Preparation method and application of SERS substrate for removing fluorescence background

Abstract

The present invention relates to the technical field of SERS substrate preparation, and specifically to a preparation method and application of a SERS substrate for removing fluorescence background. The method comprises dissolving tetrachloroauric acid in water, stirring, and sequentially adding silver nitrate and salicylic acid. After stirring for 1 hour, a gold seed solution is added under ultraviolet light irradiation, and irradiation is continued for 10 hours to obtain a solution of gold long square nanoparticles. The solution is centrifuged and washed several times, and a precipitate is dispersed in ethanol. Two conductive substrates are placed parallel to each other in the dispersion. After power is applied for a period of time, a thin film of gold long square nanoparticles is deposited on the inner side of the conductive substrate connected to the negative electrode. Dilute sulfuric acid and hydrogen peroxide are mixed to form a mixed solution, which is sprayed onto the surface of the thin film of gold long square nanoparticles. The solution is heated and then rinsed with water, and the process is repeated several times to obtain a SERS substrate for removing fluorescence background. The method for removing fluorescence background from a SERS substrate is simple and effective, does not change the morphology of the gold nanoparticles, and enhances the Raman signal intensity of the SERS substrate.

Description

Technical Field

[0001] The present invention relates to the technical field of SERS substrate preparation, in particular to a preparation method and application of a SERS substrate for removing fluorescence background. Background Art

[0002] Raman spectroscopy can identify substances based on molecular vibrations and has great potential in material identification. Traditional Raman scattering spectroscopy has weak signals and is easily interfered by fluorescence background, which is not conducive to the detection of substances. Surface-enhanced Raman spectroscopy (SERS) uses a rough metal structure to prepare a Raman substrate. Under the enhanced effect of localized surface plasmon resonance of nanoscale metal materials, the Raman signal of the analyte molecules will be greatly enhanced, which can realize the detection of trace amounts of substances and has broad prospects in the fields of food safety, environmental testing, and drug testing. However, during the scattering process of photons, only a small number of photons undergo Raman scattering, and their signal intensity is weak, only 10 of the incident laser intensity. -6 ~10 -10 At the same time, the generation of Raman scattering signals is accompanied by fluorescence signals. As an emission process of resonant absorption, the scattering cross section of fluorescence is several orders of magnitude larger than that of Raman scattering, making the fluorescence intensity much greater than the Raman scattering signal. If the influence of the fluorescence background can be eliminated during the test process, the intensity of the Raman signal will be greatly improved, thereby improving the detection sensitivity of the Raman signal.

[0003] In SERS substrates prepared using chemical synthesis techniques, a layer of organic matter inevitably coats the surface of metal nanoparticles during chemical reactions. The intensity of the fluorescence signal is closely related to the thickness of the organic matter on the metal nanoparticle surface. If this organic layer can be removed, allowing the metal surface to directly contact the analyte, the fluorescence signal of the analyte will be quenched due to electron energy transfer. A team from Southeast University published an article in (Scientific Reports 2017, 7(1): 6813). They used acetone immersion to remove organic matter from the surface of gold nanoparticles. However, due to the strong decomposition ability of acetone, it is difficult to precisely control the reaction process, resulting in deformation of the metal nanoparticles and reduced enhancement of the SERS substrate. Chinese patent CN113252637 A (application number: 202110489703.5) discloses a fluorescence background suppression system and method in Raman spectroscopy detection. It mainly modifies the Raman laser and the coded aperture fiber Raman spectroscopy analysis module, and finally uses a mathematical algorithm to perform Raman spectroscopy fluorescence background suppression on the normalized spectral signal data corresponding to each slit array. This method requires modification of the Raman spectrometer, which is relatively costly and is not conducive to the promotion of SERS substrate fluorescence background removal technology in the field of Raman detection. Summary of the Invention

[0004] To overcome the shortcomings of the above-mentioned prior art, the present invention provides a preparation method and application of a SERS substrate for removing fluorescence background. A seed-grown method is used to synthesize gold rectangular nanoparticles with a hotspot effect. These nanoparticles not only exhibit strong localized hotspot intensity at the corners of the nanoparticles, but also, due to their rectangular structure, exhibit multiple absorption peaks in their absorption spectrum curve. This not only enhances the Raman signal by providing more maximum sites, but also exhibits an enhancement effect across a wider wavelength range. Furthermore, a mixed solution of dilute sulfuric acid and hydrogen peroxide is used to etch organic matter from the surface of the gold nanoparticles in the SERS substrate, removing the fluorescence background interference of the SERS substrate without changing the morphology of the gold rectangular nanoparticles. This enhances the intensity of the Raman signal and fundamentally resolves the problem of post-processing Raman signal data caused by the fluorescence background.

