and the detection limit for rhodamine is 10 -13 M, and can detect trace amounts of methanol in blended wine.

[0030] The preparation method of the surface-enhanced Raman scattering substrate of the present invention uses the template method to prepare a substrate with good repeatability, high sensitivity, large-scale preparation, and no need for expensive equipment.

[0031] The method for detecting the methanol content in blended wine of the present invention solves the great influence of the strong background peak of ethanol on methanol analysis by adopting the analysis method of relative intensity ratio. This method is rapid, simple and reliable. Description of the Drawings

[0032] Figure 1It is a monolayer film of 1.5-μm polystyrene spheres in the preparation process of the surface-enhanced Raman scattering substrate of the present invention. The inset in the upper right corner is a side view.

[0033] Figure 2 It is an inverse opal structure in the preparation process of the surface-enhanced Raman scattering substrate of the present invention. The inset in the upper right corner is a side view.

[0034] Figure 3 It is an electron micrograph of a polystyrene layer coated in the inverse opal structure in the preparation process of the surface-enhanced Raman scattering substrate of the present invention. The inset in the upper right corner is a side view.

[0035] Figure 4 It is an electron micrograph of a monolayer of 250-nm polystyrene spheres assembled in the preparation process of the surface-enhanced Raman scattering substrate of the present invention. The inset in the upper right corner is a side view.

[0036] Figure 5 It is an electron micrograph of the "inverse sea urchin" array structure of the surface-enhanced Raman scattering substrate of the present invention. The inset in the upper right corner is a side view.

[0037] Figure 6 It is an electron micrograph of the "silver-inverse sea urchin" array structure of the electron micrograph of the surface-enhanced Raman scattering substrate of the present invention. The inset in the upper right corner is a side view.

[0038] Figure 7 It is a hydrophilicity-hydrophobicity test chart of the surface-enhanced Raman scattering substrate of the present invention.

[0039] Figure 8 They are the Raman spectra of methanol and ethanol.

[0040] Figure 9 They are the Raman spectra of the blended wine standard samples with the volume ratio V 甲醇 / V 乙醇 being (a) 0.02, (b) 0.015, (c) 0.010, and (d) 0.005.

[0041] Figure 10 They are the Raman spectra of the blended wine with the volume ratio V 甲醇 / V 乙醇 being (a) 0.02, (b) 0.015, (c) 0.010, and (d) 0.005, and the distribution of I 2837 / I 2882 for 10 measurements, where the red line represents the average intensity, and the yellow and green regions represent intensity variations of ±5% and ±5 - 10% respectively.

[0042] Figure 11 They are the volume ratio V 甲醇 / V 乙醇 of methanol and ethanol and the relative intensity ratio I甲醇 / I 乙醇 Linear fitting relationship diagram, where the red dots are the average values of the relative intensities in 10 collected spectra, and the red error bars represent the standard deviations of the relative intensities in 10 spectra.

[0043] Figure 12 This is the detection spectrum of the surface-enhanced Raman scattering substrate of the present invention for different concentrations of the probe molecule rhodamine. Detailed implementation manners

[0044] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0045] Please refer to Figures 1 - 11 . It should be noted that the diagrams provided in this embodiment only illustrate the basic concept of the present invention in a schematic manner. Therefore, only the components related to the present invention are shown in the diagrams, rather than being drawn according to the number, shape, and size of the components in actual implementation. The types, quantities, and proportions of the components in actual implementation can be arbitrarily changed, and the component layout type may also be more complex.

[0046] The first aspect of the present invention provides a surface-enhanced Raman scattering substrate, including an anti-sea urchin array structure, on which a metal thin film is modified.

[0047] The anti-sea urchin array structure in the surface-enhanced Raman scattering substrate of the present invention is composed of a number of accommodating cavities in a two-dimensional hexagonal close-packed structure. A number of conical spines are provided on the inner cavity wall of the accommodating cavity, and spheres are provided at the tops of the spines.

[0048] Among them, the two-dimensional hexagonal close-packed structure refers to the structure formed by taking a single layer of atoms on the basis of the close-packed hexagonal structure. In the present invention, the accommodating cavities are compared to atoms.

