A surface-enhanced Raman spectroscopy detection method based on aggregation restabilization strategy

Abstract

The invention is applicable to the technical field of Raman spectrum detection, and provides a surface enhanced Raman spectrum detection method based on an aggregation re-stabilization strategy, comprising the following steps: S1: uniformly mixing colloidal gold and a test object; S2: adding an aggregating agent to the system of S1 and quickly mixing them uniformly, so that the colloidal gold undergoes aggregation to generate colloidal gold aggregates; S3: adding an acrylamide-methylenebisacrylamide solution to the system of S2, and then adding a tetramethylethylenediamine solution and an ammonium persulfate solution and mixing them uniformly; S4: pouring the mixture obtained in S3 into a beaker and placing it in an incubator until a polyacrylamide hydrogel-stabilized colloidal gold aggregate SERS detection substrate is formed; S5: taking out the SERS detection substrate formed in S4 and placing it on a tin foil paper surface. The detection method of the invention improves the sensitivity of SERS detection, and greatly improves the stability and uniformity of SERS detection of gold nanoparticle aggregates.

Description

Technical Field

[0001] The invention belongs to the technical field of Raman spectroscopy detection, and in particular relates to a surface enhanced Raman spectroscopy detection method based on an aggregation restabilization strategy. Background Art

[0002] Surface enhanced Raman spectroscopy (SERS) is a molecular vibration spectroscopy technique that greatly enhances the Raman scattering signal of the target by making the target adsorb or approach the surface of a nanostructure with surface plasmons and utilizing the Raman "hot spots" generated by the nanostructure (the enhancement factor is usually greater than 10). 5 ). SERS technology has the characteristics of high sensitivity, fast detection speed, little interference from water, molecular fingerprint, and potential for non-destructive detection. It has received great attention in the research and application of food safety, environmental monitoring, medical diagnosis and other fields. In recent years, spectral instruments have developed rapidly, and various handheld Raman spectrometers have appeared one after another. Due to its advantages such as low cost and lightness, SERS technology has received great attention in the field of on-site in-situ detection. However, compared with large Raman spectrometers in laboratories, the detection performance of handheld Raman spectrometers, including sensitivity, repeatability, and stability, are relatively poor.

[0003] In order to improve the on-site in-situ detection performance of handheld Raman spectrometers, the first consideration is to obtain a substrate with excellent SERS activity. SERS substrates are generally composed of precious metal nanostructures with rough surfaces, and their enhanced performance is closely related to the composition, size, shape of the nanostructures and the gaps between adjacent nanoparticles. In recent years, domestic and foreign researchers have constructed a variety of substrates such as nanosols, solid-phase nanomaterials, and flexible nanomaterials based on the design of SERS active substrate materials; on the other hand, based on the integrated innovation of SERS detection methods, SERS is combined with solid phase extraction, molecular imprinting, immune recognition, magnetic separation, and microfluidic platforms to improve the sensitivity of SERS for trace substance detection.

[0004] As far as the design of SERS active substrate materials is concerned, although an ordered solid-phase SERS substrate can be obtained through precise nanofabrication technology, the preparation process is cumbersome and expensive, which limits its potential for practical application. Colloidal gold (gold nanoparticles, AuNPs) is simple to synthesize and is currently a commonly used SERS active substrate. However, the detection effect of dispersed colloidal gold is poor. When using colloidal gold for SERS detection, it is generally necessary to add a salt ion aggregator to aggregate the gold nanoparticles to produce more SERS "hot spots". However, the aggregation of gold nanoparticles is random, and the amount of salt ions added and the reaction time will significantly affect the aggregation process, resulting in poor stability of the resulting SERS signal. Summary of the invention

[0005] The present invention provides a surface enhanced Raman spectroscopy detection method based on an aggregation re-stabilization strategy, aiming to solve the problems of low sensitivity and poor stability of existing colloidal gold SERS detection.

[0006] The present invention is implemented as follows: a surface enhanced Raman spectroscopy detection method based on an aggregation re-stabilization strategy comprises the following steps:

[0007] S1: Mix colloidal gold and the object to be tested evenly;

[0008] S2: Add an aggregating agent to the system of S1 and mix it quickly to make the colloidal gold aggregate and generate colloidal gold aggregates. By adding an aggregating agent, the nanoparticles are induced to aggregate and generate colloidal gold aggregates, thereby generating a large number of SERS "hot spots".