[0005] The present invention is specifically implemented by the following technical solutions. According to the present invention, a method for preparing a SERS substrate for removing fluorescence background specifically comprises the following steps:

[0006] (1) Preparation of gold seed solution

[0007] Dissolve tetrachloroauric acid in deionized water to prepare a tetrachloroauric acid solution with a concentration of 0.8-1 g / L; dissolve cetyltrimethylammonium chloride in deionized water to prepare a cetyltrimethylammonium chloride solution with a concentration of 70-75 g / L; dissolve potassium borohydride in deionized water to prepare a potassium borohydride solution with a concentration of 0.56-0.8 g / L; thoroughly mix the obtained tetrachloroauric acid solution and cetyltrimethylammonium chloride solution in equal volumes, stir for 10-30 minutes, then add the potassium borohydride solution all at once and continue stirring at room temperature for 100 minutes to obtain a gold seed solution; the volume ratio of the added potassium borohydride solution to the tetrachloroauric acid solution is 1:8;

[0008] (2) Preparation of gold tetragonal nanoparticles

[0009] Add tetrachloroauric acid to a certain volume of deionized water, stir for 30 minutes, add silver nitrate thereto, continue stirring for 30 minutes, then add salicylic acid thereto, continue stirring for 1 hour, add the gold seed solution prepared in step (1) thereto under 100 W ultraviolet light irradiation, stir evenly, and then continue irradiating with ultraviolet light for 10 hours to obtain a gold tetragonal nanoparticle solution;

[0010] In this step, the mass ratio of deionized water to tetrachloroauric acid, silver nitrate, and salicylic acid is 50 mL: 190 mg: 0.2 mg: 20 mg, and the volume ratio of the added gold seed solution to the volume of deionized water in this step is 1:100;

[0011] (3) Preparation of SERS substrate

[0012] (3.1) After centrifuging the gold tetragonal nanoparticle solution prepared in step (2), the supernatant is removed, deionized water is added to the precipitate, and the solution is centrifuged again. The solution is washed by repeated centrifugation to obtain pure gold tetragonal nanoparticles; the pure gold tetragonal nanoparticles are uniformly dispersed in an ethanol solution to obtain a dispersion A, two conductive substrates of the same size are placed vertically and parallel in the dispersion A, the two conductive substrates are respectively connected to the positive and negative electrodes of a DC power supply by wires, and the two opposite surfaces of the two conductive substrates are defined as the inner side surfaces of the conductive substrates. After power is applied for a period of time, a thin film of gold tetragonal nanoparticles is deposited on the inner side surface of the conductive substrate connected to the negative electrode of the power supply;

[0013] (3.2) Mix dilute sulfuric acid and hydrogen peroxide solution to obtain a mixed solution, and evenly spray the mixed solution on the surface of the gold tetragonal nanoparticle film prepared in step (3.1). Then, place the conductive substrate on a heating table for heat treatment (with the gold tetragonal nanoparticle film facing up), and then rinse the conductive substrate with deionized water; then continue to spray the mixed solution of dilute sulfuric acid and hydrogen peroxide on the surface of the gold tetragonal nanoparticle film on the conductive substrate, place it on a heating table for heat treatment, and rinse the conductive substrate again with deionized water. Repeat the spraying-rinsing cycle 5 to 8 times, and the organic matter on the surface of the gold tetragonal nanoparticles is removed to obtain a SERS substrate with the fluorescence background removed.

[0014] In the aforementioned method for preparing a SERS substrate for removing fluorescence background, the length of the gold long square nanoparticles in the gold long square nanoparticle solution obtained in step (2) is about 25-60 nm, and the width is about 15-25 nm.

[0015] In the aforementioned method for preparing a SERS substrate for removing fluorescence background, in step (3.1), the gold long square nanoparticles are centrifuged and washed 3 to 5 times, the mass concentration of the ethanol solution is 99.5%, and the volume ratio of the ethanol solution used to the deionized water in step (2) is 1:2.

[0016] In the aforementioned method for preparing a SERS substrate for removing fluorescence background, in step (3.1), the conductive substrate is one of conductive glass, copper plate, and aluminum plate, and the two conductive substrates are placed opposite each other; the conductive glass is single-sided conductive, and the conductive surfaces of the two conductive glasses are placed opposite each other.

[0017] In the aforementioned method for preparing a SERS substrate for removing fluorescence background, in step (3.1), the voltage of the DC power supply is 10-20 V, and the power-on time is 10-30 min.

[0018] In the aforementioned method for preparing a SERS substrate for removing fluorescence background, in step (3.2), the volume ratio of the mixture of dilute sulfuric acid and hydrogen peroxide solution is 2:1 to 10:1.

[0019] Furthermore, the volume concentration of the dilute sulfuric acid is 75%, and the mass concentration of the hydrogen peroxide solution is 30%. The dilute sulfuric acid is prepared by mixing concentrated sulfuric acid (mass concentration ≥ 98%) and deionized water in a volume ratio of 3:1.