[0049] In the surface-enhanced Raman scattering substrate of the present invention, the diameter of the accommodating cavity is 1.5 - 2.0 μm, such as 1.5 - 1.6 μm, 1.6 - 1.7 μm, 1.7 - 1.8 μm, 1.8 - 1.9 μm, or 1.9 - 2.0 μm. In a preferred embodiment of the present invention, the diameter of the accommodating cavity is 1.5 μm. The bottom diameter of the conical spike is 160 nm - 210 nm, such as 160 nm - 170 nm, 170 nm - 180 nm, 180 nm - 190 nm, 190 nm - 200 nm, or 200 nm - 210 nm. In a preferred embodiment of the present invention, the bottom diameter of the conical spike is 180 nm. The height of the conical spike is 100 nm - 150 nm, such as 100 nm - 110 nm, 110 nm - 120 nm, 120 nm - 130 nm, 130 nm - 140 nm, or 140 nm - 150 nm. In a preferred embodiment of the present invention, the height of the conical spike is 120 nm. The density of the accommodating cavities in the two-dimensional hexagonal close-packed structure is 2.9*10 7 ~5.1*10 7 pcs / cm 2 For example, it is 2.9*10 7 ~3.2*10 7 pcs / cm 2 、3.2*10 7 ~3.6*10 7 pcs / cm 2 、3.6*10 7 ~4.0*10 7 pcs / cm 2 、4.0*10 7 ~4.5*10 7 pcs / cm 2 、4.5*10 7 ~5.1*10 7 pcs / cm 2 。In a preferred embodiment of the present invention, the density of the accommodating cavities is 5.1*10 7 pcs / cm 2 。The diameter of the sphere is 50 nm - 100 nm, such as 50 nm - 60 nm, 60 nm - 70 nm, 70 nm - 80 nm, 80 nm - 90 nm, or 90 nm - 100 nm. The material of the accommodating cavity is SiO 2 。

[0050] In the surface-enhanced Raman scattering substrate of the present invention, the metal film modified on the anti-sea urchin array structure is a silver thin film, a gold thin film, or a copper thin film. In a preferred embodiment of the present application, the metal film modified on the anti-sea urchin array structure is a silver thin film.

[0051] The second aspect of the present invention provides a method for preparing a surface-enhanced Raman scattering substrate, which at least includes the following steps:

[0052] 1), forming a monolayer film of polystyrene spheres on a substrate;

[0053] 2), preparing an inverse opal array structure on the basis of the monolayer film of polystyrene spheres prepared in step 1);

[0054] 3), preparing an etching layer on the inverse opal array structure prepared in step 2);

[0055] 4), etching to obtain an inverse sea urchin array structure;

[0056] 5), coating to obtain a metal-inverse sea urchin array structure, that is, a surface-enhanced Raman scattering substrate.

[0057] In the method for preparing the surface-enhanced Raman scattering substrate of the present invention, the monolayer film of polystyrene spheres is prepared by the interfacial self-assembly method. By using the interfacial self-assembly method, the polystyrene spheres are transferred onto the substrate to form a hexagonal close-packed monolayer film of polystyrene spheres. The specific process is as follows: Select a substrate for assembling the monolayer film of polystyrene spheres, and clean and sterilize it before use; then slowly inject the polystyrene sphere emulsion onto the water surface of a petri dish, so that the spheres cover the entire liquid surface, and add an aqueous solution of sodium dodecyl sulfate to induce the formation of a dense monolayer film at the air-water interface; tilt the substrate and immerse it in water, slowly transfer the polystyrene monolayer film to the substrate surface, pick it up and suck off the water layer between the polystyrene spheres and the substrate, and place the substrate at room temperature for drying, thus completing the preparation of the monolayer film of polystyrene spheres.

[0058] Among them, the substrate is selected as a glass substrate. In a preferred embodiment of the present invention, glass with a size of 1 cm * 1 cm is selected as the substrate for assembling the monolayer film of polystyrene spheres. For the cleaning and sterilization of the glass substrate, a glass water cleaner, deionized water, and ethanol are used to clean the glass substrate, preferably ultrasonic cleaning, and the ultrasonic time is preferably 15 minutes; preferably, ultraviolet ozone irradiation is used for sterilization, and the sterilization time is preferably 10 minutes.

[0059] The polystyrene sphere emulsion is obtained by diluting a polystyrene sphere emulsion with a diameter of 1.5 μm and a mass fraction of 5 wt%. In a preferred embodiment of the present invention, the polystyrene sphere emulsion is diluted with ethanol and deionized water according to a volume ratio of 3:5:5 of the polystyrene sphere emulsion, ethanol, and deionized water, and the diluted polystyrene sphere emulsion is dispersed by the ultrasonic method.

[0060] The polystyrene sphere emulsion is slowly injected onto the water surface of a petri dish along a drainage sheet using a syringe. In a preferred embodiment of the present invention, a petri dish with a diameter of 8 cm is selected.

[0061] The sodium dodecyl sulfate aqueous solution can make the polystyrene balls more closely arranged on the water surface of the culture dish. In a preferred embodiment of the present invention, the concentration of the sodium dodecyl sulfate aqueous solution is 1 wt %.

