Stable paper-based SERS (Surface Enhanced Raman Scattering) substrate as well as preparation method and application

Abstract

The invention discloses a stable paper-based SERS (Surface Enhanced Raman Scattering) substrate as well as a preparation method and application thereof, and belongs to the technical field of detection, filter paper is immersed into a KH-560 aqueous solution, dried at room temperature for 30-60 minutes and then immersed into a cysteamine aqueous solution, and thiol modified filter paper is obtained; agNPs sol, a tetrabutylammonium nitrate aqueous solution and dichloromethane are evenly mixed, AgNPs are arranged into a single layer on a water-gas interface, then cyclohexane is added, AgNPs are migrated to a water-cyclohexane interface, and a densely-stacked silver nanoparticle single layer is generated; after cyclohexane is removed, thiol modified filter paper is immersed below the silver nanoparticle single layer and then taken out to form the stable paper-based SERS substrate, and the stable paper-based SERS substrate is high in stability and reproducibility and can be used for rapid, accurate and trace detection of methylene blue in a water body.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a stable paper-based SERS substrate, its preparation method, and its application. Background Technology

[0002] Industrial emissions of the cationic dye methylene blue (MB) can lead to ecotoxicity in water bodies and bioaccumulation through the food chain. Therefore, detecting MB in water bodies is crucial for maintaining aquatic ecological security and public health. As a result, developing highly sensitive MB detection technologies is a key focus in the field of aquatic environmental analysis.

[0003] Significant progress has been made in current methods for detecting micromolecular molecules (MB), including: ① Spectrophotometry, a standard method that is simple to operate and low in cost, but susceptible to interference from coexisting substances, and has limited sensitivity and selectivity; ② Chromatography (such as HPLC-MS), which has high separation efficiency and accuracy, and is suitable for trace detection in complex matrices, but the equipment is expensive and the pretreatment is complex; ③ Electrochemical methods, which utilize nanomaterials (such as graphene and metal oxides) to modify electrodes to achieve high sensitivity and rapid response, and are convenient for portable on-site detection, but the stability and antifouling properties of the electrodes still need to be improved; ④ Biosensing methods, which achieve specific detection through biomolecular recognition elements, are environmentally friendly, but their stability and practicality face challenges.

[0004] Among the latest detection methods, surface-enhanced Raman scattering (SERS) technology, compared to traditional methods (such as spectrophotometry which is susceptible to interference, chromatography which uses expensive equipment, and electrochemical methods which suffer from insufficient stability), can achieve ultrasensitive detection of MB molecules through the localized surface plasmon resonance effect generated by noble metal nanostructures (such as gold / silver nanoparticles). The sensitivity can reach trace or even single-molecule levels, and it has excellent fingerprint recognition capabilities, enabling specific analysis in complex water matrices. Paper-based SERS substrates are widely used in rapid on-site detection due to their unique advantages. Their core advantages are: First, paper materials (such as filter paper and chromatographic paper) are extremely low in cost, flexible, and easy to process. They can be miniaturized and self-driven by samples (relying on capillary action) through cutting and folding, which greatly reduces detection costs and simplifies operation. Second, the three-dimensional porous fiber structure of paper provides a large specific surface area for loading noble metal nanoparticles (such as gold and silver nanosols), which not only enhances the hot spot density but also efficiently enriches target molecules, thereby significantly improving detection sensitivity. However, the porous structure of current paper-based SERS leads to uneven distribution of nanoparticles, poor hot spot reproducibility compared to rigid substrates, and large fluctuations in Raman signals. In addition, its mechanical strength is relatively weak, and fiber swelling or nanoparticle shedding may occur in complex liquid environments. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a stable paper-based SERS substrate, its preparation method, and its application, thereby solving the problems of poor reproducibility, fiber swelling, or nanoparticle shedding. It is stable, highly reproducible, and can be used for rapid, accurate, and trace detection of methylene blue in water.