[0009] S3: Add acrylamide-methylenebisacrylamide solution to the system of S2, then add tetramethylethylenediamine solution and ammonium persulfate solution and mix them evenly to promote the rapid formation of polyacrylamide hydrogel. Based on the binding effect of polyacrylamide hydrogel, excessive aggregation and sedimentation of colloidal gold are avoided, and the stability of colloidal gold aggregates and the distribution of SERS "hot spots" are effectively maintained;

[0010] S4: pour the mixture obtained in S3 into a beaker and place it in an incubator until a polyacrylamide hydrogel-stabilized colloidal gold aggregate SERS detection substrate is formed;

[0011] S5: Take out the SERS detection substrate formed by S4, place it on the surface of the tin foil, align the Raman probe with the hydrogel surface, adjust the detection distance, collect multiple Raman spectra at different positions on the hydrogel surface, calculate the average spectrum for SERS detection.

[0012] Preferably, step S1 specifically comprises: mixing 1 mL of colloidal gold with a concentration of 0.05 to 0.6 nM with the object to be tested evenly using a vortex oscillator, and leaving the mixture at room temperature for 15 to 30 minutes.

[0013] Preferably, step S2 specifically comprises: adding 60 to 100 μL of an aggregating agent solution having a mass concentration of 1 M to the system of S1, and rapidly mixing the solution to uniformly aggregate the colloidal gold to generate colloidal gold aggregates.

[0014] Preferably, step S3 is specifically as follows: 0.25-2 mL of a 30% mass concentration acrylamide-methylenebisacrylamide solution is added to the system of S2, wherein the mass ratio of acrylamide to methylenebisacrylamide is 29:1, and then 2-4 μL of tetramethylethylenediamine solution and 20 μL of ammonium persulfate aqueous solution are added with a pipette and mixed evenly.

[0015] Preferably, in step S4, the temperature of the incubator is 30-40° C., and the placement time is 5 minutes.

[0016] Preferably, the colloidal gold in step S1 is spherical, has a particle size of 30 to 100 nm, and has SERS activity.

[0017] Preferably, the aggregating agent in step S2 includes one or more combinations of sodium chloride, sodium nitrate, potassium chloride, potassium nitrate, and hydrochloric acid, and the aggregating agent can shield the electrostatic interaction between nanoparticles and induce the chemical substance of nanoparticles to aggregate.

[0018] Preferably, after the aggregating agent in step S2 is added and quickly mixed, it is left for 10 to 30 seconds, which can not only promote the aggregation of nanoparticles, but also avoid the formation of large-size nanoclusters before the polyacrylamide hydrogel is generated.

[0019] Preferably, the ammonium persulfate aqueous solution in step S3 needs to be prepared and used immediately, with a mass concentration of 10-15%.

[0020] Preferably, the polyacrylamide hydrogel-stabilized colloidal gold aggregate SERS detection substrate formed in step S4 has a gel strength of 328 to 6206 g, and the colloidal gold aggregates are coated inside the polyacrylamide hydrogel network structure.

[0021] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0022] 1. The SERS detection method based on PAH-AuNAs substrate provided by the present invention has a large number of gold nanoparticle aggregates coated inside the polyacrylamide hydrogel, and strong plasma resonance coupling can be formed between adjacent gold nanoparticles, which has excellent Raman enhancement effect and can be used for trace detection.

[0023] 2. The present invention utilizes the rapid gelation of polyacrylamide hydrogel to effectively stabilize gold nanoparticle aggregates, avoid excessive aggregation or sedimentation of gold nanoparticles induced by aggregating agents, and ensure exposure of a large number of SERS "hot spots", which not only improves the sensitivity of SERS detection, but also greatly improves the stability and uniformity of gold nanoparticle aggregates for SERS detection;

[0024] 3. The SERS detection method based on PAH-AuNAs substrate provided by the present invention uses conventional medicines as reagents, does not require the use of complex instruments and equipment, has a simple material preparation process, is low in cost, and is easy to use in combination with a handheld Raman spectrometer. It is suitable for on-site rapid detection of chemical pollutants such as pigments, pesticides and veterinary drugs, and toxins in food and the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1A flow chart of a surface enhanced Raman spectroscopy detection method based on an aggregation restabilization strategy provided by the present invention;

