A SERS silent zone self-calibration substrate and its preparation method and application

Abstract

The invention discloses a SERS silent zone self-calibration substrate and a preparation method and application thereof, belonging to the technical field of analysis and detection. The invention adopts a water-oil two-phase liquid-liquid interface self-assembly method to prepare a SERS silent zone self-calibration substrate, and calibrates the sample to be tested by the SERS signal of the internal standard 4-MBN molecule, the internal standard molecule is located at 2223 cm ‑1 The Raman characteristic peak is located in the silent zone of biological molecules and will not affect the detection; the SiO2 shell can not only isolate the sample from the substrate, but also effectively protect the calibration substrate so that it can be stored in the air for more than half a year. The preparation method of the SERS silent zone self-calibration substrate provided by the present invention is simple; SERS detection is convenient; and it has high SERS activity, detection stability and repeatability.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a SERS silent zone self-calibration substrate and a preparation method and application thereof. Background Art

[0002] Antibiotics are widely used in the fields of medicine, aquatic products, livestock and poultry farming, etc. However, the widespread use of antibiotics has led to the increase of antibiotic resistance and promoted the proliferation of resistant bacteria, posing direct and indirect threats to humans and ecosystems. Therefore, there is an increasing demand for rapid, sensitive and low-cost detection methods for screening antibiotic residues in the environment. Conventional methods for detecting antibiotics in the environment include chromatography, microbial screening and immunoassays, which have good sensitivity and specificity. However, these methods are limited by expensive instruments, complex detection processes and time-consuming analytical procedures.

[0003] Surface Enhanced Raman Spectroscopy (SERS) is an emerging detection technology with special advantages such as extremely high surface detection sensitivity and providing rich chemical structure information. It has been widely used in the detection of materials, chemistry, environment and biomedicine. Only a few metals such as gold, silver and copper have good SERS enhancement effect, and the enhancement of SERS signal depends largely on the nanostructure morphology of the substrate, including the size, shape and gap of nanoparticles. Often, the inhomogeneity of nanoparticles will cause the SERS signal to fluctuate by several orders of magnitude and affect the results of trace detection.

[0004] Therefore, there is an urgent need to develop a SERS substrate with high stability and high repeatability. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a SERS silent zone self-calibration substrate and a preparation method and application thereof. The present invention prepares the SERS silent zone self-calibration substrate by a water-oil two-phase liquid-liquid interface self-assembly method, which can achieve high-sensitivity and high-accuracy detection of the object to be tested.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention:

[0008] A method for preparing a SERS silent zone self-calibration substrate comprises the following steps:

[0009] A 3-aminopropyltrimethoxysilane (APTMS) solution is added to a gold nanoparticle (Au NPs) sol, and after stirring, a 4-mercaptobenzonitrile (4-MBN) solution and a sodium silicate solution are added, and after stirring again, the mixture is reacted in a water bath to obtain a sol containing Au@4-MBN@SiO2 nanoparticles;

[0010] The sol containing Au@4-MBN@SiO2 nanoparticles is centrifuged, and the obtained precipitate is dispersed in a polyvinyl pyrrolidone (PVP) ethanol solution, and the sol is centrifuged for the second time after ultrasonication, and the obtained precipitate is dispersed in ethanol to obtain a PVP-coated Au@4-MBN@SiO2 sol;

[0011] The PVP-coated Au@4-MBN@SiO2 sol is added to a centrifuge tube, and after adding dichloromethane and water, the tube is shaken. After adding n-hexane along the wall of the centrifuge tube, a monolayer film is formed at the n-hexane / water interface after the centrifuge tube is tilted. After removing the upper n-hexane layer, the monolayer film is taken out using a single crystal silicon wafer to obtain the SERS silent zone self-calibration substrate.

[0012] Preferably, the preparation method of the Au NPs sol comprises the following steps: mixing chloroauric acid (HAuCl4) solution with water, heating, adding sodium citrate solution when heated to boiling, stopping heating after the solution turns brick red for a period of time, and obtaining Au NPs sol after cooling.

[0013] Preferably, the concentration of the sodium citrate solution is 1 wt %;

[0014] The concentration of the chloroauric acid solution is 1 wt %.