[0020] In the aforementioned method for preparing a SERS substrate for removing fluorescence background, in step (3.2), the temperature of the heating treatment is 50-80°C.

[0021] In the aforementioned method for preparing a SERS substrate for removing fluorescence background, in step (3.2), the number of times a mixed solution of dilute sulfuric acid and hydrogen peroxide is sprayed onto the surface of the gold long square nanoparticle film is 5 to 8 times, and the ratio of the volume of the mixed solution of dilute sulfuric acid and hydrogen peroxide sprayed each time to the area of ​​the inner side of the conductive substrate is 0.5 mL: 1 cm 2 .

[0022] The present invention also provides an application of a SERS substrate obtained by the above-mentioned preparation method and having a fluorescence background removed therefrom in wastewater detection, in particular in detecting rhodamine 6G in wastewater.

[0023] Compared with the prior art, the present invention has obvious advantages and beneficial effects. By means of the above technical solution, the present invention can achieve considerable technological advancement and practicality, and has a wide range of utilization value. It has at least the following advantages:

[0024] The present invention adopts the seed growth method to synthesize a kind of gold rectangular nanoparticles with hotspot effect. This structure not only has strong local hotspot intensity at the corners of the nanoparticles, but also because of its rectangular structure, its absorption spectrum curve will have multiple absorption peaks, thereby not only bringing more maximum sites to the enhancement of Raman signals, but also having an enhancement effect in a wider band range.

[0025] On this basis, the present invention provides a method for removing the fluorescence background in the SERS substrate, using a mixed solution of dilute sulfuric acid and hydrogen peroxide to etch the organic matter on the surface of the gold long square nanoparticles in the SERS substrate. Under the acidic condition of dilute sulfuric acid, the electrode potential of hydrogen peroxide will increase, thereby improving the etching ability of hydrogen peroxide on organic matter. In addition, the H +The etching rate of organic matter by hydrogen peroxide can be reduced, preventing the morphology of the gold tetragonal nanoparticles from being altered by the rapid etching of hydrogen peroxide. The etching rate can be controlled by adjusting the volume ratio of the dilute sulfuric acid to hydrogen peroxide solution, preventing the surface of the gold nanoparticles from being etched simultaneously with the organic matter, thus ensuring that the morphology of the gold nanoparticles remains unchanged.

[0026] Compared to traditional methods that remove fluorescence background by modifying the Raman spectrometer, the present invention's fluorescence background removal method is much simpler. It only requires etching the organic matter on the SERS substrate surface, and then utilizes electron transfer between the metal surface and the analyte in direct contact to achieve fluorescence quenching. Because there is a conflicting relationship between Raman spectral intensity and fluorescence background, removing the fluorescence background further enhances the Raman signal intensity, fundamentally resolving the post-processing issues associated with the fluorescence background. While maintaining the original rectangular morphology of the gold nanoparticles, it also completely removes the organic matter on their surface, resulting in a Raman signal spectrum free of fluorescence background interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a transmission electron microscope (TEM) image of the gold tetragonal nanoparticle solution prepared in step (2) of Example 1.

[0028] Figure 2 This is the absorption spectrum curve of the gold tetragonal nanoparticle solution prepared in step (2) of Example 1 in the range of 400-1100 nm.

[0029] Figure 3 This is a scanning electron microscope (SEM) image of the surface of the SERS substrate prepared in Example 1 with the fluorescence background removed.

[0030] Figure 4 This is a Raman spectrum of the SERS substrate (sample A) without removing the fluorescence background in the control example.

[0031] Figure 5 This is the Raman spectrum of the SERS substrate (sample B) after removing the fluorescence background in Example 1.

[0032] Figure 6 Schematic diagram of the electron transfer process when the metal surface directly contacts the analyte R6G dye.

[0033] Figure 7 Schematic diagram of the electron transfer process when the metal coated with organic matter comes into contact with the analyte R6G dye. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0035] The present invention is described in detail below with reference to specific examples. In the following examples, if no specific conditions are specified, the experiments were carried out according to conventional conditions or those recommended by the manufacturer. Raw materials and reagents used, if the manufacturer is not specified, are all commercially available conventional products. Example 1

[0036] (1) Preparation of gold seed solution:

[0037] Place 20 mL of deionized water in a 50 mL beaker A, add 0.016 g of tetrachloroauric acid, and stir magnetically at 500 rpm for about 30 min until the solution is completely dissolved to obtain a tetrachloroauric acid solution.

[0038] Place 20 mL of deionized water in a 50 mL beaker B, add 1.456 g of hexadecyltrimethylammonium chloride (CTAC), and stir magnetically at 700 rpm for about 30 min until the CTAC is completely dissolved to obtain a CTAC solution.

[0039] Place 2.5 mL of deionized water in a 5 mL beaker C, add 1.4 mg of potassium borohydride, place beaker C in an ice bath, and magnetically stir the mixture at 500 rpm for about 1 h to obtain a potassium borohydride solution.