[0026] Figure 2 are electron micrographs of gold nanoparticles, A and B correspond to the transmission electron micrographs of AuNPs and AuNAs, and C corresponds to the scanning electron micrograph of PAH-AuNAs;

[0027] Figure 3 The SERS detection performance diagram of PAH-AuNAs prepared with different concentrations of colloidal gold;

[0028] Figure 4 The SERS detection performance diagram of PAH-AuNAs corresponding to different concentrations of polyacrylamide gel;

[0029] Figure 5 The results of the SERS detection stability test of PAH-AuNAs at different time periods;

[0030] Figure 6 This is the SERS detection uniformity test result diagram of PAH-AuNAs in different regions;

[0031] Figure 7 The SERS detection results of PAH-AuNAs to 4-MBA probe molecules with different concentrations;

[0032] Figure 8 The SERS detection results of PAH-AuNAs on R6G pigments with different concentrations;

[0033] Fig. 9 This is the SERS detection result of PAH-AuNAs to different concentrations of thiram pesticide. DETAILED DESCRIPTION

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of this application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0035] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0036] like Figure 1 As shown, the technical solution adopted by the present invention includes the following steps:

[0037] S1: Mix colloidal gold and the object to be tested evenly;

[0038] S2: Add an aggregating agent to the system of S1 and mix it quickly to make the colloidal gold aggregate and generate colloidal gold aggregates. By adding an aggregating agent, the nanoparticles are induced to aggregate and generate colloidal gold aggregates, thereby generating a large number of SERS "hot spots".

[0039] S3: Add acrylamide-methylenebisacrylamide solution to the system of S2, then add tetramethylethylenediamine solution and ammonium persulfate solution and mix them evenly to promote the rapid formation of polyacrylamide hydrogel. Based on the binding effect of polyacrylamide hydrogel, excessive aggregation and sedimentation of colloidal gold are avoided, and the stability of colloidal gold aggregates and the distribution of SERS "hot spots" are effectively maintained;

[0040] S4: pour the mixture obtained in S3 into a beaker and place it in an incubator until a polyacrylamide hydrogel-stabilized colloidal gold aggregate SERS detection substrate is formed;

[0041] S5: Take out the SERS detection substrate formed in S4, place it on the surface of the tin foil, align the Raman probe with the hydrogel surface, adjust the detection distance, collect multiple Raman spectra at different positions on the hydrogel surface, calculate the average spectrum for SERS detection;

[0042] Example 1: Detection of 4-MBA probe molecules by gold nanoparticles at different concentrations

[0043] The embodiment of the present invention provides a surface enhanced Raman spectroscopy detection method based on an aggregation re-stabilization strategy, comprising the following steps:

[0044] S1: Mix 1 mL of 0.05-0.6 nM colloidal gold (gold nanoparticles) with 20 μL of 10 -5 M of 4-MBA was mixed evenly using a vortex shaker and left at room temperature for 15 min;

[0045] S2: Add 60 μL of 1 M sodium chloride solution into the system, mix quickly with a vortexer, and leave for 10 seconds to promote the aggregation of gold nanoparticles to produce colloidal gold (gold nanoparticle) aggregates (AuNAs);

[0046] S3: Add 0.5 mL of 30% acrylamide-methylenebisacrylamide solution to the solution system of S2, wherein the mass ratio of acrylamide to methylenebisacrylamide is 29:1, use a pipette to transfer 2 μL of tetramethylethylenediamine solution and mix it evenly with the solution in S1, then add 20 μL of 10% ammonium persulfate aqueous solution and mix thoroughly;

[0047] S4: Pour the solution in S3 into a small beaker and place it in an incubator at 30°C for 5 min to form a polyacrylamide hydrogel-stabilized colloidal gold aggregate (PAH-AuNAs) SERS detection substrate;

[0048] S5: Take out the formed PAH-AuNAs substrate and place it on the surface of tin foil. Aim the Raman probe of the handheld Raman spectrometer at the hydrogel surface, adjust the optimal detection distance, and collect SERS spectra at different positions on the hydrogel surface. The test uses a Ruhai Optoelectronics RMS1000 handheld Raman spectrometer, with an excitation wavelength of 785nm laser light source, a laser power of 30mW, and a collection time of 2s.