[0015] Preferably, when the chloroauric acid solution is mixed with water, the volume ratio of the water to the chloroauric acid solution is 4:0.0485;

[0016] The volume ratio of the chloroauric acid solution to the sodium citrate solution is 4.85:3.

[0017] More preferably, the preparation method of the Au NPs sol is: add 1.2125 mL of 1 wt% HAuCl4 solution to 100 mL of water, then heat to boiling and add 0.75 mL of 1 wt% sodium citrate solution. After adding the sodium citrate solution, the HAuCl4 solution will gradually change from light yellow to black. After the color of the solution turns to brick red, continue heating for 20 minutes, then stop heating and cool to room temperature to obtain a 55 nm Au NPs sol.

[0018] Preferably, the concentration of the APTMS solution is 1 mM; the concentration of the 4-MBN solution is 0.05-0.1 mM; the concentration of the sodium silicate solution is 0.54-1.08 wt%; and the concentration of the PVP ethanol solution is 1-2 wt%.

[0019] Preferably, the volume ratio of the APTMS solution, the 4-MBN solution and the sodium silicate solution is 1:0.75:8.

[0020] Preferably, when the reaction is carried out in a water bath, the temperature of the water bath is 99° C. and the reaction time is 30 min.

[0021] Preferably, when the centrifuge tube is tilted to form a monolayer film at the n-hexane / water interface, the centrifuge tube is slightly tilted, and more preferably the tilt angle of the centrifuge tube is (10°-15°).

[0022] Since high-performance SERS spectroscopy cannot be separated from a SERS substrate with good performance as support, the type, morphology and structure of the substrate material will strongly affect the SERS performance. In order to improve the performance of the SERS substrate, the present invention provides a method for preparing a SERS silent zone self-calibration substrate, by assembling a uniform single layer of Au@4-MBN@SiO2 on a single crystal silicon wafer substrate as a substrate calibration unit, using 4-MBN as an internal standard molecule, and placing it at 2223 cm -1 The characteristic peak at is used as the calibration peak. The calibration peak is located in the Raman silent zone and does not interfere with the trace detection of the sample to be tested. At the same time, the coated SiO2 shell can effectively isolate the sample to be tested from the substrate, improve the hydrophobicity of the substrate, and weaken the coffee ring effect. After the gold nanosol is mixed with the solution to be tested and dripped on the substrate, a three-dimensional interlayer hot spot will be generated between the calibration unit and the detection unit, and a great SERS enhancement will be generated for the molecules to be tested and the internal standard molecules. However, the random distribution of the upper nanoparticles will lead to uneven distribution of hot spots, and detection of different points will obtain detection results with large differences, but there is only a slight difference in the ratio of the characteristic peak intensity of the molecule to be tested and the characteristic peak intensity of the internal standard molecule. The ratio can effectively solve the detection difference caused by the uneven distribution of hot spots and improve the accuracy of its detection results. The present invention only needs to simply mix the gold nanosol with the molecule to be tested and then drip it on the detection substrate, combining the characteristic peak intensity ratio of the molecule to be tested and the internal standard molecule to achieve quantitative detection. More preferably, the preparation method of the SERS silent zone self-calibration substrate is:

[0023] 1. Dissolve solid chloroauric acid in ultrapure water, with the mass ratio of solid chloroauric acid to water being 1:100, to obtain a chloroauric acid solution with a concentration of 1 wt%. Place 100 mL of water and 1.2125 mL of the prepared chloroauric acid solution in a round-bottom flask, set up a condensation reflux device, start magnetic stirring and heating, and heat the solution.

[0024] 2. Dissolve 1.1396 g of sodium citrate in 100 mL of ultrapure water to obtain a sodium citrate solution with a concentration of 1 wt%. When the above chloroauric acid solution is heated to boiling, quickly add 0.75 mL of sodium citrate solution. After the sodium citrate solution is added, the chloroauric acid solution will gradually change from light yellow to black. After the solution color turns brick red, continue heating for 20 min, stop heating, and cool to room temperature to obtain 55 nm Au NPs.