[0062] After the polystyrene monolayer film is transferred to the substrate surface, there is a thick water layer between the freshly picked up polystyrene balls and the glass substrate. This water layer will destroy the self-assembly of the polystyrene balls on the glass. The glass can be slightly tilted and a corner of the glass can be gently touched with a wet paper towel. The paper towel can quickly absorb the water layer between the polystyrene balls and the glass.

[0063] In the preparation method of the surface enhanced Raman scattering substrate of the present invention, the inverse opal array structure is prepared by a template-assisted spin coating method. The specific process is: the prepared tetraethyl orthosilicate precursor solution is dropped on the polystyrene ball monolayer film template, the solution penetrates through the gaps of the polystyrene balls and contacts the underlying substrate, and the spin coating is performed using a spin coater, and then the template is placed on a heating plate and heated to form a highly ordered SiO 2 Inverse opal nanoarray. The template is heated by a hot plate, and the tetraethyl orthosilicate precursor solution is converted into SiO 2 At the same time, the polystyrene beads are thermally decomposed into CO 2 and H 2 In a preferred embodiment of the present invention, the temperature of the heating plate is 500° C. and the heating time is 1 minute.

[0064] The tetraethyl orthosilicate precursor solution is prepared by mixing analytically pure tetraethyl orthosilicate, HCl and ethanol in a volume ratio of 2:2:3 and ultrasonically treating for 30 minutes. In a preferred embodiment of the present invention, the concentration of HCl is 37 wt % and the ethanol is anhydrous ethanol.

[0065] During the spin coater rotation, the precursor solution on the surface of the polystyrene beads is separated from the surface by centrifugal force, leaving only the lower hemisphere of the polystyrene bead array with the precursor solution. In a preferred embodiment of the present invention, the spin coater rotates at a speed of 3000 rpm for 20 seconds.

[0066] In the method for preparing the surface enhanced Raman scattering substrate of the present invention, the inverse opal array structure is bowl-shaped, the diameter of the bowl mouth is consistent with the diameter of the single-layer polystyrene ball template, and the height is half of the diameter.

[0067] In the method for preparing the surface-enhanced Raman scattering substrate of the present invention, the etching layer includes a polystyrene layer and a polystyrene microsphere layer. Starting from the inverse opal array structure, they are a polystyrene layer and a polystyrene microsphere layer in sequence. The specific preparation process of the etching layer is as follows: First, spin-coat a layer of polystyrene solution on the inverse opal structure array, and then, through the interfacial self-assembly method, secondarily assemble a monolayer film of polystyrene microspheres and transfer it to the inverse opal array covered with the polystyrene layer, and that's it.

[0068] Among them, the layer of polystyrene solution spin-coated on the inverse opal structure array is an auxiliary layer for etching. The concentration of the polystyrene solution of the auxiliary layer is 3.0 wt% to 5.0 wt%. In a preferred embodiment of the present invention, the concentration of the polystyrene solution of the auxiliary layer is 3.0 wt%, 3.6 wt%, 4.7 wt%. In a preferred embodiment of the present invention, the concentration of the polystyrene solution of the auxiliary layer is 3.6 wt%. In a preferred embodiment of the present invention, the conditions of the spin coater during spin coating are to rotate at 6000 rmp for 20 s. In a preferred embodiment of the present invention, the thickness of the etching auxiliary layer is 150 nm to 170 nm. In a preferred embodiment of the present invention, the thickness of the etching auxiliary layer is 150 nm.

[0069] The preparation method of the second monolayer film of polystyrene microspheres assembled is the same interfacial self-assembly method as that used in step 1) for preparing the template of the monolayer film of polystyrene microspheres. In a preferred embodiment of the present invention, a polystyrene microsphere emulsion with a diameter of 250 nm and a mass fraction of 5 wt% is used, and it is diluted according to the volume ratio of polystyrene microsphere emulsion: ethanol: deionized water of 3:5:5, and ultrasonically prepared for use.

[0070] In the method for preparing the surface-enhanced Raman scattering substrate of the present invention, the etching is carried out by oxygen plasma etching. The specific process is as follows: The oxygen plasma etching instrument etches the etching layer to form a morphology in which a nanosphere is attached above a nanocone in the bowl of the inverse opal array, that is, an "inverse sea urchin" nanoarray structure.

[0071] Among them, the conditions of the oxygen plasma etcher are an oxygen flow rate of 2 sccm, a power of 40 W, and a frequency of 20 KHz. When treating polystyrene microspheres with an oxygen plasma etcher, excessive power and oxygen flow rate will cause a sharp increase in charged ions in the cavity, resulting in the surface charging of polystyrene microspheres, causing adjacent microspheres to repel or attract each other, and destroying their original morphology. In a preferred embodiment of the present invention, the etching time is 90 min to 120 min. For example, it is 90 min to 100 min, 100 min to 110 min, or 110 min to 120 min. In a preferred embodiment of the present invention, the etching time is 120 min. When etching for 60 min, the polystyrene microspheres at the top are slightly etched, only exposing a small area of the polystyrene layer, and no cone structure is formed. When etching for 150 min, the polystyrene microspheres are completely etched off the single molecular layer, the polystyrene layer is completely exposed, and the etching is thorough, forming sparse nanocones.

