Preparation method and application of 3D hydrogel SERS (Surface Enhanced Raman Scattering) substrate

Abstract

The invention belongs to the field of sweat analysis and detection, and particularly discloses a preparation method and application of a 3D hydrogel SERS (Surface Enhanced Raman Scattering) substrate, and the preparation method comprises the following steps: firstly synthesizing a gold nanocluster, then generating a gold core by the gold nanocluster, modifying the gold core by using 4-MBN, and preparing gold core 4-MBN particles; on the basis of the prepared gold core coated 4-MBN particle, continuously growing a gold nano petal-shaped structure to form an interstitial structure, and then modifying a 4-MBA / 3-MPBA probe molecule to prepare a gold nano petal interstitial particle coated 4-MBA / 3-MPBA with an internal standard function; and the prepared gold nano petal gap particles (at) 4-MBA / 3-MPBA and hydrogel are mixed and freeze-dried, and then the 3D hydrogel SERS substrate (at) 4-MBA / 3-MPBA is prepared. According to the method, probe molecules (4-MBN) are encapsulated in nanoscale gaps to form gold nanoflower-shaped nano-labels with internal standard, and then the gold nanoflower-shaped nano-labels are combined with agarose hydrogel to prepare the 3D flexible SERS substrate, so that the repeatability and stability of sweat analysis can be remarkably improved, and the method has important application potential in the aspect of analyzing the pH value and glucose in human sweat.

Description

Technical Field

[0001] The invention belongs to the field of sweat analysis and detection, and specifically relates to a preparation method and application of a 3D hydrogel SERS substrate. Background Art

[0002] Sweat analysis has gained increasing attention as a direct and non-invasive method for analyzing biological samples. Current biological sample detection technologies, including blood tests and tissue biopsies, are often invasive and time-consuming. In contrast, biomarkers in sweat, such as pH, glucose, and lactate, provide a non-invasive method and provide real-time data that more accurately reflects immediate physiological changes. For example, sweat pH can indicate an individual's acid-base balance, and abnormal levels may be associated with metabolic acidosis or alkalosis, among others. In addition, monitoring glucose in sweat can provide a supplementary assessment of blood glucose levels for diabetic patients, helping to better control blood sugar. Therefore, detecting pH and glucose in sweat is essential for understanding physiological status and has great application potential in sports, health management, and other areas.

[0003] Surface-enhanced Raman spectroscopy (SERS) is a molecular vibrational fingerprinting technique known for its ultrahigh sensitivity, label-free nature, and specificity. SERS achieves this by enhancing the Raman scattering signal of molecules adsorbed on nanostructured metal surfaces, enabling the detection of low-concentration analytes in sweat, with great potential for applications in health monitoring and disease diagnosis. Despite its great potential, SERS still faces several challenges in sweat analysis. For example, constructing SERS sensors on rigid substrates may not be suitable for flexible skin, so flexible and stretchable substrate materials need to be developed. In addition, interference from the sweat matrix can affect the stability, repeatability, and uniformity of the SERS substrate. In addition, the SERS sweat detection platform must incorporate the functionality of biofluids, facilitate the capture of analytes in sweat to hotspots, and integrate SERS into portable devices for field testing, which poses significant challenges. In summary, addressing issues related to substrate uniformity and stability, standardizing protocols, and mitigating matrix interference are critical to promoting the widespread application of SERS technology in biochemical analysis settings. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects of the prior art and provide a preparation method and application of a 3D hydrogel SERS substrate, which can significantly improve the repeatability and stability of sweat analysis by encapsulating probe molecules (4-MBN) in nanoscale gaps to form gold nanoflower-like nanotags with internal standards, and then combining them with agarose hydrogel to prepare a 3D flexible SERS substrate. The gold nanopetal gap particles modified with probe molecules are uniformly dispersed in the porous structure of the hydrogel, showing that the ratio-based 3D hydrogel SERS substrate has convenient, reliable and rapid detection capabilities, providing a non-invasive solution for real-time monitoring of pH and glucose levels in sweat.

[0005] In order to achieve the above objectives, one of the technical solutions of the present invention is: a method for preparing a 3D hydrogel SERS substrate, comprising the following steps:

[0006] S1: First, synthesize gold nanoclusters, then generate gold cores from the gold nanoclusters, and use 4-MBN to modify the gold cores to prepare gold core@4-MBN particles;

[0007] S2: On the basis of the gold core@4-MBN particles prepared in step S1, gold nanopetal-like structures are further grown to form gap structures, and then 4-MBA or 3-MPBA probe molecules are modified to prepare gold nanopetal gap particles@4-MBA or gold nanopetal gap particles@3-MPBA with internal standard function;

[0008] S3: The gold nanopetal gap particles @4-MBA or 3-MPBA prepared in step S2 are mixed with the hydrogel and freeze-dried to prepare a 3D hydrogel SERS substrate @4-MBA or a 3D hydrogel SERS substrate @3-MPBA.