[0049] like Figure 2 As shown, the gold nanoparticles used in this example have a particle size of about 40 nm. The SERS substrates prepared with gold nanoparticles of different concentrations detect the 4-MBA probe molecules. Figure 3 As shown, the SERS substrate prepared with gold nanoparticles at a concentration of 0.2 nM has dense SERS "hot spots" formed, which is more conducive to SERS detection.

[0050] Example 2: Detection of 4-MBA probe molecules at different acrylamide concentrations

[0051] The embodiment of the present invention provides a surface enhanced Raman spectroscopy detection method based on an aggregation re-stabilization strategy, comprising the following steps:

[0052] S1: Mix 1 mL of 0.2 nM colloidal gold (gold nanoparticles) with 20 μL of 10 -5 M of 4-MBA was mixed evenly using a vortex shaker and left at room temperature for 20 min;

[0053] S2: Add 60 μL of 1 M sodium chloride solution into the system, mix quickly with a vortexer, and leave for 15 seconds to promote the aggregation of gold nanoparticles to produce colloidal gold (gold nanoparticle) aggregates (AuNAs);

[0054] S3: Add 0.25-2.5 mL of 30% acrylamide-methylenebisacrylamide solution to the solution system of S2, wherein the mass ratio of acrylamide to methylenebisacrylamide is 29:1, use a pipette to transfer 2 μL of tetramethylethylenediamine solution to the solution in S1 and mix evenly, then add 20 μL of 10% ammonium persulfate aqueous solution and mix thoroughly;

[0055] S4: Pour the solution in S3 into a small beaker and place it in an incubator at 32°C for 5 minutes to form a polyacrylamide hydrogel-stabilized colloidal gold aggregate (PAH-AuNAs) SERS detection substrate;

[0056] S5: Take out the formed PAH-AuNAs substrate, place it on the surface of the tin foil, aim the Raman probe of the handheld Raman spectrometer at the hydrogel surface, adjust the optimal detection distance, and collect SERS spectra at different positions on the hydrogel surface. The test uses a Ruhai Optoelectronics RMS1000 handheld Raman spectrometer, with an excitation wavelength of 785nm laser light source, a laser power of 30mW, and a collection time of 2s.

[0057] The concentration of the formed polyacrylamide gel is 6% to 22%, and the gel strength is 328 to 6206 g. The detection effect of polyacrylamide gel of different concentrations on 4-MBA is as follows Figure 4 As shown in Figure 3, high concentrations of polyacrylamide may shield the SERS “hot spots” between gold nanoparticle aggregates and affect SERS detection.

[0058] Example 3: Stability of PAH-AuNAs for 4-MBA probe molecule detection

[0059] The embodiment of the present invention provides a surface enhanced Raman spectroscopy detection method based on an aggregation re-stabilization strategy, comprising the following steps:

[0060] S1: Mix 1 mL of 0.2 nM colloidal gold (gold nanoparticles) with 20 μL of 10 -5 M of 4-MBA was mixed evenly using a vortex shaker and left at room temperature for 20 min;

[0061] S2: Add 60 μL of 1 M sodium chloride solution into the system, mix quickly with a vortexer, and leave for 15 seconds to promote the aggregation of gold nanoparticles to produce colloidal gold (gold nanoparticle) aggregates (AuNAs);

[0062] S3: Add 0.5 mL of 30% acrylamide-methylenebisacrylamide solution to the solution system of S2, wherein the mass ratio of acrylamide to methylenebisacrylamide is 29:1, use a pipette to transfer 2 μL of tetramethylethylenediamine solution and mix it evenly with the solution in S1, then add 20 μL of 10% ammonium persulfate aqueous solution and mix thoroughly;

[0063] S4: Pour the solution in S3 into a small beaker and place it in an incubator at 35°C for 5 minutes to form a polyacrylamide hydrogel-stabilized colloidal gold aggregate (PAH-AuNAs) SERS detection substrate;

[0064] S5: Take out the formed PAH-AuNAs substrate and place it on the surface of tin foil. Aim the Raman probe of the handheld Raman spectrometer at the hydrogel surface, adjust the optimal detection distance, collect 1 SERS spectrum every 1 minute at different positions on the hydrogel surface, and evaluate the signal stability of PAH-AuNAs within the 1-hour detection interval. The test uses a Ruhai Optoelectronics RMS1000 handheld Raman spectrometer, with an excitation wavelength of 785nm laser light source, a laser power of 30mW, and a collection time of 2s.