[0072] In the method for preparing the surface-enhanced Raman scattering substrate of the present invention, the metal in the metal--anti-sea urchin array structure can be gold, silver, or copper. In a preferred embodiment of the present invention, the metal is silver.

[0073] In the method for preparing the surface-enhanced Raman scattering substrate of the present invention, the coating is performed by thermal evaporation coating. The specific process is as follows: Use a thermal evaporation coater to evaporate a metal film on the "anti-sea urchin" nanoarray structure to obtain a "metal-anti-sea urchin" nanoarray structure template. In a preferred embodiment of the present invention, the metal film is a 40-nm silver thin film. The evaporation rate is The vacuum degree is 8x10 -4 Pa.

[0074] The third aspect of the present invention provides a use of a surface-enhanced Raman scattering substrate, which is used for detecting the methanol content in blended liquor.

[0075] The fourth aspect of the present invention provides a method for detecting the methanol content in blended liquor, which at least includes the following steps:

[0076] Collect the Raman spectrum of the blended liquor sample by using the above surface-enhanced Raman scattering substrate of the present invention, and analyze the volume ratio V of methanol and ethanol in the blended liquor sample through the relative intensity ratio I of the methanol analysis peak and the ethanol analysis peak obtained from the Raman spectrum of the blended liquor sample 甲醇 / I 乙醇 to quantitatively analyze the methanol content in the blended liquor. 甲醇 / V 乙醇

[0077] In the method for detecting the methanol content in blended liquor of the present invention, the standard curve is obtained by the following steps:

[0078] S1. Collect the Raman spectrum of the blended liquor standard sample by using the above surface-enhanced Raman scattering substrate of the present invention;

[0079] S2. For the relative intensity ratio I of the methanol analysis peak and the ethanol analysis peak in the blended wine label sample 甲醇 / I 乙醇 and the volume ratio V of methanol to ethanol 甲醇 / V 乙醇 perform linear fitting to obtain a linear graph.

[0080] In the method for detecting the methanol content in blended wine of the present invention, in steps S1 and S2, the blended wine label sample is a blended wine with different methanol / ethanol volume ratios V 甲醇 / V 乙醇 . Specifically: The blended wine label sample is prepared from 67° Laobaigan sold on the market and analytical pure methanol in the laboratory. Since the methanol content specified in the national safety standard is calculated relative to 100% alcohol, when preparing the methanol-Laobaigan solution, we need to consider the alcohol content of Laobaigan. First, it is necessary to calculate the volume of ethanol contained in 100 μL of the Laobaigan solution: 100 μL * 67% = 67 μL. For the blended wine prepared by mixing 1 mL of methanol with 100 mL of Laobaigan wine, the volume ratio of methanol to Laobaigan is 1:100, and the actual volume ratio of methanol to ethanol is 1:67.

[0081] The specific process of collecting the Raman spectrum of the blended wine label sample using the surface-enhanced Raman scattering substrate is as follows: Place the surface-enhanced Raman scattering substrate prepared by the present invention on the stage of the Raman spectrometer, drop 30 μL of the blended wine label sample on the substrate, and immediately collect the Raman spectrum through the excitation light. In the preferred embodiment of the present invention, the excitation light of 532 nm is used, and the effective power of the laser source is 15 mW. Since both methanol and ethanol are volatile and have different volatilization rates, when testing, we need to quickly collect the Raman spectrum to minimize the inaccuracy of the detection results caused by solvent volatilization.

[0082] In the method for detecting the methanol content in blended wine of the present invention, in steps S1 and S2, the methanol analysis peak is the characteristic peak at 2837 cm -1 of methanol, and the ethanol analysis peak is the characteristic peak at 2882 cm -1 of ethanol. First, it is necessary to collect the Raman spectra of methanol and ethanol to determine the characteristic peaks of methanol and ethanol, so as to further determine the analysis peaks of methanol and ethanol. Both methanol and ethanol are of analytical purity. Through the collected spectra, the characteristic peaks of methanol are located at 1040 cm -1 , 1440 cm -1 , 2837 cm -1 , 2946 cm -1 , and the characteristic peaks of ethanol are located at 886 cm -1 , 1040 cm -1 , 1457 cm -1 , 2882 cm-1 , 2933 cm -1 and 2977 cm -1 . From the appendix Figure 8 it can be seen that the peaks with stronger intensities for both methanol and ethanol are between 2800 cm -1 -3000 cm -1 . Select the characteristic peak at 2837 cm -1 (marked by a red star) as the analysis peak for methanol, which corresponds to the symmetric stretching vibration of -CH 3 . The content of ethanol in the blended wine is much greater than that of methanol, and the strong ethanol signal will affect the subsequent quantitative analysis of methanol by selecting 2837 cm -1 as the analysis peak for methanol. In the blended wine, as the methanol content increases, the intensity of the characteristic peak at 2837 cm -1 will increase, and the relative intensity with the adjacent ethanol peak at 2882 cm -1 will change. Therefore, select the peak at 2882 cm -1 (marked by a blue triangle) as the analysis peak for ethanol, which corresponds to the symmetric stretching vibration of -CH 2 .