[0025] 3. Add 18.5 μL APTMS to 100 mL ultrapure water to obtain an APTMS solution with a concentration of 1 mmol / L. Add 0.675 mg 4-MBN to 100 mL ultrapure water to obtain a 4-MBN solution with a concentration of 0.05 mmol / L. Add 2 mL sodium silicate to 100 mL ultrapure water to obtain a sodium silicate solution with a concentration of 0.54 wt%. Add 30 mL of the prepared 55 nm Au NPs and 400 μL of the prepared APTMS solution to a round-bottom flask, stir for 15 min, add 300 μL of the prepared 4-MBN solution and 3.2 mL of the prepared sodium silicate solution, stir for 5 min, then transfer to a 99°C water bath for 30 min, take out and immediately put in an ice water bath to terminate the reaction, and obtain a solution containing Au@4-MBN@SiO2 nanoparticles.

[0026] 4. Add 1g PVP to 100 mL ethanol to obtain a 1 wt% PVP ethanol solution. Centrifuge the above-prepared solution containing Au@4-MBN@SiO2 nanoparticles at 5000 rpm for 10 minutes, remove the supernatant, and disperse the precipitate in 1 mL of the above PVP ethanol solution. Ultrasonicate for 60 min and centrifuge for the second time. After removing the supernatant, disperse the precipitate in 100 μL ethanol to obtain a PVP-coated Au@4-MBN@SiO2 solution. Next, 100 μL of PVP-coated Au@4-MBN@SiO2 solution was added to a centrifuge tube, followed by 1 mL of dichloromethane and 1.8 mL of ultrapure water. The mixed solution was vigorously shaken for 1 min, and then 400 μL of n-hexane was slowly added along the wall of the centrifuge tube. After the container was slightly tilted, a golden monolayer film was formed on the n-hexane / water interface. After removing the upper layer of n-hexane, the monolayer film was removed using a single crystal silicon wafer to obtain the SERS silent zone self-calibration substrate.

[0027] The second technical solution of the present invention:

[0028] The invention provides a SERS silent zone self-calibration substrate prepared by the preparation method.

[0029] The third technical solution of the present invention:

[0030] The invention provides application of the SERS silent zone self-calibration substrate in detecting dyes or antibiotics.

[0031] The SERS silent zone self-calibration substrate of the present invention can detect dyes or antibiotics with high efficiency and high sensitivity, and can be used for the detection of a single antibiotic or a mixed detection of multiple antibiotics. The preferred dye is rhodamine 6G (R6G); the preferred antibiotic is penicillin potassium, tetracycline hydrochloride and / or levofloxacin.

[0032] The fourth technical solution of the present invention:

[0033] The invention provides a method for highly sensitively detecting dyes or antibiotics: a sample solution to be tested is mixed with a gold nanoparticle sol, the obtained mixed solution is dripped on the SERS silent zone self-calibration substrate, and after air drying, is placed in a Raman instrument for detection.

[0034] Preferably, when the detection is performed in a Raman instrument, the Raman spectrum excitation wavelength is 633 nm and the output power is 0.326 mW / cm 2 , the exposure time is 10 s.

[0035] Compared with the prior art, the present invention has the following advantages and technical effects:

[0036] The present invention uses shell-isolated nanoparticles containing internal standard molecules as calibration substrates, and calibrates the sample to be tested through the SERS signal of the internal standard molecules. The internal standard molecules are located at 2223 cm -1 The Raman characteristic peak is located in the silent zone of biological molecules and will not affect the detection, and the SiO2 shell can effectively isolate the sample from the substrate. The SERS silent zone self-calibration substrate preparation method provided by the present invention is simple; SERS detection is convenient; it has high SERS activity, detection stability and repeatability, and can detect multiple substances simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0038] Figure 1 Schematic diagram of the preparation and detection process of the SERS silent zone self-calibration substrate of the present invention.

[0039] Figure 2 This is a scanning electron microscope image of the SERS silent zone self-calibration substrate prepared in Example 1 of the present invention.

[0040] Figure 3The SERS intensities of 10 random detection points of the SERS silent zone self-calibration substrate prepared in Example 1 of the present invention.

[0041] Figure 4 This is the substrate strength of the SERS silent zone self-calibration substrate prepared in Example 1 of the present invention within 6 months after preparation.

[0042] Figure 5 The Raman spectra (left) and working curves (right) of the SERS silent zone self-calibration substrate prepared in Example 1 of the present invention for testing different concentrations of Rhodamine 6G (R6G). The "blank" in the Raman spectrum refers to the detection of ultrapure water mixed with gold nanoparticles and then dropped on the substrate.