[0065] like Figure 5 As shown, the signal intensity of AuNAs without adding polyacrylamide gel fluctuates greatly over time within the detection interval of 1 hour, and the relative standard deviation (RSD) of the signal within 0-20 minutes is 6.97%, the RSD within 20-40 minutes is 11.33%, and the RSD within 40-60 minutes is 20.44%. The signal intensity of the PAH-AuNAs substrate prepared by the present invention is relatively stable within the detection interval of 1 hour, and the RSD is less than 3%, indicating that the present invention greatly improves the signal stability of SERS detection compared with traditional colloidal gold detection, and has extremely high application value for on-site in-situ detection of handheld Raman spectrometers.

[0066] Example 4: Uniformity of PAH-AuNAs Detection of 4-MBA Probe Molecules

[0067] The embodiment of the present invention provides a surface enhanced Raman spectroscopy detection method based on an aggregation re-stabilization strategy, comprising the following steps:

[0068] S1: Mix 1 mL of 0.2 nM colloidal gold (gold nanoparticles) with 20 μL of 10 -5 M of 4-MBA was mixed evenly using a vortex shaker and left at room temperature for 20 min;

[0069] S2: Add 60 μL of 1 M sodium chloride solution into the system, mix quickly with a vortexer, and leave for 10 seconds to promote the aggregation of gold nanoparticles to produce colloidal gold (gold nanoparticle) aggregates (AuNAs);

[0070] S3: Add 0.5 mL of 30% acrylamide-methylenebisacrylamide solution to the solution system of S2, wherein the mass ratio of acrylamide to methylenebisacrylamide is 29:1, use a pipette to transfer 2 μL of tetramethylethylenediamine solution and mix it evenly with the solution in S1, then add 20 μL of 10% ammonium persulfate aqueous solution and mix thoroughly;

[0071] S4: Pour the solution in S3 into a small beaker and place it in an incubator at 38°C for 5 minutes to form a polyacrylamide hydrogel-stabilized colloidal gold aggregate (PAH-AuNAs) SERS detection substrate;

[0072] S5: Take out the formed PAH-AuNAs substrate and place it on the surface of tin foil. Align the Raman probe of the handheld Raman spectrometer to the hydrogel surface, adjust the optimal detection distance, and randomly collect 20 SERS spectra at different positions on the hydrogel surface to evaluate the uniformity of the detection signal of the PAH-AuNAs substrate. The test uses a Ruhai Optoelectronics RMS1000 handheld Raman spectrometer, with an excitation wavelength of 785nm laser light source, a laser power of 30mW, and a collection time of 2s.

[0073] like Figure 6 As shown, the SERS spectra collected at different positions of the PAH-AuNAs substrate have good consistency, without obvious SERS signal intensity fluctuations, and the RSD of the SERS spectra at 20 points is less than 5%, indicating that the PAH-AuNAs substrate prepared by the present invention has high signal uniformity when used for SERS detection.

[0074] Example 5: Detection of 4-MBA probe molecules at different concentrations by PAH-AuNAs substrate

[0075] The embodiment of the present invention provides a surface enhanced Raman spectroscopy detection method based on an aggregation re-stabilization strategy, comprising the following steps:

[0076] S1: Mix 1 mL of 0.2 nM colloidal gold (gold nanoparticles) and 20 μL of 4-MBA using a vortex oscillator and place at room temperature for 20 min;

[0077] S2: Add 60 μL of 1 M sodium chloride solution into the system, mix quickly with a vortexer, and leave for 15 seconds to promote the aggregation of gold nanoparticles to produce colloidal gold (gold nanoparticle) aggregates (AuNAs);

[0078] S3: Add 0.5 mL of 30% acrylamide-methylenebisacrylamide solution to the solution system of S2, wherein the mass ratio of acrylamide to methylenebisacrylamide is 29:1, use a pipette to transfer 2 μL of tetramethylethylenediamine solution and mix it evenly with the solution in S1, then add 20 μL of 10% ammonium persulfate aqueous solution and mix thoroughly;

[0079] S4: Pour the solution in S3 into a small beaker and place it in an incubator at 30°C for 5 min to form a polyacrylamide hydrogel-stabilized colloidal gold aggregate (PAH-AuNAs) SERS detection substrate;

[0080] S5: Take out the formed PAH-AuNAs substrate, place it on the surface of the tin foil, aim the Raman probe of the handheld Raman spectrometer at the hydrogel surface, adjust the optimal detection distance, and collect SERS spectra at different positions on the hydrogel surface. The test uses a Ruhai Optoelectronics RMS1000 handheld Raman spectrometer, with an excitation wavelength of 785nm laser light source, a laser power of 30mW, and a collection time of 2s.