[0083] The present invention utilizes a monolayer film of polystyrene spheres to prepare an inverse opal structure, coats and secondarily assembles a polystyrene layer and polystyrene spheres on the inverse opal structure, and then prepares a "reverse sea urchin" periodic nanoarray structure covered with metal through oxygen plasma etching and thermal evaporation coating. Using the template of this structure as a surface-enhanced Raman scattering substrate, the Raman spectrum of the blended wine is collected. And the content of methanol in the blended wine is quantitatively analyzed by analyzing the relative intensity ratio of the characteristic peak of methanol to the characteristic peak of ethanol in the blended wine.

[0084] Example 1

[0085] Prepare a surface-enhanced Raman scattering substrate:

[0086] 1. Select a glass with a size of 1 cm * 1 cm as the substrate for assembling a monolayer of polystyrene spheres. Before use, ultrasonically clean it with glass water cleaner, deionized water, and ethanol for 15 minutes each, and irradiate it with ultraviolet ozone for 10 minutes. Select a commercial polystyrene sphere emulsion with a diameter of 1.5 μm and a mass fraction of 5 wt%. Dilute the polystyrene sphere emulsion with ethanol and deionized water according to a volume ratio of 3:5:5, and then disperse the diluted polystyrene sphere emulsion by ultrasonic method. Slowly inject the diluted polystyrene sphere emulsion onto the water surface of a petri dish with a diameter of 8 cm along the drainage strip using a syringe until the spheres cover the entire liquid surface. Add a few drops of 1 wt% sodium dodecyl sulfate aqueous solution at the notch of the petri dish liquid surface to induce the formation of a dense monolayer film of spheres at the air-water interface. Then tilt the cleaned glass substrate and immerse it in water, and slowly transfer the polystyrene monolayer film to its surface. There is a relatively thick water layer between the freshly picked up polystyrene spheres and the glass substrate, and this water layer will disrupt the self-assembly of polystyrene spheres on the glass. We tilt the glass slightly and gently touch the wet paper towel with a corner of the glass, and the paper towel will quickly absorb the water layer between the polystyrene spheres and the glass. Finally, place the glass vertically and dry it at room temperature to obtain a monolayer film of polystyrene spheres( Figure 1 ).

[0087] 2. Mix analytical pure tetraethyl orthosilicate, 37 wt% HCl, and absolute ethanol together according to a volume ratio of 2:2:3, and prepare a tetraethyl orthosilicate precursor solution after ultrasonic treatment for 30 minutes. Drop 50 μL of the precursor solution on the prepared monolayer film template of polystyrene spheres, and the solution penetrates through the gaps between the polystyrene spheres and contacts the underlying glass. Use a spin coater in a common laboratory environment and spin it at a speed of 3000 rmp for 20 s. During the spinning process, the precursor solution on the surface of the polystyrene spheres is driven away by centrifugal force, and only the precursor solution retained by the lower hemisphere of the polystyrene sphere array remains. Then place the spin-coated template on a hot plate and heat it at a high temperature of 500 °C for 1 minute. The polystyrene thermally decomposes into CO2 and H2O, and the precursor solution is converted into SiO2 to form a bowl-shaped inverse opal array. The diameter of its bowl mouth is the same as the diameter of the monolayer polystyrene sphere template, and the height is half of its diameter( Figure 2 ).