[0006] This invention is achieved through the following technical solution: A method for preparing a stable paper-based SERS substrate includes the following steps: Step 1: Immerse the filter paper in KH-560 aqueous solution, dry it at room temperature for 30-60 min, and then immerse it in cysteine ​​aqueous solution to obtain thiol-modified filter paper. AgNPs sol, tetrabutylammonium nitrate aqueous solution and dichloromethane were mixed evenly. AgNPs arranged into a monolayer at the water-air interface. Then cyclohexane was added. AgNPs migrated to the water-cyclohexane interface and produced a densely packed monolayer of silver nanoparticles. Step 2: After removing cyclohexane, thiol-modified filter paper is immersed below the silver nanoparticle monolayer and then removed to form a stable paper-based SERS substrate.

[0007] A further improvement of the present invention is that: In the KH-560 aqueous solution described in step 1, the mass ratio of KH-560 to deionized water is 1:40, and the concentration of the cysteamine aqueous solution is 5~10 mM.

[0008] Step 1: Immerse the filter paper in a KH-560 aqueous solution at 55-65°C for 10-14 hours, dry it, and then immerse it in a cysteine ​​aqueous solution at 55-65°C for 1-3 hours to obtain thiol-modified filter paper.

[0009] The volume ratio of AgNPs sol, tetrabutylammonium nitrate aqueous solution and dichloromethane in step 1 is (5000~10000):(10~20):(5000~10000), and the concentration ratio of AgNPs sol to tetrabutylammonium nitrate aqueous solution is 0.1:10000.

[0010] Step 1: AgNPs sol, tetrabutylammonium nitrate aqueous solution and dichloromethane are mixed in a centrifuge tube at an angle >60° and a shaking frequency >4 times per second for 30-60 seconds. After that, AgNPs are arranged into a monolayer at the water-air interface.

[0011] Step 1: Add cyclohexane along the wall of the centrifuge tube. The volume ratio of cyclohexane to dichloromethane is (2~4):(5~10).

[0012] After removing cyclohexane in step 2, tilt the centrifuge tube and immerse the thiol-modified filter paper below the silver nanoparticle monolayer for 3-5 seconds. After removing it, let it air dry for 1-2 minutes to obtain a stable paper-based SERS substrate.

[0013] A stable paper-based SERS substrate obtained by the method for preparing a stable paper-based SERS substrate as described in any one of the above.

[0014] Application of stable paper-based SERS substrates in the detection of methylene blue in water.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects: This invention discloses a method for preparing a stable paper-based SERS substrate. In the KH-560 molecule, the three methoxy groups are replaced by hydroxyl groups in the presence of water, generating highly reactive silanol groups. Due to the abundant hydroxyl groups on the filter paper surface, when the two meet, a condensation reaction occurs, removing one molecule of water and forming a stable covalent bond CO-Si on the filter paper surface, containing the epoxy groups of the KH560 molecule itself. Because of the strong covalent interaction between the epoxy groups and the amino groups of cysteine, a strong and durable covalent bond is formed through an efficient ring-opening addition reaction. This imparts a certain degree of hydrophobicity to the filter paper while grafting thiol functional groups onto the paper surface. This can be accomplished with only two simple impregnation steps, enabling mass production. Using the oil (cyclohexane) / water (AgNPs sol) / oil (dichloromethane) three-phase interfacial tension gradient method, AgNPs can first be arranged into a monolayer at the water-gas interface, and then migrate to the water-cyclohexane interface, producing a densely packed monolayer of silver nanoparticles. When the modified filter paper is immersed in the solution beneath the tightly packed AgNPs monolayer membrane, the -SH atom tends to lose a proton, transforming into a more nucleophilic sulfide anion. After the filter paper is removed from the air-water interface, the AgNPs monolayer membrane rapidly transfers to the surface of the modified filter paper. The negatively charged sulfur atom injects its lone pair electron into the empty orbital of the silver atom, forming a strong coordinate covalent bond, the Ag-S bond, thus creating a stable AgNPs@FP substrate without fiber swelling or nanoparticle detachment. The SERS substrate prepared by this invention exhibits good reproducibility (RSD < 20%), and the analytical results are accurate and reliable. No complex sample preparation steps are required; after immersing the substrate in the target water solution for 30 min, the presence of the cationic dye methylene blue can be detected within 5 s. The operation is simple, highly reproducible, and suitable for rapid and accurate screening of methylene blue content in water. Attached Figure Description

[0016] Figure 1a XPS full spectrum of KH560 modified filter paper and KH560@Cysteamine modified filter paper.