[0081] Detection of different concentrations of 4-MBA using PAH-AuNAs substrate Figure 7 As shown, the detection range is 5×10 -9 ~5×10 -7 M, the calibration curve is Y = 10.01 + 0.844X, R 2 =0.964.

[0082] Example 6: Detection of R6G pigments at different concentrations by PAH-AuNAs substrate

[0083] The embodiment of the present invention provides a surface enhanced Raman spectroscopy detection method based on an aggregation re-stabilization strategy, comprising the following steps:

[0084] S1: Mix 1 mL of 0.2 nM colloidal gold (gold nanoparticles) and 20 μL R6G pigment using a vortex oscillator and place at room temperature for 20 min.

[0085] S2: Add 60 μL of 1 M sodium chloride solution into the system, mix quickly with a vortexer, and leave for 20 seconds to promote the aggregation of gold nanoparticles to produce colloidal gold (gold nanoparticle) aggregates (AuNAs);

[0086] S3: Add 0.5 mL of 30% acrylamide-methylenebisacrylamide solution to the solution system of S2, wherein the mass ratio of acrylamide to methylenebisacrylamide is 29:1, use a pipette to transfer 2 μL of tetramethylethylenediamine solution and mix it evenly with the solution in S1, then add 20 μL of 10% ammonium persulfate aqueous solution and mix thoroughly;

[0087] S4: Pour the solution in S3 into a small beaker and place it in an incubator at 30°C for 5 min to form a polyacrylamide hydrogel-stabilized colloidal gold aggregate (PAH-AuNAs) SERS detection substrate;

[0088] S5: Take out the formed PAH-AuNAs substrate, place it on the surface of the tin foil, aim the Raman probe of the handheld Raman spectrometer at the hydrogel surface, adjust the optimal detection distance, and collect SERS spectra at different positions on the hydrogel surface. The test uses a Ruhai Optoelectronics RMS1000 handheld Raman spectrometer, with an excitation wavelength of 785nm laser light source, a laser power of 30mW, and a collection time of 2s.

[0089] Detection of different concentrations of R6G using PAH-AuNAs substrate Figure 8 As shown, the detection range is 1×10 -8 ~1×10 -6 M, the calibration curve is Y = 11.59 + 1.12X, R 2 =0.972.

[0090] Example 7: Detection of Thiram pesticide at different concentrations using PAH-AuNAs substrate

[0091] The embodiment of the present invention provides a surface enhanced Raman spectroscopy detection method based on an aggregation re-stabilization strategy, comprising the following steps:

[0092] S1: Mix 1 mL of 0.2 nM colloidal gold (gold nanoparticles) and 20 μL of thiram solution using a vortex oscillator and place at room temperature for 20 min;

[0093] S2: Add 60 μL of 1 M sodium chloride solution into the system, mix quickly with a vortexer, and leave for 30 seconds to promote the aggregation of gold nanoparticles to produce colloidal gold (gold nanoparticle) aggregates (AuNAs);

[0094] S3: Add 0.5 mL of 30% acrylamide-methylenebisacrylamide solution to the solution system of S2, wherein the mass ratio of acrylamide to methylenebisacrylamide is 29:1, use a pipette to transfer 2 μL of tetramethylethylenediamine solution and mix it evenly with the solution in S1, then add 20 μL of 10% ammonium persulfate aqueous solution and mix thoroughly;

[0095] S4: Pour the solution in S3 into a small beaker and place it in an incubator at 35°C for 5 minutes to form a polyacrylamide hydrogel-stabilized colloidal gold aggregate (PAH-AuNAs) SERS detection substrate;

[0096] S5: Take out the formed PAH-AuNAs substrate and place it on the surface of tin foil. Aim the Raman probe of the handheld Raman spectrometer at the hydrogel surface, adjust the optimal detection distance, and collect SERS spectra at different positions on the hydrogel surface.