[0040] Thoroughly mix 20 mL of tetrachloroauric acid solution and 20 mL of CTAC solution, stir for about 10 min, then add 2.5 mL of prepared potassium borohydride solution all at once and continue stirring at room temperature for 100 min to obtain a gold seed solution;

[0041] (2) Preparation of gold tetragonal nanoparticles:

[0042] 50 mL of deionized water was placed in a 100 mL beaker, and 190 mg of tetrachloroauric acid was added thereto while stirring rapidly. After stirring for 30 min, 0.2 mg of silver nitrate was added thereto, and stirring was continued for 30 min. Then, 20 mg of salicylic acid was added thereto. After continuous stirring for 1 h, 500 μL of the gold seed solution prepared in step (1) was added thereto under 100 W ultraviolet light irradiation, and the mixture was stirred evenly. Then, ultraviolet light was continued to be irradiated for 10 h to obtain a gold tetragonal nanoparticle solution.

[0043] (3) Preparation of surface-enhanced Raman scattering substrate:

[0044] (3.1) After centrifuging the gold tetragonal nanoparticle solution prepared in step (2), the supernatant was removed, deionized water was added to the precipitate after centrifugation, and the mixture was centrifuged again. The centrifugation and washing were repeated three times to obtain pure gold tetragonal nanoparticles. The pure gold tetragonal nanoparticles were uniformly dispersed in 25 mL of 99.5% ethanol solution, and then two conductive glasses (1 cm × 3 cm) of the same size were placed vertically and parallel in the ethanol dispersion of the gold tetragonal nanoparticles. The conductive glasses were single-sided conductive, and the conductive surfaces of the two conductive glasses were placed opposite each other (the conductive surfaces were the inner surfaces). The two conductive glasses were connected to the positive and negative poles of a DC power supply with wires, respectively. Under the action of a 15V voltage, after 20 minutes of power supply, a thin film of gold tetragonal nanoparticles was deposited on the conductive surface of the conductive glass connected to the negative pole of the power supply.

[0045] (3.2) Dilute sulfuric acid with a volume concentration of 75% and hydrogen peroxide solution with a mass concentration of 30% were mixed in a volume ratio of 4:1 to obtain a mixed solution. 1.5 mL of the mixed solution was evenly sprayed on the surface of the gold tetragonal nanoparticle film prepared in step (3.1) with the help of a nanosprayer. The film was then placed on a heating table at 60°C for heating treatment (with the gold tetragonal nanoparticle film facing upward). Under the influence of temperature, the dilute sulfuric acid and hydrogen peroxide in the mixed solution etched the organic matter on the surface of the gold tetragonal nanoparticles for 10 min. The conductive glass was then rinsed with deionized water; then a mixed solution of dilute sulfuric acid and hydrogen peroxide was sprayed on the surface of the gold tetragonal nanoparticle film, and the film was placed on a heating table and heated to 60°C. After etching for 10 minutes, the conductive glass was rinsed with deionized water again. The spraying-rinsing cycle was repeated 5 times, with the volume of the mixed solution of dilute sulfuric acid and hydrogen peroxide sprayed each time being 1.5 mL. This removed the organic matter on the surface of the gold tetragonal nanoparticles and obtained a SERS substrate with the fluorescence background removed.

[0046] Figure 1 This is a transmission electron microscope (TEM) image of the gold tetragonal nanoparticle solution prepared in step (2) of Example 1. It can be clearly seen that the gold nanoparticles are in the shape of a tetragon, with a length of about 25 to 60 nm and a width of about 15 to 25 nm.

[0047] Figure 2 FIG1 is the absorption spectrum curve of the gold tetragonal nanoparticle solution prepared in step (2) of Example 1 in the range of 400-1100 nm. It can be seen that the gold tetragonal nanoparticles have three absorption peaks in the range of 400-1100 nm, and the maximum absorption wavelength is 850 nm.

[0048] Figure 3 This is a scanning electron microscope (SEM) image of the SERS substrate prepared in Example 1 with the fluorescence background removed. It can be seen that the rectangular shape of the gold nanoparticles is not changed after the organic layer on the surface of the gold nanoparticles is removed.

[0049] Comparative Example:

[0050] The conductive glass deposited with a thin film of gold elongated tetragonal nanoparticles prepared in step (3.1) of Example 1 was used as a comparative example. The thin film of gold elongated tetragonal nanoparticles in this comparative example was not etched with a mixed solution of dilute sulfuric acid and hydrogen peroxide, so the SERS substrate obtained was not free of fluorescent background, and was recorded as sample A.