[0017] Figure 1b for Figure 1a S in 2P Detailed spectrum.

[0018] Figure 1c for Figure 1a N in 1S Detailed spectrum.

[0019] Figure 2a Transmission electron microscopy (TEM) images of AgNPs synthesized using the classic Lee and Meisel method.

[0020] Figure 2b UV spectra of AgNPs synthesized based on the classic Lee and Meisel method (b).

[0021] Figure 2c This is a scanning electron microscope image of a tightly packed monolayer of AgNPs prepared by the three-phase interface gradient tension method. The scale bar is 500 nm.

[0022] Figure 2d This is a size statistics chart of AgNPs particles.

[0023] Figure 3 The images show the physical images of the prepared paper-based AgNPs substrate, where (a), (c), and (e) are the unmodified filter paper, the filter paper modified by impregnation with 0.3 M NaCl, and the substrate of the present invention, respectively. (b), (d), and (f) are the physical images of (a), (c), and (e) after being impregnated with 10 mL of deionized water, respectively.

[0024] Figure 4a Raman spectra of circular qualitative filter paper prepared in different batches (10 batches, n = 10) for reproducibility.

[0025] Figure 4b Raman spectra of the stability of circular qualitative filter paper prepared in different batches after 30 days of storage at room temperature in a sealed, light-protected environment.

[0026] Figure 5 This is a graph showing the linear relationship between the logarithmic SERS response intensity and the logarithmic methylene blue concentration. Detailed Implementation

[0027] The principles and advantages of this invention will be explained and illustrated below through specific embodiments to enable those skilled in the art to better understand this invention. The following description is merely exemplary and does not limit its scope.

[0028] instrument FA1004 electronic balance (Shimadzu Philippines Manufacturing Co., Ltd.), BPG-9070A precision forced-air drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.), laboratory ultra-deionized water system (Shanghai Hetai Instruments Co., Ltd.), laser confocal micro Raman imaging spectrometer (PERS-RH1700, Xiamen Pushi Nanotechnology Co., Ltd.), SU8100 high-resolution scanning electron microscope (Bruker GmbH, Germany), X-ray photoelectron spectroscopy equipped with monochromatic Al Kα radiation source (XPS, AXIS SUPRA, UK), field emission transmission electron microscope (Tecnai G2 F20 S-TWIN, FEI), X-6 ultraviolet spectrophotometer (Shanghai Yuanxi Instruments Co., Ltd.).

[0029] reagents Analytical grade silver nitrate (AgNO3, 99.8%), sodium citrate (98%), methylene blue (98%), dichloromethane (CH2Cl2), and cyclohexane were purchased from Sinopharm Chemical Reagent Co., Ltd. Tetrabutylammonium nitrate (TBA) + NO3 - 98%), 3-glycidoxypropyltrimethoxysilane (C9H 20 O5Si (KH-560, 97%) and cysteine ​​(95%) were purchased from Aladdin Chemical Company. Qualitative filter paper was from Hangzhou Special Paper Co., Ltd. (Hangzhou, China). All reagents were ready for use without further purification.

[0030] This invention provides a method for preparing a stable paper-based SERS substrate, specifically including the following steps: Step 1) First, immerse the circular qualitative filter paper (FP) in a KH-560 aqueous solution (the mass ratio of KH-560 to deionized water is 1:40) at 55~65°C for 10~14 h, take it out and dry it at room temperature for 30~60 min; then immerse it in a 5~10 mM cysteine ​​aqueous solution at 55~65°C for 1~3 h to obtain thiol-modified filter paper. In the KH-560 molecule, the three methoxy groups are replaced by hydroxyl groups in the presence of water, generating highly reactive silanol groups. Due to the abundant hydroxyl groups naturally present on the paper surface, when the two meet, a condensation reaction occurs, removing one molecule of water (H₂O) and forming a stable covalent bond CO-Si on the paper surface, containing the epoxy groups inherent in the KH560 molecule itself. Because of the strong covalent interaction between the epoxy groups and the amino group of cysteine ​​(one end is amino, the other is thiol), a strong and durable covalent bond is formed through an efficient ring-opening addition reaction. This imparts a certain degree of hydrophobicity to the filter paper while grafting thiol (-SH) functional groups onto the paper surface.