[0097] The detection of different concentrations of thiram using PAH-AuNAs substrate is shown in Figure 2. Fig. 9 As shown, the detection range is 5×10 -9 ~2×10 -7 M, the calibration curve is Y = 12.13 + 1.09X, R 2 =0.955.

[0098] In summary, the present invention provides a surface enhanced Raman spectroscopy detection method based on an aggregation and then stabilization strategy. Through the strategy of "aggregation first and then stabilization" of gold nanoparticles, the sensitivity of SERS detection of gold nanoparticles is guaranteed and the stability of SERS detection is improved. First, colloidal gold and the object to be detected are evenly mixed, and then a certain amount of aggregating agent is added to induce the aggregation of nanoparticles to produce gold nanoparticle aggregates and form a large number of SERS "hot spots". Then, acrylamide-methylenebisacrylamide solution, a small amount of tetramethylethylenediamine solution and ammonium persulfate solution are added to promote the rapid formation of polyacrylamide hydrogel. Based on the binding effect of polyacrylamide hydrogel, excessive aggregation and sedimentation of gold nanoparticles are avoided, and the stability of gold nanoparticle aggregates and the distribution of SERS "hot spots" are effectively maintained, forming a SERS substrate with excellent enhancement performance and stable Raman signals for SERS spectral detection of target objects;

[0099] From the perspective of improving the stability of traditional colloidal gold SERS detection, it is of great significance to promote the application of SERS technology in on-site in-situ detection by ensuring the generation of sufficient SERS "hot spots" and maintaining the consistency of SERS signal intensity within the detection time.

[0100] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the present invention according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the present invention in essence, and these technical solutions also belong to the scope of protection of the present invention.

Claims

1. A surface enhanced Raman spectroscopy detection method based on aggregation re-stabilization strategy, characterized in that: The steps include: S1: Mix 1 mL of 0.2 nM colloidal gold and the test substance evenly using a vortex oscillator and place at room temperature for 15-30 minutes; S2: Add 60-100 μL of 1M aggregating agent solution to the system of S1, and mix quickly and evenly to aggregate the colloidal gold to produce colloidal gold aggregates; S3: Add 0.25-2 mL of 30% acrylamide-methylenebisacrylamide solution to the system of S2, wherein the mass ratio of acrylamide to methylenebisacrylamide is 29:1, and then add 2-4 μL of tetramethylethylenediamine solution and 20 μL of ammonium persulfate aqueous solution with a pipette and mix well; S4: pour the mixture obtained in S3 into a beaker and place it in an incubator until a polyacrylamide hydrogel-stabilized colloidal gold aggregate SERS detection substrate is formed; S5: Take out the SERS detection substrate formed by S4, place it on the surface of the tin foil, align the Raman probe with the hydrogel surface, adjust the detection distance, collect multiple Raman spectra at different positions on the hydrogel surface, calculate the average spectrum for SERS detection.

2. The surface enhanced Raman spectroscopy detection method based on the aggregation re-stabilization strategy according to claim 1, characterized in that: In step S4, the temperature of the incubator is 30-40°C and the placement time is 5 minutes.

3. The surface enhanced Raman spectroscopy detection method based on the aggregation re-stabilization strategy according to claim 1, characterized in that: The colloidal gold in step S1 is spherical, has a particle size of 30-100 nm, and has SERS activity.

4. The surface enhanced Raman spectroscopy detection method based on the aggregation re-stabilization strategy according to claim 1, characterized in that: The aggregating agent in step S2 includes one or more combinations of sodium chloride, sodium nitrate, potassium chloride, potassium nitrate, and hydrochloric acid.

5. The surface enhanced Raman spectroscopy detection method based on the aggregation re-stabilization strategy according to claim 4, characterized in that: After the aggregating agent in step S2 is added and quickly mixed, it is left to stand for 10 to 30 seconds.

6. The surface enhanced Raman spectroscopy detection method based on aggregation re-stabilization strategy according to claim 1, characterized in that: The ammonium persulfate aqueous solution in step S3 needs to be prepared and used immediately, and the mass concentration is 10-15%.

7. The surface enhanced Raman spectroscopy detection method based on aggregation re-stabilization strategy according to claim 1, characterized in that: The polyacrylamide hydrogel-stabilized colloidal gold aggregate SERS detection substrate formed in step S4 has a gel strength of 328-6206 g, and the colloidal gold aggregates are coated inside the polyacrylamide hydrogel network structure.

Images