[0051] The SERS substrate of the comparative example (sample A) was compared with the SERS substrate with fluorescence background removed prepared in step (3.2) of Example 1 (denoted as sample B) to verify the Raman signal enhancement effect of the SERS substrate prepared in Example 1, as follows:

[0052] Rhodamine 6G (R6G) dye was dissolved in a methanol solution to obtain an R6G dye solution, wherein the mass concentration of the methanol solution was 99% and the concentration of R6G in the methanol solution was 1×10 -9 mol / L. Take a piece of sample A and a piece of sample B respectively and immerse them vertically in the above R6G dye solution (vertical immersion is adopted for the convenience of placement and removal, horizontal immersion is also possible) for 3 hours. Then, take out sample A and sample B and dry them at room temperature. The dried samples A and sample B are tested for Raman spectra using a Raman spectrometer.

[0053] Figure 4 This is the Raman spectrum test result of the SERS substrate (sample A) without removing the fluorescence background. Figure 5 This is the Raman spectrum test result of the SERS substrate (sample B) with the fluorescence background removed in Example 1. Figure 4 and Figure 5 It can be seen that the fluorescence background-removed SERS substrate (sample B) prepared in Example 1 has its fluorescence background removed for the Raman spectrum of the R6G dye, while the SERS substrate (sample A) in the comparative example, which was not etched with a mixed solution of dilute sulfuric acid and hydrogen peroxide, has a strong fluorescence background in the Raman spectrum of the R6G dye due to the organic matter on the surface of the gold nanoparticles. In this case, data post-processing is generally required to remove the fluorescence background. In addition, there is a contradictory relationship between the Raman spectrum intensity and the fluorescence background. The smaller the fluorescence background, the stronger the Raman peak obtained. Figure 5 shown.

[0054] Figure 6This is a schematic diagram of the electron transfer process when the metal surface directly contacts the analyte R6G dye. Figure 7 This is a schematic diagram of the electron transfer process when the metal coated with organic matter comes into contact with the analyte R6G dye. It can be seen that when the metal surface is in direct contact with the analyte, the electrons will jump from the conduction band to the molecular surface and quench, and no fluorescence will be generated. Figure 6 When organic matter exists on the metal surface, the organic layer will block the electrons from jumping from the conduction band to the metal surface, and the electrons can only jump to the valence band, thus generating fluorescence, as shown in Figure 2. Figure 7 shown.

[0055] The above comparative tests show that the method of the present invention can effectively remove the fluorescence background of the SERS substrate without changing the shape of the gold nanoparticles. In addition, the present invention prepares gold nanoparticles with a rectangular shape. This structure not only has a strong local hot spot intensity at the corners of the nanoparticles, but also has multiple absorption peaks in its absorption spectrum curve due to its rectangular structure (such as Figure 2 As shown in Figure 2, the enhancement of Raman signals not only brings more maximum sites but also has an enhancement effect in a wider wavelength range. Example 2

[0056] (1) Preparation of gold seed solution, same as step (1) of Example 1;

[0057] (2) Preparation of gold tetragonal nanoparticles, same as step (2) of Example 1;

[0058] (3) Preparation of surface-enhanced Raman scattering substrate:

[0059] (3.1) After centrifuging the gold tetragonal nanoparticle solution prepared in step (2), the supernatant was removed, deionized water was added to the precipitate, and the mixture was centrifuged again. The centrifugal washing was repeated 4 times to obtain pure gold tetragonal nanoparticles. The pure gold tetragonal nanoparticles were uniformly dispersed in 25 mL of 99.5% ethanol solution. Two pieces of conductive glass (1 cm × 3 cm) of the same size were placed vertically and parallel in the ethanol dispersion of the gold tetragonal nanoparticles. The conductive glass was single-sided conductive, and the conductive surfaces of the two pieces of conductive glass were placed opposite each other (the conductive surfaces were the inner sides). The two pieces of conductive glass were connected to the positive and negative poles of a DC power supply with wires, respectively. Under the action of a 20 V voltage, after 15 minutes of power supply, a thin film of gold tetragonal nanoparticles was deposited on the conductive surface of the conductive glass connected to the negative pole of the power supply.

[0060] (3.2) Dilute sulfuric acid with a volume concentration of 75% and hydrogen peroxide solution with a mass concentration of 30% were mixed in a volume ratio of 2:1 to obtain a mixed solution. 1.5 mL of the mixed solution was evenly sprayed on the surface of the gold tetragonal nanoparticle film prepared in step (3.1) with the help of a nanosprayer, and then placed on a heating table at 70°C for heating treatment (the gold tetragonal nanoparticle film was facing up). Under the influence of temperature, the dilute sulfuric acid and hydrogen peroxide in the mixed solution etched the organic matter on the surface of the gold tetragonal nanoparticles for 10 min, and then the conductive glass was rinsed with deionized water; then the mixed solution of dilute sulfuric acid and hydrogen peroxide was continued to be sprayed on the surface of the gold tetragonal nanoparticle film, and placed on a heating table and heated to 70°C for 10 min. The conductive glass was rinsed with deionized water again. The spraying-rinsing cycle was repeated 7 times, and the volume of the mixed solution of dilute sulfuric acid and hydrogen peroxide sprayed each time was 1.5 mL. The organic matter on the surface of the gold tetragonal nanoparticles was removed to obtain a SERS substrate with removed fluorescence background. Example 3