[0043] Figure 6 The Raman spectra and working curves of the SERS silent zone self-calibration substrate prepared in Example 2 of the present invention tested on single solutions of three different antibiotics (penicillin potassium, tetracycline hydrochloride and levofloxacin), wherein a is the Raman spectrum of the SERS silent zone self-calibration substrate tested on penicillin potassium solution, b is the Raman spectrum of the SERS silent zone self-calibration substrate tested on tetracycline hydrochloride solution, c is the Raman spectrum of the SERS silent zone self-calibration substrate tested on levofloxacin solution, d is the working curve of the SERS silent zone self-calibration substrate tested on penicillin potassium solution, e is the working curve of the SERS silent zone self-calibration substrate tested on tetracycline hydrochloride solution, and f is the working curve of the SERS silent zone self-calibration substrate tested on levofloxacin solution.

[0044] Figure 7 Raman spectra and working curves of the SERS silent zone self-calibration substrate prepared in Example 3 of the present invention tested on a mixed solution of three different antibiotics (penicillin potassium, tetracycline hydrochloride and levofloxacin), a is the Raman spectrum, b is the working curve, and the "blank" in the Raman spectrum refers to the detection after ultrapure water and gold nanoparticles are mixed and dropped on the substrate. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0047] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0048] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] Unless otherwise specified, the room temperature in the present invention is 25±2°C.

[0051] All raw materials used in the embodiments of the present invention are purchased from the market. As an example, single crystal silicon wafers are purchased from Zhejiang Lijing Optoelectronics Technology Co., Ltd.

[0052] The schematic diagram of the preparation and detection process of the SERS silent zone self-calibration substrate of the present invention is shown in Figure 1 .

[0053] The technical solution of the present invention is further illustrated by the following embodiments.

[0054] Example 1

[0055] 1. Dissolve solid chloroauric acid in ultrapure water, with the mass ratio of solid chloroauric acid to water being 1:100, to obtain a chloroauric acid solution with a concentration of 1 wt%. Place 100 mL of water and 1.2125 mL of the prepared chloroauric acid solution in a round-bottom flask, set up a condensation reflux device, start magnetic stirring and heating, and heat the solution.

[0056] 2. Dissolve 1.1396 g of sodium citrate in 100 mL of ultrapure water to obtain a sodium citrate solution with a concentration of 1 wt%. When the above chloroauric acid solution is heated to boiling, quickly add 0.75 mL of sodium citrate solution. After the sodium citrate solution is added, the chloroauric acid solution will gradually change from light yellow to black. After the solution color turns brick red, continue heating for 20 min, stop heating, and cool to room temperature to obtain 55 nm Au NPs.

[0057] 3. Add 18.5 μL 3-aminopropyltrimethoxysilane (APTMS) to 100 mL ultrapure water to obtain an APTMS solution with a concentration of 1 mmol / L. Add 0.675 mg 4-mercaptobenzonitrile (4-MBN) to 100 mL ultrapure water to obtain a 4-MBN solution with a concentration of 0.05 mmol / L. Add 2 mL of 27 wt% sodium silicate solution to 100 mL ultrapure water to obtain a sodium silicate solution with a concentration of 0.54 wt%. Add 30 mL of the prepared 55 nm Au NPs and 400 μL of the prepared APTMS solution to a round-bottom flask, stir for 15 min, add 300 μL of the prepared 4-MBN solution and 3.2 mL of the prepared sodium silicate solution, stir for 5 min, then transfer to a 99°C water bath for 30 min, take out and immediately put in an ice water bath to terminate the reaction, and obtain a solution containing Au@4-MBN@SiO2 nanoparticles.