[0031] Step 2) Based on the classic Lee and Meisel method (by reducing silver nitrate with sodium citrate), AgNPs are synthesized. 45 mg of AgNO3 is added to 250 mL of ultrapure water, and then the mixture is boiled with vigorous stirring in a flask. Subsequently, 5 mL of 1% sodium citrate solution is added, and the mixture is boiled for 1 h and then allowed to cool naturally. At this point, the system is brownish-green and forms a 0.1 nM AgNPs sol.

[0032] Then, a tightly packed AgNPs (silver nanoparticles) monolayer was prepared using the three-phase interfacial tension gradient method of oil (cyclohexane) / water (AgNPs sol) / oil (dichloromethane): Add 5-10 mL of AgNPs sol and 10-20 μL of 10 mM TBA to a polypropylene centrifuge tube. + NO3 - An aqueous solution (as an inducer) was mixed with 5-10 mL of CH2Cl2 and then vigorously shaken (hand shaking frequency > 4 times / second, container tilt angle > 60°) for 30-60 s to promote the interaction between the AgNPs sol (aqueous phase) and the organic phase (dichloromethane). The dichloromethane was located at the bottom of the centrifuge tube, and the AgNPs sol was located on top of the dichloromethane. Due to the hydrophobicity of the centrifuge tube, the AgNPs nanoparticles arranged into a monolayer at the water-air interface. Subsequently, 2-4 mL of cyclohexane was added along the tube wall. The n-hexane floated on the aqueous phase, driving the AgNPs nanoparticles to migrate to the water-cyclohexane interface, producing a densely packed monolayer of nanoparticles.

[0033] Step 3) Preparation of paper-based AgNPs; After slowly removing cyclohexane using a pipette, tilt the centrifuge tube and immerse the modified filter paper in the aqueous phase below the silver nanoparticle monolayer for 3-5 seconds. Then, extract it from the air-water interface to allow AgNP to be deposited tightly and uniformly on FP. Allow it to air dry for 1-2 minutes.

[0034] After the filter paper undergoes the aforementioned two impregnation treatments, it carries -SH. When the modified filter paper is tilted and immersed in the solution under the tightly packed AgNPs monolayer membrane, the -SH tends to lose a proton and transform into a sulfide anion with stronger nucleophilic ability. After the filter paper is extracted from the air-water interface, the AgNPs monolayer membrane quickly transfers to the surface of the modified filter paper. The negatively charged sulfur atom throws its lone pair electron into the empty orbital of the silver atom, forming a strong coordinate covalent bond Ag-S bond, thus forming the AgNPs@FP substrate.

[0035] Example 1 Step 1, Thiol modification of filter paper First, a circular qualitative filter paper (5 cm in diameter) was immersed in a KH-560 aqueous solution (1:40 ratio) at 60°C for 12 h. After that, it was taken out and dried at room temperature for 45 min. Then, it was immersed in an 8 mM cysteine ​​aqueous solution at 60°C for 2 h.

[0036] Figure 1a The presence of characteristic Si peaks in the full XPS spectrum confirms the successful surface modification of the paper substrate by KH-560. The fine XPS spectrum further demonstrates this. Figure 1b 163.2 eV (s) 2p )and Figure 1c 399.2 eV (N) 1s The characteristic binding energy observed at the ) site proves that cysteine ​​was successfully grafted onto the paper matrix, achieving thiol modification of the paper matrix.