[0061] (1) Preparation of gold seed solution, same as step (1) of Example 1;

[0062] (2) Preparation of gold tetragonal nanoparticles, same as step (2) of Example 1;

[0063] (3) Preparation of surface-enhanced Raman scattering substrate:

[0064] (3.1) After centrifuging the gold tetragonal nanoparticle solution prepared in step (2), the supernatant was removed, deionized water was added to the precipitate, and the mixture was centrifuged again. The centrifugal washing was repeated 5 times to obtain pure gold tetragonal nanoparticles. The pure gold tetragonal nanoparticles were uniformly dispersed in 25 mL of 99.5% ethanol solution. Two pieces of conductive glass (1 cm × 3 cm) of the same size were placed vertically and parallel in the ethanol dispersion of the gold tetragonal nanoparticles. The conductive glass was single-sided conductive, and the conductive surfaces of the two pieces of conductive glass were placed opposite each other (the conductive surfaces were the inner sides). The two pieces of conductive glass were connected to the positive and negative poles of a DC power supply with wires, respectively. Under the action of a 10 V voltage, after 30 minutes of power supply, a thin film of gold tetragonal nanoparticles was deposited on the conductive surface of the conductive glass connected to the negative pole of the power supply.

[0065] (3.2) Dilute sulfuric acid with a volume concentration of 75% and hydrogen peroxide solution with a mass concentration of 30% were mixed in a volume ratio of 6:1 to obtain a mixed solution. 1.5 mL of the mixed solution was evenly sprayed on the surface of the gold tetragonal nanoparticle film prepared in step (3.1) with the help of a nanosprayer, and then placed on a heating table at 50°C for heating treatment (the gold tetragonal nanoparticle film was facing up). Under the influence of temperature, the dilute sulfuric acid and hydrogen peroxide in the mixed solution etched the organic matter on the surface of the gold tetragonal nanoparticles for 10 min, and then the conductive glass was rinsed with deionized water; then the mixed solution of dilute sulfuric acid and hydrogen peroxide was continued to be sprayed on the surface of the gold tetragonal nanoparticle film, and heated to 50°C, etched for 10 min, and the conductive glass was rinsed with deionized water again. The spraying-rinsing cycle was repeated 5 times, and the volume of the mixed solution of dilute sulfuric acid and hydrogen peroxide sprayed each time was 1.5 mL, so that the organic matter on the surface of the gold tetragonal nanoparticles could be removed to obtain a SERS substrate with removed fluorescence background. Example 4

[0066] (1) Preparation of gold seed solution, same as step (1) of Example 1;

[0067] (2) Preparation of gold tetragonal nanoparticles, same as step (2) of Example 1;

[0068] (3) Preparation of surface-enhanced Raman scattering substrate:

[0069] (3.1) After centrifuging the gold tetragonal nanoparticle solution prepared in step (2), the supernatant was removed, deionized water was added to the precipitate, and the mixture was centrifuged again. The centrifugal washing was repeated 4 times to obtain pure gold tetragonal nanoparticles. The pure gold tetragonal nanoparticles were uniformly dispersed in 25 mL of 99.5% ethanol solution. Two pieces of conductive glass (1 cm × 3 cm) of the same size were placed vertically and parallel in the ethanol dispersion of the gold tetragonal nanoparticles. The conductive glass was single-sided conductive, and the conductive surfaces of the two pieces of conductive glass were placed opposite each other (the conductive surfaces were the inner sides). The two pieces of conductive glass were connected to the positive and negative poles of a DC power supply with wires, respectively. Under the action of a 15 V voltage, after 20 minutes of power supply, a thin film of gold tetragonal nanoparticles was deposited on the conductive surface of the conductive glass connected to the negative pole of the power supply.

[0070] (3.2) Dilute sulfuric acid with a volume concentration of 75% and hydrogen peroxide solution with a mass concentration of 30% were mixed in a volume ratio of 8:1 to obtain a mixed solution. 1.5 mL of the mixed solution was evenly sprayed on the surface of the gold tetragonal nanoparticle film prepared in step (3.1) with the help of a nanosprayer, and then placed on a heating table at 60°C for heating treatment (the gold tetragonal nanoparticle film was facing up). Under the influence of temperature, the dilute sulfuric acid and hydrogen peroxide in the mixed solution etched the organic matter on the surface of the gold tetragonal nanoparticles for 10 minutes, and then the conductive glass was rinsed with deionized water; then the mixed solution of dilute sulfuric acid and hydrogen peroxide was continued to be sprayed on the surface of the gold tetragonal nanoparticle film, and heated to 60°C, etched for 10 minutes, and the conductive glass was rinsed with deionized water again. The spraying-rinsing cycle was repeated 6 times, and the volume of the mixed solution of dilute sulfuric acid and hydrogen peroxide sprayed each time was 1.5 mL. The organic matter on the surface of the gold tetragonal nanoparticles was removed to obtain a SERS substrate with removed fluorescence background. Example 5