[0058] 4. Add 1g of polyvinyl pyrrolidone (PVP) to 100 mL of ethanol to obtain a PVP ethanol solution with a concentration of 1 wt%. Centrifuge the above-prepared solution containing Au@4-MBN@SiO2 nanoparticles at 5000 rpm for 10 minutes, remove the supernatant, and disperse the precipitate in 1 mL of the above PVP ethanol solution. After ultrasonication for 60 min, centrifuge for the second time, remove the supernatant, and disperse the precipitate in 100 μL of ethanol to obtain a PVP-coated Au@4-MBN@SiO2 solution. Next, 100 μL of PVP-coated Au@4-MBN@SiO2 solution was added to a centrifuge tube, followed by 1 mL of dichloromethane and 1.8 mL of ultrapure water. The mixed solution was vigorously shaken for 1 min, and then 400 μL of n-hexane was slowly added along the wall of the centrifuge tube. After the container was slightly tilted (the tilt angle was 10°), a golden monolayer film was formed on the n-hexane / water interface. After removing the upper layer of n-hexane, the monolayer film was removed using a single crystal silicon wafer to obtain the SERS silent zone self-calibration substrate.

[0059] The scanning electron microscope image of the SERS silent zone self-calibration substrate prepared in this example is shown in Figure 2 The nanoparticle film is densely and evenly stacked without obvious aggregation and voids, indicating that the substrate of the present invention is well prepared.

[0060] The SERS intensities of 10 random detection points of the SERS silent zone self-calibration substrate prepared in this example are shown in FIG. Figure 3 (The intensity test standard refers to R6G solution detection). There is no obvious difference in the intensity of the Raman characteristic peak, which shows that the SERS silent zone self-calibration substrate has good repeatability and uniformity.

[0061] The SERS silent zone self-calibration substrate prepared in this example has a substrate strength of 6 months after preparation. Figure 4 (The strength test standard refers to the R6G solution test). After being stored in air at room temperature for 24 weeks, the SERS signal of the substrate did not change significantly, indicating that the substrate of the present invention has good time stability.

[0062] 5 μL of different concentrations (10 -8 , 10 -7 , 10 -6 , 10 -5 and 10 -4 mol / L) R6G (rhodamine 6G) solution was mixed with 5 μL of the 55 nm Au NPs prepared above and stirred evenly to obtain a mixed solution. 2 μL of the mixed solution was dropped on the SERS silent zone self-calibration substrate and naturally air-dried for SERS detection. During the test, the Raman spectrum excitation wavelength was 633 nm and the output power was 0.326 mW / cm 2 , the exposure time is 10 s.

[0063] The Raman spectra (left) and working curves (right) of the SERS silent zone self-calibration substrate prepared in this example for different concentrations of R6G are shown in Figure 5 , in R6G solution 10 -4 -10 -8 mol / L concentration range, the intensity of the R6G Raman characteristic peak increases with the increase of concentration. In order to further explore the relationship between the probe molecule concentration and the SERS signal intensity, the R6G molecule at 1511 cm -1 The relationship between the intensity of the Raman characteristic peak attributed to the vibration of the aromatic ring and the molecular concentration, such as Figure 5 As shown in Figure 2, direct analysis of the characteristic peak intensity shows that due to the irregular aggregation of nanoparticles, the detection results have large errors and poor linearity. -1 The peak intensity is similar to that of the base 2223 cm -1 The peak intensity comparison shows that the ratio error is small and it shows excellent concentration linear dependence. The linear equation is y=7.74+0.95x(R 2 =0.998), and the minimum detection limit (LOD) was calculated according to the 3σ principle to be 18 nmol / L.

[0064] Example 2

[0065] 1. Dissolve solid chloroauric acid in ultrapure water, with the mass ratio of solid chloroauric acid to water being 1:100, to obtain a chloroauric acid solution with a concentration of 1 wt%. Place 100 mL of water and 1.2125 mL of the prepared chloroauric acid solution in a round-bottom flask, set up a condensation reflux device, start magnetic stirring and heating, and heat the solution.

[0066] 2. Dissolve 1.1396 g of sodium citrate in 100 mL of ultrapure water to obtain a sodium citrate solution with a concentration of 1 wt%. When the above chloroauric acid solution is heated to boiling, quickly add 0.75 mL of sodium citrate solution. After the sodium citrate solution is added, the chloroauric acid solution will gradually change from light yellow to black. After the solution color turns brick red, continue heating for 20 min, stop heating, and cool to room temperature to obtain 55 nm Au NPs.