[0037] Step 2, Preparation of filter paper-based AgNPs (AgNPs@FP): Add 45 mg AgNO3 to 250 mL of ultrapure water, then boil in a flask with vigorous stirring. Then add 5 mL of 1% sodium citrate solution and boil for 1 h. After cooling naturally, the system is brownish-green in color. Figure 2a High-resolution transmission electron microscopy revealed that the synthesized AgNPs sol exhibited a morphology of quasi-spherical and partially anisotropic rod-like structures. Figure 2b The visible absorption peak at 430 nm is due to localized surface plasmon resonance of AgNPs.

[0038] Add 8 mL of Ag sol and 15 μL of 10 mM TBA to a polypropylene centrifuge tube. + NO3 - The inducing agent was mixed with 7 mL of CH2Cl2 and then shaken vigorously for 45 s (5 shakes / second, container tilt angle 70°). The nanoparticles arranged into a monolayer at the water-air interface. Then, 3 mL of cyclohexane was added along the tube wall to drive the nanoparticles to migrate to the water-cyclohexane boundary, producing a densely packed monolayer of nanoparticles.

[0039] After slowly removing cyclohexane using a pipette, the centrifuge tube was tilted, and the modified filter paper was immersed in the aqueous phase below the silver nanoparticle monolayer for 4 seconds. Then it was extracted from the air-water interface, allowing AgNPs to be deposited tightly and uniformly on FP. After natural drying for 2 minutes, AgNPs@FP substrate was obtained.

[0040] pass Figure 2c Morphological characterization performed by SEM, combined with Figure 2d The average particle size of the synthesized silver nanoparticles was 89±11.5 nm.

[0041] Comparison and explanation: Literature reports that chloride ions have a greater affinity for AgNPs than citrate ions. Treating filter paper by immersing it in a NaCl solution of a certain concentration, followed by immersion in AgNP sol, can more effectively retain AgNPs in the filter paper. Therefore, three preparation methods were compared: (1) Load a single layer of AgNP directly onto unmodified filter paper using the method described above; (2) After pretreatment with sodium chloride (soaking in 0.3 M NaCl for 5 min), a monolayer of AgNP was loaded according to the above method; (3) AgNPs@FP substrate of Example 1.

[0042] After drying at 60℃ for 1 h, all substrates were subjected to aqueous ultrasonic treatment in 10 mL of deionized water for 15 min. The experimental results are as follows: Figure 3 In (a), (c), (e), (b), (d), and (f), under ultrasonic impregnation conditions, some AgNPs in the non-thiol-modified paper were dispersed in the water, while the surface modified only by thiol maintained the integrity of the nanoparticles after ultrasonic treatment. This proves the necessity of thiolization for the formation of a stable and uniform AgNP monolayer.

[0043] The prepared substrate was immersed in 10 -8 After soaking in M ​​methylene blue solution for 30 min, the sample was removed and allowed to air dry for half an hour before testing. The parameters of the laser confocal micro Raman imaging spectrometer were as follows: excitation wavelength: 785 nm, laser energy: 10 mW, 50 × objective lens (using a microscope objective lens with a magnification of 50x), integration time: 5 s, and wavenumber scan range: 200-1800 cm⁻¹. -1 .

[0044] like Figure 4a As shown, 10 substrate pairs (10 samples) obtained according to the method of Example 1 can be seen. -8 The reproducibility of the M methylene blue solution was good, with an RSD of 14.63%. The substrate was stored at room temperature in a sealed environment for 30 days, and measurements were taken every 3 days at 1620 cm⁻¹. Figure 4b As shown, it exhibits good stability with an RSD of 6.44%.

[0045] Step 4) Application of AgNPs@FP substrate in detecting methylene blue in real water samples Set the following parameters in the laser confocal micro Raman imaging spectrometer: Excitation wavelength: 785 nm, laser energy: 10 mW, 50 × objective lens (using a microscope objective lens with 50x magnification), integration time: 5 s, wavenumber scan range: 200-1800 cm⁻¹-1 .