[0071] (1) Preparation of gold seed solution, same as step (1) of Example 1;

[0072] (2) Preparation of gold tetragonal nanoparticles, same as step (2) of Example 1;

[0073] (3) Preparation of surface-enhanced Raman scattering substrate:

[0074] (3.1) After centrifuging the gold tetragonal nanoparticle solution prepared in step (2), the supernatant was removed, deionized water was added to the precipitate, and the mixture was centrifuged again. The centrifugation was repeated 3 times to obtain pure gold tetragonal nanoparticles. The pure gold tetragonal nanoparticles were uniformly dispersed in 25 mL of 99.5% ethanol solution. Two pieces of conductive glass (1 cm × 3 cm) of the same size were placed vertically and parallel in the ethanol dispersion of the gold tetragonal nanoparticles. The conductive glass was single-sided conductive, and the conductive surfaces of the two pieces of conductive glass were placed opposite each other (the conductive surfaces were the inner sides). The two pieces of conductive glass were connected to the positive and negative poles of a DC power supply with wires, respectively. Under the action of a 20 V voltage, after 15 minutes of power supply, a thin film of gold tetragonal nanoparticles was deposited on the conductive surface of the conductive glass connected to the negative pole of the power supply.

[0075] (3.2) Dilute sulfuric acid with a volume concentration of 75% and hydrogen peroxide solution with a mass concentration of 30% were mixed in a volume ratio of 10:1 to obtain a mixed solution. 1.5 mL of the mixed solution was evenly sprayed on the surface of the gold tetragonal nanoparticle film prepared in step (3.1) with the help of a nanosprayer, and then placed on a heating table at 60°C for heating treatment (the gold tetragonal nanoparticle film was facing up). Under the influence of temperature, the dilute sulfuric acid and hydrogen peroxide in the mixed solution etched the organic matter on the surface of the gold tetragonal nanoparticles for 10 min, and then the conductive glass was rinsed with deionized water; then the mixed solution of dilute sulfuric acid and hydrogen peroxide was continued to be sprayed on the surface of the gold tetragonal nanoparticle film, and heated to 60°C, etched for 10 min, and the conductive glass was rinsed with deionized water again. The spraying-rinsing cycle was repeated 5 times, and the volume of the mixed solution of dilute sulfuric acid and hydrogen peroxide sprayed each time was 1.5 mL, so that the organic matter on the surface of the gold tetragonal nanoparticles could be removed to obtain a SERS substrate with removed fluorescence background. Example 6

[0076] The conductive glass was replaced with a copper plate. The two vertically parallel copper plates in step (3.1) were connected to the positive and negative electrodes of a DC power supply with wires, respectively. A potential difference was formed between the two opposing surfaces of the two copper plates. These two opposing surfaces were defined as the inner side surfaces of the copper plates. Finally, a thin film of gold long square nanoparticles was deposited on the inner side surface of the copper plate connected to the negative electrode of the power supply. The remaining operations were the same as in Example 1, and a SERS substrate with the fluorescence background removed was finally obtained. Example 7

[0077] The conductive glass was replaced with an aluminum plate. The two vertically parallel aluminum plates in step (3.1) were connected to the positive and negative electrodes of a DC power supply with wires, respectively, to form a potential difference between the two opposing surfaces of the two aluminum plates. These two opposing surfaces were defined as the inner side surfaces of the aluminum plates. Finally, a thin film of gold long square nanoparticles was deposited on the inner side surface of the aluminum plate connected to the negative electrode of the power supply. The remaining operations were the same as in Example 1, and a SERS substrate with the fluorescence background removed was finally obtained.