[0088] 3. Add different masses of polystyrene to anhydrous chlorobenzene and stir for 12 h to completely dissolve it to prepare a 3.6 wt% polystyrene solution. Drop 30 μL of the polystyrene solution on the inverse opal array structure, and use a spin coater in a common laboratory environment and spin it at 6000 rmp for 20 s. The thickness of the polystyrene layer is about 150 nm, meeting the requirements for subsequent etching into nano-cones( Figure 3)。Select a commercial polystyrene microsphere emulsion with a diameter of 250 nm and a mass fraction of 5 wt%. Dilute the polystyrene microsphere emulsion, ethanol, and deionized water according to a volume ratio of 3:5:5, and ultrasonically disperse it for later use. Assemble the 250-nm-diameter polystyrene microspheres onto the polystyrene layer of the inverse opal array structure using the same interfacial self-assembly method as for preparing the polystyrene monolayer film template. The 250-nm-diameter polystyrene microspheres are deposited along the curved surface of the inverse opal array structure to form a monolayer ( Figure 4 )。

[0089] 4. Use an oxygen plasma etcher to etch the polystyrene microspheres and the polystyrene layer under the conditions of an oxygen flow rate of 2 sccm, a power of 40 W, and a frequency of 20 kHz. After etching for 120 min, a morphology with a nanosphere attached above the nanocone is formed in the bowl of the inverse opal array, namely, the "inverse sea urchin" nanoarray structure ( Figure 5 )。

[0090] 5. Evaporate a 40-nm silver thin film on the "inverse sea urchin" nanoarray structure using a thermal evaporation coater. The evaporation rate is The vacuum degree is 8x10 -4 Pa, and the "silver-inverse sea urchin" nanoarray structure, that is, the surface-enhanced Raman scattering substrate, is obtained.

[0091] Example 2

[0092] Prepare a surface-enhanced Raman scattering substrate: Compared with Example 1, the difference is that in step 1, a commercial polystyrene microsphere emulsion with a diameter of 2.0 μm and a mass fraction of 5 wt% is selected. Dilute the polystyrene microsphere emulsion, ethanol, and deionized water according to a volume ratio of 3:5:5 for the polystyrene microsphere emulsion, and then disperse the diluted polystyrene microsphere emulsion by the ultrasonic method. The remaining preparation steps are the same as in Example 1.

[0093] Example 3

[0094] Detect the methanol content in blended liquor using the surface-enhanced Raman scattering substrate prepared in Example 1:

[0095] 1. Preparation of blended liquor standard samples:

[0096] Use 67° Laobaigan sold on the market and laboratory analytical pure methanol to prepare blended liquor standard samples. Prepare methanol-Laobaigan blended liquor standard samples with the following volume ratios (see Table 1):

[0097] Table 1

[0098] Methanol / ml Laobaigan / ml V Methanol:V Laobaigan V Methanol:V Ethanol 13.40 1000 0.0134 0.020 10.00 1000 0.0100 0.015 6.70 1000 0.0067 0.010 3.35 1000 0.0034 0.005

[0099] 2. Collect the Raman spectra of the blended liquor standard samples:

[0100] The surface-enhanced Raman scattering substrate prepared in Example 1 was cut into a square of 5 mm * 5 mm with a glass cutter. The substrate was placed on the stage of a Raman spectrometer, and 532 nm excitation light was used with an effective power of the laser source of 15 mW. 30 μL of blended liquor was dropped on the substrate, and the Raman spectrum was immediately collected. The collection time for a single spectrum was 3 s, and the number of times was 3. To avoid the contingency of testing and improve the accuracy of the detection results, 10 spectral collections were performed on the standard samples of 4 volume ratios respectively.

[0101] 3. Determine the methanol analysis peak and ethanol analysis peak:

[0102] Collect the Raman spectra of analytical pure methanol and ethanol in the same way as collecting the blended liquor standard samples in Step 2 to determine their characteristic peaks ( Figure 8 ). Select the characteristic peak at 2837 cm -1 (marked with a red star) as the methanol analysis peak; select the peak at 2882 cm -1 (marked with a blue triangle) as the ethanol analysis peak.

[0103] 4. Analysis of the spectra of blended liquor standard samples:

[0104] Analyze the Raman spectra of the 4 volume ratios of blended liquor in Table 1, and select the relative intensity ratio I 2837 / I 2882 of the methanol peak and ethanol peak to analyze the component content of the blended liquor. To clearly obtain the intensities of the methanol and ethanol characteristic peaks, Gaussian peak fitting was performed on the measured spectra using origin ( Figure 9 ). As Figure 9 , the black spectral line is the Raman spectrum of the actually measured blended liquor, the green is the single-peak spectral line after Gaussian fitting, and the red is the fitted spectral line. Four peaks appear in the figure. The first peak is the characteristic peak of methanol, the second and fourth are the characteristic peaks of ethanol. The peaks at 2946 cm -1 of methanol and 2933 cm -1 of ethanol are too close to be distinguished, so the third peak is considered to be the common characteristic peak of methanol and ethanol. The red star mark is the selected methanol analysis peak, and the blue triangle mark is the selected ethanol analysis peak. As the volume ratio of methanol in the blended liquor decreases, the relative intensity I -1 / I -1 between the characteristic peak at 2837 cm 2837 / I 2882 decreases.