[0046] Prepare 10 in deionized water -4 Methylene blue stock solution (0.0032 g methylene blue dissolved in 10 mL deionized water), then diluted with deionized water to a final volume of 10 mL. -5 10 -6 10 -7 10 -8 10 -9 10 -10 and 10 -11 M, the seven AgNPs@FP substrates obtained by the method in Example 1 were immersed for 30 min, then removed and air-dried for half an hour, and analyzed within 5 s. The logarithmic SERS response intensity of methylene blue (450 cm⁻¹) was plotted on the x-axis as the logarithmic concentration of methylene blue. -1 Using ) as the ordinate, 450 cm⁻¹ is also a characteristic peak of methylene blue, corresponding to the C–N–C skeletal bending vibration of the methylene blue molecule. Compared to the C–C stretching vibration of the benzene ring (1620 cm⁻¹), the peak intensity is significantly higher, approximately 1.5 times higher. Figure 5 As shown, it has a concentration range of 10 -7 ~10- 11 The relationship between M and R is linear. 2 =0.9922.

[0047] The limit of detection (LOD) was calculated using 3.3δ / S, where δ is the standard deviation of the blank sample (n=6), and S is the slope value of 0.56 obtained from the linear curve equation. LOD = 2.18 × 10⁻⁶ -12 M (corresponding to 0.697 ng / L). Then, water samples were randomly collected from the Bahe River and Weihe River around Xi'an, as well as from the artificial lake within Shaanxi University of Science and Technology (n=45, 45 samples). After soaking for 30 min, the samples were taken out, and the logarithmic SERS response intensity was analyzed within 5 s. Substituting the values ​​into the equation y=0.56x+7.65, the results showed that methylene blue was detected in all samples, but the content x was less than 50 μg / L after conversion, which is lower than the limit standard for methylene blue in water bodies in the "Environmental Protection Law of the People's Republic of China".

[0048] Example 2 Step 1, Thiol modification of filter paper First, a circular qualitative filter paper (5 cm in diameter) was immersed in a KH-560 aqueous solution (material-liquid ratio 1:40) at 55°C for 14 h, then removed and dried at room temperature for 30 min. Next, it was immersed in a 5 mM cysteine ​​aqueous solution at 55°C for 3 h.

[0049] Step 2, Preparation of filter paper-based AgNPs (AgNPs@FP): Add 45 mg AgNO3 to 250 mL of ultrapure water, then boil in a flask with vigorous stirring. Then add 5 mL of 1% sodium citrate solution and boil for 1 h. After cooling naturally, the system is brownish-green in color. Step 3: Add 5 mL of Ag sol and 10 μL of 10 mM TBA to a polypropylene centrifuge tube. + NO3 - The inducing agent was mixed with 5 mL of CH2Cl2 and then shaken vigorously for 30 s (5 shakes / second, container tilt angle 70°). The nanoparticles arranged into a monolayer at the water-air interface. Then, 2 mL of cyclohexane was added along the tube wall to drive the nanoparticles to migrate to the water-cyclohexane boundary, producing a densely packed monolayer of nanoparticles.

[0050] Step 4: After slowly removing cyclohexane using a pipette, tilt the centrifuge tube and immerse the modified filter paper in the aqueous phase below the silver nanoparticle monolayer for 3 seconds. Then, extract it from the air-water interface to allow AgNPs to be deposited tightly and uniformly on FP. Allow it to air dry for 1 minute to obtain the AgNPs@FP substrate.

[0051] Example 3 Step 1, Thiol modification of filter paper First, a circular qualitative filter paper (5 cm in diameter) was immersed in a KH-560 aqueous solution (material-to-liquid ratio 1:40) at 65°C for 10 h. After being removed and dried at room temperature for 45 min, it was further immersed in a 10 mM cysteine ​​aqueous solution at 65°C for 1 h.