[0078] The above description is merely an embodiment of the present invention and does not constitute any form of limitation to the present invention. The present invention may also have other forms of embodiments based on the above structures and functions, which are not listed here one by one. Therefore, any simple modification, equivalent changes, and modifications made to the above embodiments by any person skilled in the art in accordance with the technical essence of the present invention without departing from the scope of the technical solution of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a SERS substrate for removing fluorescence background, characterized in that: The following steps are involved: (1) Preparation of gold seed solution Dissolve tetrachloroauric acid in deionized water to prepare a tetrachloroauric acid solution with a concentration of 0.8-1 g / L; dissolve cetyltrimethylammonium chloride in deionized water to prepare a cetyltrimethylammonium chloride solution with a concentration of 70-75 g / L; dissolve potassium borohydride in deionized water to prepare a potassium borohydride solution with a concentration of 0.56-0.8 g / L; thoroughly mix the obtained tetrachloroauric acid solution and cetyltrimethylammonium chloride solution in equal volumes, stir for 10-30 minutes, then add the potassium borohydride solution all at once and continue stirring at room temperature for 100 minutes to obtain a gold seed solution; the volume ratio of the added potassium borohydride solution to the tetrachloroauric acid solution is 1:8; (2) Preparation of gold tetragonal nanoparticles Add tetrachloroauric acid to a certain volume of deionized water, stir for 30 minutes, add silver nitrate thereto, continue stirring for 30 minutes, then add salicylic acid thereto, continue stirring for 1 hour, add the gold seed solution prepared in step (1) thereto under 100 W ultraviolet light irradiation, stir evenly, and then continue irradiating with ultraviolet light for 10 hours to obtain a gold tetragonal nanoparticle solution; In this step, the mass ratio of deionized water to tetrachloroauric acid, silver nitrate, and salicylic acid is 50 mL: 190 mg: 0.2 mg: 20 mg, and the volume ratio of the added gold seed solution to the volume of deionized water in this step is 1:100; (3) Preparation of SERS substrate (3.1) After centrifuging the gold tetragonal nanoparticle solution prepared in step (2), the supernatant is removed, deionized water is added to the precipitate, and the solution is centrifuged again. The solution is washed by repeated centrifugation to obtain pure gold tetragonal nanoparticles; the pure gold tetragonal nanoparticles are uniformly dispersed in an ethanol solution to obtain a dispersion A, two conductive substrates of the same size are placed vertically and parallel in the dispersion A, the two conductive substrates are respectively connected to the positive and negative electrodes of a DC power supply by wires, and the two opposite surfaces of the two conductive substrates are defined as the inner side surfaces of the conductive substrates. After power is applied for a period of time, a thin film of gold tetragonal nanoparticles is deposited on the inner side surface of the conductive substrate connected to the negative electrode of the power supply; (3.2) Dilute sulfuric acid and hydrogen peroxide solution are mixed to obtain a mixed solution, and the mixed solution is evenly sprayed on the surface of the gold elongated tetragonal nanoparticle film prepared in step (3.1), and then the conductive substrate is placed on a heating table for heating treatment, and then the conductive substrate is rinsed with deionized water; the mixed solution is continued to be sprayed on the surface of the gold elongated tetragonal nanoparticle film on the conductive substrate, and the conductive substrate is placed on a heating table for heating treatment, and the conductive substrate is rinsed with deionized water again. This cycle is repeated 5 to 8 times, and the organic matter on the surface of the gold elongated tetragonal nanoparticles is removed to obtain a SERS substrate with the fluorescence background removed.

2. The method for preparing a SERS substrate for removing fluorescence background according to claim 1, wherein: The length of the gold long tetragonal nanoparticles in the gold long tetragonal nanoparticle solution obtained in step (2) is 25-60 nm, and the width is 15-25 nm.

3. The method for preparing a SERS substrate for removing fluorescence background according to claim 1, wherein: In step (3.1), the gold long square nanoparticles are centrifugally washed 3 to 5 times, the mass concentration of the ethanol solution is 99.5%, and the volume ratio of the ethanol solution used to the deionized water in step (2) is 1:

2.

4. The method for preparing a SERS substrate for removing fluorescence background according to claim 1, wherein: In step (3.1), the conductive substrate is one of conductive glass, copper plate, and aluminum plate, and two conductive substrates are placed opposite to each other.

5. The method for preparing a SERS substrate for removing fluorescence background according to claim 1, wherein: In step (3.1), the voltage of the DC power supply is 10~20V, and the power-on time is 10~30 minutes.

6. The method for preparing a SERS substrate for removing fluorescence background according to claim 1, wherein: In step (3.2), the volume ratio of the mixture of dilute sulfuric acid and hydrogen peroxide solution is 2:1 to 10:

1.

7. The method for preparing a SERS substrate for removing fluorescence background according to claim 1 or 6, wherein: The volume concentration of dilute sulfuric acid is 75%, and the mass concentration of hydrogen peroxide solution is 30%.

8. The method for preparing a SERS substrate for removing fluorescence background according to claim 1, wherein: In step (3.2), the temperature of the heating treatment is 50~80℃.

9. The method for preparing a SERS substrate for removing fluorescence background according to claim 1, wherein: In step (3.2), the number of times the mixed solution of dilute sulfuric acid and hydrogen peroxide is sprayed on the surface of the gold long square nanoparticle film is 5 to 8 times, and the ratio of the volume of the mixed solution of dilute sulfuric acid and hydrogen peroxide sprayed each time to the area of ​​the inner side of the conductive substrate is 0.5 mL: 1 cm 2 .

10. Use of the SERS substrate with fluorescence background removed obtained by the preparation method according to claim 1 in wastewater detection.

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