[0105] To avoid the contingency of testing and improve the accuracy of the detection results, 10 spectral collections were performed on the samples of 4 volume ratios respectively, and the relative intensity I 2837 / I2882 Bar chart of distribution ( Figure 10 ). The red line in the figure represents the average intensity of the relative intensity in 10 acquisitions. The yellow and green areas represent the intensity changes relative to the average intensity of ±5% and ±5 - 10% respectively. By calculating the 10 spectra collected, the relative standard deviations of their relative intensities are 5.46%, 10.16%, 2.96%, and 6.42% respectively, all less than 15%, which can well illustrate the scientific nature of the test results.

[0106] 5. Quantitative analysis of methanol content in blended wine samples

[0107] For the relative intensity ratio I 甲醇 / I 乙醇 of the methanol analysis peak and ethanol analysis peak in the blended wine standard sample and the volume ratio V 甲醇 / V 乙醇 a linear fitting is performed to obtain a linear relationship diagram of the volume ratio and the relative intensity ratio ( Figure 11 ). Figure 11 The volume ratio of methanol to ethanol in the blended wine standard sample and the relationship with the relative intensity of I 2837 / I 2882 are given. The red dots in the figure are the average values of the relative intensities in 10 collected spectra, and the red error bars represent the standard deviations of the relative intensities in 10 spectra.

[0108] It can be seen from Figure 11 that within the range of the relatively small volume ratio selected in the present invention, the volume ratio and the relative intensity are positively correlated.

[0109] From the appendix Figure 11It can be known that the detection limits of the surface-enhanced Raman scattering substrate prepared in Example 1 of the present invention for blended liquor are a methanol-to-ethanol ratio of 0.005 and a methanol-to-Laobaigan ratio of 0.0034. Taking the lowest methanol content that can be detected by the present invention as an example, there is 3.4 ml of methanol in each liter of blended liquor. The density of methanol is 0.79, and when converted to mass, there is 2.686 g of methanol in each liter of blended white liquor. When the methanol intake reaches 4 g, it will cause human poisoning. That is, at this detection limit concentration, an adult drinking 1.5 L of this 67° blended white liquor will cause poisoning, 3.7 L will cause blindness, and 8.8 L will cause death. This drinking amount exceeds the normal human's one-time drinking amount, that is, normal drinking of liquor at this detection limit concentration (methanol-to-ethanol ratio of 0.005, methanol-to-Laobaigan ratio of 0.0034) will not cause poisoning or death. If drinking blended liquor with a concentration lower than this limit, drinking more than 1.5 L will cause poisoning, and drinking more than 8.8 L will cause death. If the methanol content in the blended liquor is relatively high, higher than the methanol-to-ethanol ratio of 0.005 (methanol-to-Laobaigan ratio of 0.0034), the detection method of the methanol content in the blended liquor of the present invention can detect the methanol content. Therefore, the method for detecting the methanol content in blended liquor of the present invention has good application value.

[0110] Example 4

[0111] Hydrophobicity experiment of the surface-enhanced Raman scattering substrate:

[0112] A contact angle measuring instrument is used to verify the hydrophilicity and hydrophobicity of the substrate. The specific process is as follows: Place the surface-enhanced Raman scattering substrate prepared in Example 1 on the sample stage of the contact angle measuring instrument, and use the sessile drop method to drop water on the sample surface, controlling the liquid drop volume at 1 - 5 microliters; then, the image of the liquid drop appears on the computer screen and is quickly stored to prevent the subsequent evaporation from affecting the experimental results; finally, the contact angle of the sample is obtained through the measurement and analysis of the contact angle measuring instrument software. ( Figure 7 )

[0113] As can be seen from the appendix Figure 7 it can be known that the contact angle of this substrate is 132.5°, indicating that the surface-enhanced Raman scattering substrate prepared in Example 1 has good hydrophobicity, and the hydrophobic property can achieve rapid detection and reduce the error influence caused by the different volatilization rates of different substances in liquor during the experimental process.

[0114] Example 5

[0115] Determination of the detection limit of the surface-enhanced Raman scattering substrate for the probe molecule rhodamine:

[0116] Using the dye molecule rhodamine as the probe molecule to investigate the sensitivity of the surface-enhanced Raman scattering substrate. The specific process is as follows: The concentration is from 10 -8 M to 10 -13Rhodamine of M was dropped onto the surface-enhanced Raman scattering substrate prepared in Example 1, dried in an oven, and then the substrate adsorbed with probe molecules was placed on the sample stage of a Raman spectrometer, and Raman spectra were collected with a 532 nm laser. As Figure 12 shown.