[0067] 3. Add 18.5 μL APTMS to 100 mL ultrapure water to obtain an APTMS solution with a concentration of 1 mmol / L. Add 1.35 mg 4-MBN to 100 mL ultrapure water to obtain a 4-MBN solution with a concentration of 0.1 mmol / L. Add 4 mL of 27 wt% sodium silicate solution to 100 mL ultrapure water to obtain a sodium silicate solution with a concentration of 1.08 wt%. Add 30 mL of the prepared 55 nm Au NPs and 400 μL of the prepared APTMS solution to a round-bottom flask and stir for 15 min. Add 300 μL of the prepared 4-MBN solution and 3.2 mL of the prepared sodium silicate solution, stir for 5 min and transfer to a 99°C water bath for 30 min. Immediately take out and place in an ice water bath to terminate the reaction. A solution containing Au@4-MBN@SiO2 nanoparticles is obtained.

[0068] 4. Add 2g of PVP to 100 mL of ethanol to obtain a PVP ethanol solution with a concentration of 2 wt%. Centrifuge the above-prepared solution containing Au@4-MBN@SiO2 nanoparticles at 5000 rpm for 10 minutes, remove the supernatant, and disperse the precipitate in 1 mL of the above PVP ethanol solution. After ultrasonication for 60 min, centrifuge for the second time. After removing the supernatant, disperse the precipitate in 100 μL of ethanol to obtain a PVP-coated Au@4-MBN@SiO2 solution. Next, add 100 μL of PVP-coated Au@4-MBN@SiO2 solution to a centrifuge tube, followed by 1 mL of dichloromethane and 1.8 mL of ultrapure water. Vigorously shake the mixed solution for 1 min, and then slowly add 400 μL of n-hexane along the wall of the centrifuge tube. After the container is slightly tilted (the tilt angle is 15°), a golden monolayer film will be formed on the n-hexane / water interface. After removing the upper layer of n-hexane, the monolayer film is taken out using a single crystal silicon wafer to obtain the SERS silent zone self-calibration substrate.

[0069] 5 μL of different concentrations of single antibiotic solutions (penicillin potassium solution, tetracycline hydrochloride solution and levofloxacin solution, the concentration of the three antibiotic solutions is 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M and 10 -7 M) was mixed with 5 μL of 55 nm AuNPs and stirred to obtain a mixed solution. 2 μL of the mixed solution was dropped on the SERS silent zone self-calibration substrate and air-dried for SERS detection. During the test, the Raman spectrum excitation wavelength was 633 nm and the output power was 3.26 mW / cm 2 , the exposure time is 10s.

[0070] The Raman spectra and working curves of the SERS silent zone self-calibration substrate prepared in this example for the single solution test of three different antibiotics (penicillin potassium, tetracycline hydrochloride and levofloxacin) are shown in FIG. Figure 6 , penicillin potassium, tetracycline hydrochloride and levofloxacin at 10 -3 -10 -7 M and 10 -4 -10 -8 The concentration-dependent SERS spectra in the range of 10 nM are shown in Figure 2. Compared with the SERS spectra of the substrate, the presence of these three antibiotics results in some new Raman characteristic peaks. The intensity of these characteristic peaks increases with the increase of sample concentration and shows a good linear dependence in the concentration range. The LODs are 26.9, 28.2 and 2.4 nM, respectively. It can be seen that the present invention has high performance in the quantitative SERS detection of trace antibiotics.

[0071] Example 3

[0072] 1. Dissolve solid chloroauric acid in ultrapure water, with the mass ratio of solid chloroauric acid to water being 1:100, to obtain a chloroauric acid solution with a concentration of 1 wt%. Place 100 mL of water and 1.2125 mL of the prepared chloroauric acid solution in a round-bottom flask, set up a condensation reflux device, start magnetic stirring and heating, and heat the solution.

[0073] 2. Dissolve 1.1396 g of sodium citrate in 100 mL of ultrapure water to obtain a sodium citrate solution with a concentration of 1 wt%. When the above chloroauric acid solution is heated to boiling, quickly add 0.75 mL of sodium citrate solution. After the sodium citrate solution is added, the chloroauric acid solution will gradually change from light yellow to black. After the solution color turns brick red, continue heating for 20 min, stop heating, and cool to room temperature to obtain 55 nm Au NPs.