[0052] Step 2, Preparation of filter paper-based AgNPs (AgNPs@FP): Add 45 mg AgNO3 to 250 mL of ultrapure water, then boil in a flask with vigorous stirring. Then add 5 mL of 1% sodium citrate solution and boil for 1 h. After cooling naturally, the system is brownish-green in color. Step 3: In a polypropylene centrifuge tube, add 10 mL of Ag sol and 20 μL of 10 mM TBA. + NO3 - The inducing agent was mixed with 10 mL of CH2Cl2 and then shaken vigorously for 60 s (hand shaking frequency 5 times / second, container tilt angle 70°). The nanoparticles arranged into a monolayer at the water-air interface. Subsequently, 4 mL of cyclohexane was added along the tube wall to drive the nanoparticles to migrate to the water-cyclohexane boundary, producing a densely packed monolayer of nanoparticles.

[0053] Step 4: After slowly removing cyclohexane using a pipette, tilt the centrifuge tube and immerse the modified filter paper in the aqueous phase below the silver nanoparticle monolayer for 5 seconds. Then, extract it from the air-water interface to allow AgNPs to be deposited tightly and uniformly on FP. Allow it to air dry for 2 minutes to obtain the AgNPs@FP substrate.

[0054] Furthermore, the above-described embodiments of the present invention are examples, and any technical solutions that have the same technical concept as those in the claims of the present invention and achieve the same effect are all included within the present invention.

Claims

1. A method for preparing a stable paper-based SERS substrate, characterized in that, Includes the following steps: S1, filter paper is immersed in KH-560 aqueous solution, dried, and then immersed in cysteine ​​aqueous solution to obtain thiol-modified filter paper; S2, AgNPs sol, tetrabutylammonium nitrate aqueous solution and dichloromethane are mixed evenly. AgNPs are arranged into a monolayer at the water-air interface. Then cyclohexane is added. AgNPs migrate to the water-cyclohexane interface and generate a densely packed monolayer of silver nanoparticles. S3, after removing cyclohexane, thiol-modified filter paper is immersed below the silver nanoparticle monolayer, and then removed to form a stable paper-based SERS substrate.

2. The method for preparing a stable paper-based SERS substrate according to claim 1, characterized in that, In the KH-560 aqueous solution, the mass ratio of KH-560 to deionized water is 1:40, and the concentration of the cysteamine aqueous solution is 5~10 mM.

3. The method for preparing a stable paper-based SERS substrate according to claim 2, characterized in that, S1. The filter paper is immersed in the KH-560 aqueous solution at 55-65°C for 10-14 hours, dried, and then immersed in the cysteine ​​aqueous solution at 55-65°C for 1-3 hours to obtain thiol-modified filter paper.

4. The method for preparing a stable paper-based SERS substrate according to claim 2, characterized in that, The drying process is performed at room temperature for 30-60 minutes.

5. The method for preparing a stable paper-based SERS substrate according to claim 1, characterized in that, The volume ratio of AgNPs sol, tetrabutylammonium nitrate aqueous solution and dichloromethane in S2 is (5000~10000):(10~20):(5000~10000), and the concentration ratio of AgNPs sol to tetrabutylammonium nitrate aqueous solution is 0.1:10000.

6. The method for preparing a stable paper-based SERS substrate according to claim 5, characterized in that, S2 involves mixing AgNPs sol, tetrabutylammonium nitrate aqueous solution, and dichloromethane in a centrifuge tube at an angle greater than 60° and a shaking frequency of greater than 4 times per second for 30-60 seconds. After that, AgNPs are arranged into a monolayer at the water-air interface.

7. The method for preparing a stable paper-based SERS substrate according to claim 6, characterized in that, S2 adds cyclohexane along the wall of the centrifuge tube, with the volume ratio of cyclohexane to dichloromethane being (2~4):(5~10).

8. The method for preparing a stable paper-based SERS substrate according to claim 7, characterized in that, After removing cyclohexane in S3, tilt the centrifuge tube and immerse the thiol-modified filter paper below the monolayer of silver nanoparticles for 3-5 seconds. After removing it, let it air dry for 1-2 minutes to obtain a stable paper-based SERS substrate.

9. A stable paper-based SERS substrate obtained by the preparation method of the stable paper-based SERS substrate according to any one of claims 1 to 8.

10. The application of the stable paper-based SERS substrate as described in claim 9 in the detection of methylene blue in water.

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