[0117] It can be seen from the attached Figure 12 that as the concentration of rhodamine decreases, the signal peak intensity decreases until the rhodamine concentration drops to 10 -13 M, and the signal peak intensity is extremely weak. Therefore, we determined that the detection limit of this substrate for rhodamine is 10 -13 M.

[0118] In summary, the surface-enhanced Raman scattering substrate of the present invention has good preparation repeatability, high sensitivity, can be prepared on a large scale, and is fast, simple and reliable when applied to detect the methanol content in blended wine, which is of great significance to the development of food safety. Therefore, the present invention effectively overcomes various disadvantages in the prior art and has high industrial utilization value.

[0119] The above embodiments are only illustrative of the principles and effects of the present invention, and are not used to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A surface-enhanced Raman scattering substrate, characterized in that, it includes an anti-urchin array structure, on which a metal thin film is modified; the anti-urchin array structure is composed of a number of accommodating cavities in a two-dimensional hexagonal close-packed structure, and a number of conical spines are provided on the inner cavity wall of the accommodating cavity, and a sphere is provided at the top of the spine.

2. The surface-enhanced Raman scattering substrate according to claim 1, characterized in that, the metal thin film is a silver thin film, a gold thin film or a copper thin film.

3. The surface-enhanced Raman scattering substrate according to claim 2, characterized in that, the diameter of the accommodating cavity is 1.5 - 2.0 um; and / or, the bottom diameter of the conical spine is 160 nm - 210 nm, and the height is 100 nm - 150 nm; And / or, the density of the accommodating cavities of the two-dimensional hexagonal close-packed structure is 2.9*10 7 ~5.1*10 7 Pieces / cm 2 ; and / or, the diameter of the sphere is 50 - 100 nm; And / or, the material of the accommodating cavity is SiO 2 .

4. The preparation method of the surface-enhanced Raman scattering substrate according to any one of claims 1 - 3, characterized in that, at least includes the following steps: 1), forming a monolayer film of polystyrene microspheres on a substrate; 2), preparing an inverse opal array structure on the basis of the monolayer film of polystyrene microspheres prepared in step 1); 3), preparing an etching layer on the inverse opal array structure prepared in step 2); 4), etching to obtain an anti-urchin array structure; 5), coating to obtain a metal - anti-urchin array structure, that is, a surface-enhanced Raman scattering substrate.

5. The preparation method of the surface-enhanced Raman scattering substrate according to claim 4, characterized in that, the monolayer film of polystyrene microspheres formed on the substrate in step 1) is prepared by the interface self-assembly method; and / or, the inverse opal array structure in step 2) is prepared by the template-assisted spin coating method.

6. The preparation method of the surface-enhanced Raman scattering substrate according to claim 4, characterized in that, the etching layer in step 3) includes a polystyrene layer and a polystyrene microsphere layer, and from the inverse opal array structure, they are the polystyrene layer and the polystyrene microsphere layer in sequence.

7. The preparation method of the surface-enhanced Raman scattering substrate according to claim 4, characterized in that, the etching in step 4) is carried out by oxygen plasma etching; and / or, the coating in step 5) is carried out by thermal evaporation coating; and / or, the metal in step 5) is gold, silver or copper.

8. The use of the surface-enhanced Raman scattering substrate according to any one of claims 1 - 3 is for detecting the methanol content in blended liquor.

9. A method for detecting the methanol content in blended liquor, characterized in that, at least includes the following steps: Collect the Raman spectrum of the blended liquor sample using the surface-enhanced Raman scattering substrate according to any one of claims 1 to 3. By obtaining the relative intensity ratio I 甲醇 / I 乙醇 of the methanol analysis peak and the ethanol analysis peak of the blended liquor sample, analyze the volume ratio V 甲醇 / V 乙醇 of methanol and ethanol in the blended liquor sample using a standard curve to quantitatively analyze the methanol content in the blended liquor.

10. The method for detecting the methanol content in blended liquor according to claim 9, characterized in that, the standard curve is obtained by the following steps: S1, collecting the Raman spectrum of the blended liquor standard sample by using the surface-enhanced Raman scattering substrate according to any one of claims 1 - 3; S2. Analyze the relative intensity ratio I of the methanol analysis peak and the ethanol analysis peak in the blended wine label sample 甲醇 / I 乙醇 and the volume ratio V of methanol and ethanol 甲醇 / V 乙醇 Perform linear fitting to obtain a linear graph.

11. The method for detecting the methanol content in blended liquor according to claim 10, characterized in that, The blended wine label samples described in steps S1 and S2 are blended wines prepared with different methanol / ethanol volume ratios V 甲醇 / V 乙醇 ; and / or, the methanol analysis peak is the characteristic peak at 2837 cm -1 of methanol; the ethanol analysis peak is the characteristic peak at 2882 cm -1 of ethanol.

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