[0074] 3. Add 18.5 μL APTMS to 100 mL ultrapure water to obtain an APTMS solution with a concentration of 1 mmol / L. Add 1.35 mg 4-MBN to 100 mL ultrapure water to obtain a 4-MBN solution with a concentration of 0.1 mmol / L. Add 2 mL of 27% sodium silicate solution to 100 mL ultrapure water to obtain a sodium silicate solution with a concentration of 0.54 wt%. Add 30 mL of the prepared 55 nm Au NPs and 400 μL of the prepared APTMS solution to a round-bottom flask and stir for 15 min. Add 300 μL of the prepared 4-MBN solution and 3.2 mL of the prepared sodium silicate solution, stir for 5 min and transfer to a 99°C water bath for 30 min. Immediately take out and place in an ice water bath to terminate the reaction. A solution containing Au@4-MBN@SiO2 nanoparticles is obtained.

[0075] 4. Add 1g of PVP to 100 mL of ethanol to obtain a PVP ethanol solution with a concentration of 1 wt%. Centrifuge the solution containing Au@4-MBN@SiO2 nanoparticles prepared above at 5000 rpm for 10 minutes, remove the supernatant, disperse the precipitate in 1 mL of the above PVP ethanol solution, and perform a second centrifugation after ultrasonication for 60 min. After removing the supernatant, disperse the precipitate in 100 μL of ethanol to obtain a PVP-coated Au@4-MBN@SiO2 solution. Next, add 100 μL of PVP-coated Au@4-MBN@SiO2 solution to a centrifuge tube, followed by 1 mL of dichloromethane and 1.8 mL of ultrapure water. Vigorously shake the mixed solution for 1 min, and then slowly add 400 μL of n-hexane along the wall of the centrifuge tube. After the container is slightly tilted (the tilt angle is 15°), a golden monolayer film will be formed on the n-hexane / water interface. After removing the upper layer of n-hexane, the monolayer film is taken out using a single crystal silicon wafer to obtain the SERS silent zone self-calibration substrate.

[0076] 5 μL of mixed antibiotic solutions of different concentrations (a mixed solution of penicillin potassium, tetracycline hydrochloride and levofloxacin (volume ratio of 1:1:1), the concentrations of the mixed solution are 1 mg / L, 5 mg / L, 10 mg / L, 50 mg / L and 100 mg / L) and 5 μL of the prepared 55 nm Au NPs were mixed and stirred to obtain a mixed solution. 2 μL of the mixed solution was dropped on the SERS silent zone self-calibration substrate and naturally air-dried for SERS detection. During the test, the Raman spectrum excitation wavelength was 633 nm and the output power was 3.26 mW / cm 2 , the exposure time is 10 s.

[0077] The Raman spectra and working curves of the SERS silent zone self-calibration substrate prepared in this example for the mixed solution test of three different antibiotics (penicillin potassium, tetracycline hydrochloride and levofloxacin) are shown in FIG. Figure 7 (where a is the Raman spectrum and b is the working curve). The detection of mixed solutions of different antibiotics shows that we have successfully detected the Raman characteristic peaks of three antibiotic molecules. The peak positions are consistent with the characteristic peak positions when a single antibiotic is detected. Quantitative analysis was performed based on the intensity of the characteristic peaks of each antibiotic. The results showed that when quantitative analysis of multiple antibiotics was performed simultaneously, the peak intensity could also show good linearity within the concentration range of 1 mg / L-100 mg / L.

[0078] Comparative Example 1

[0079] The detection method is the same as in Example 1, except that the mixture of 55 nm Au NPs and R6G is directly dropped on the single crystal silicon wafer. The detection results show that the mixed solution drops on the silicon wafer substrate produce a strong coffee ring effect, the detection difference at different points is large, and the SERS intensity and detection sensitivity are lower than those in Example 1.

[0080] Comparative Example 2

[0081] The prepared 55 nm Au NPs sol was centrifuged at 5000 rpm for 10 minutes, the supernatant was removed, and the precipitate was dispersed in 1 mL PVP ethanol solution. After ultrasonication for 60 min, a second centrifugation was performed. After removing the supernatant, the precipitate was dispersed in 100 μL ethanol to obtain PVP-coated 55 nm Au NPs. Next, 100 μL PVP-coated 55 nm AuNPs were added to a centrifuge tube, followed by 1 mL dichloromethane and 1.8 mL ultrapure water. The mixed solution was vigorously shaken for 1 min, and then 400 μL n-hexane was slowly added along the wall of the centrifuge tube. After the container was slightly tilted (the tilt angle was 15°), a golden monolayer film was formed on the n-hexane / water interface. After removing the upper n-hexane, the monolayer film was removed with a single crystal silicon wafer to obtain a 55 nm Au NPs substrate. The above 55 nm Au NPs substrate was then soaked in 10 mL R6G solution. The detection results show that the detection differences at different points cannot be calibrated because no calibration molecules are introduced, and the linearity of the standard working curve is poor; and the SERS intensity and detection sensitivity are both lower than those in Example 1; at the same time, the effective time limit of the substrate is short, and the SERS signal intensity decreases significantly within one week after preparation.

[0082] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. Application of a SERS silent zone self-calibration substrate in detecting dyes or antibiotics, characterized in that: The method for preparing the SERS silent zone self-calibration substrate comprises the following steps: (1) Dissolve solid chloroauric acid in ultrapure water at a mass ratio of solid chloroauric acid to water of 1:100 to obtain a chloroauric acid solution with a concentration of 1 wt%. Place 100 mL of water and 1.2125 mL of the prepared chloroauric acid solution in a round-bottom flask, set up a condensation reflux device, start magnetic stirring and heating, and heat the solution. (2) Dissolve 1.1396 g of sodium citrate in 100 mL of ultrapure water to obtain a sodium citrate solution with a concentration of 1 wt%. When the above chloroauric acid solution is heated to boiling, quickly add 0.75 mL of sodium citrate solution. After the sodium citrate solution is added, the chloroauric acid solution will gradually change from light yellow to black. After the solution color turns to brick red, continue heating for 20 min, stop heating, and cool to room temperature to obtain 55 nm Au NPs. (3) Add 18.5 μL of APTMS to 100 mL of ultrapure water to obtain an APTMS solution with a concentration of 1 mmol / L. Add 0.675 mg of 4-MBN to 100 mL of ultrapure water to obtain a 4-MBN solution with a concentration of 0.05 mmol / L. Add 2 mL of sodium silicate to 100 mL of ultrapure water to obtain a sodium silicate solution with a concentration of 0.54 wt%. Add 30 mL of the prepared 55 nm Au NPs and 400 μL of the prepared APTMS solution to a round-bottom flask and stir for 15 min. Add 300 μL of the prepared 4-MBN solution and 3.2 mL of the prepared sodium silicate solution. Stir for 5 min and transfer to a 99°C water bath for 30 min. Take out and immediately place in an ice water bath to terminate the reaction. A solution containing Au@4-MBN@SiO2 nanoparticles is obtained. (4) 1 g of PVP was added to 100 mL of ethanol to obtain a 1 wt% PVP ethanol solution. The solution containing Au@4-MBN@SiO2 nanoparticles prepared above was centrifuged at 5000 rpm for 10 min, the supernatant was removed, and the precipitate was dispersed in 1 mL of the above PVP ethanol solution. After ultrasonication for 60 min, a second centrifugation was performed. After removing the supernatant, the precipitate was dispersed in 100 μL of ethanol to obtain a PVP-coated Au@4-MBN@SiO2 solution. Next, 100 μL of the PVP-coated Au@4-MBN@SiO2 solution was added to a centrifuge tube, followed by the addition of 1 mL of dichloromethane and 1.8 mL of ultrapure water. The mixed solution was vigorously shaken for 1 min, and then 400 μL of n-hexane is slowly added along the wall of the centrifuge tube. After the container is slightly tilted, a golden monolayer film will be formed on the n-hexane / water interface. After removing the upper n-hexane, the monolayer film is taken out with a single crystal silicon wafer to obtain the SERS silent zone self-calibration substrate. The minimum detection limit of the SERS silent zone self-calibration substrate is 18 nmol / L.

2. A method for highly sensitive detection of dyes or antibiotics, characterized in that: The sample solution to be tested is mixed with the gold nanoparticle sol, and the obtained mixed solution is dropped on the SERS silent zone self-calibration substrate in claim 1, and after air drying, is placed in a Raman instrument for detection.

Images