[0009] In a preferred embodiment of the present invention, the gold core @ 4-MBN particles in step S1 are prepared by the following method: first, a gold nanocluster solution is prepared, chloroauric acid and NaBH4 solution are added to a CTAC (tri(hexadecyl)methylammonium chloride) solution, and ultrasonic treatment is performed until fully mixed, and then the mixture is allowed to stand for 0.5-1.5 hours to generate a gold nanocluster solution; then, chloroauric acid solution, ascorbic acid (AA) solution and gold nanocluster solution are added to the CTAC solution to obtain gold nanospheres; after the gold nanospheres are synthesized, 4-MBN solution is added for modification, ultrasonication is performed at room temperature, and the gold core @ 4-MBN particles are prepared.

[0010] Further preferably, the molar ratio of the CTAC solution, chloroauric acid, and NaBH4 solution for generating the gold nanocluster solution is 100-2000:1-20:5-30.

[0011] Further preferably, the molar ratio of the CTAC solution, chloroauric acid, ascorbic acid and gold nanocluster solution for preparing the gold nanospheres is 100-2000:1-20:10-200, and the ratio of the gold nanospheres and the 4-MBN solution for modifying the gold nanospheres is 1-100 μL:0.1-5 mmol.

[0012] Further preferably, the ultrasonic time is 5-30 min, and centrifugal washing is performed after ultrasonication, and the number of washing times is 1-3 times.

[0013] In a preferred embodiment of the present invention, the gold nanopetal gap particles @4-MBA or gold nanopetal gap particles @3-MPBA in step S2 are prepared by the following method: the gold core @4-MBN particles prepared in step (1) are added to a mixture of CTAC solution, ascorbic acid and HAuCl4 to grow gold nanopetals to form a gap structure, and then 4-MBA or 3-MPBA solution is added to react for 0.5-1.5h, centrifuged and washed, and finally resuspended in CTAC solution for washing to prepare gold nanopetal gap particles @4-MBA or gold nanopetal gap particles @3-MPBA with internal standard function.

[0014] Further preferably, the molar ratio of the CTAC solution, AA, HAuCl4, 4-MBA or 3-MPBA solution is 10-1000: 1-20: 0.1-10: 10-500.

[0015] Further preferably, the concentration ratio of the CTAC solution for washing to the CTAC solution for growing the gold nano-petal-shaped gap structure is 0.01-1:1-50, and the volume ratio of the two is 1:1.

[0016] In a preferred embodiment of the present invention, the 3D hydrogel SERS substrate @4-MBA or 3D hydrogel SERS substrate @3-MPBA in step S3 is prepared by the following method: the concentrate of gold nanopetal gap particles @4-MBA or gold nanopetal gap particles @3-MPBA concentrated 5-50 times is mixed with an agarose solution with a concentration of 0.1-5wt% in a volume ratio of 1:1-1:5 and poured into a mold, followed by freeze drying for 3-30 hours to obtain a novel 3D hydrogel SERS substrate @4-MBA or 3D hydrogel SERS substrate @3-MPBA.

[0017] In order to achieve the above objectives, the second technical solution of the present invention is: a 3D hydrogel SERS substrate prepared by a preparation method of a 3D hydrogel SERS substrate.

[0018] In order to achieve the above objectives, the third technical solution of the present invention is: a 3D hydrogel SERS substrate is used to detect sweat pH and H2O2 and glucose in sweat.

[0019] In a preferred embodiment of the present invention, the method for applying the 3D hydrogel SERS substrate to detect the pH of sweat is as follows: placing the 3D hydrogel SERS substrate@4-MBA on a glass slide, then adding 10-1000 μL of sweat sample to the substrate, and using a portable Raman spectrometer to collect spectra.

[0020] In a preferred embodiment of the present invention, the method for applying the 3D hydrogel SERS substrate to detect H2O2 and glucose in sweat is as follows: immersing the 3D hydrogel SERS substrate @3-MPBA in 10-1000 μL sweat sample, incubating it at an incubation temperature of 25-45°C for 5-60 minutes, and then placing it on a microscope slide, and using a portable Raman spectrometer to collect spectra.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. Compared with ordinary gold nanoparticles, the nanogap marker particles embedded with internal standard molecules in the present invention can provide stronger signals for probe molecules; the nanogap marker particles embedded with internal standard molecules are evenly distributed in the agarose hydrogel, showing properties similar to those of liquid SERS substrates, effectively eliminating the "coffee ring" effect; therefore, the hydrogel SERS substrate shows good reproducibility;

[0023] 2. The present invention is located in the three-key silent zone (2225cm -1 ) nanogaps to avoid external environmental interference. Calibration using these internal standard molecules can improve linearity and quantitative detection.

[0024] 3. The freeze-drying process used in the present invention enables the agarose hydrogel to have good water absorption capacity, improves the detection sensitivity, and effectively enables the signal probe particles to avoid many interferences in the actual sample, and can directly perform SERS detection in a variety of liquid samples without complicated pretreatment;

[0025] 4. The present invention combines a portable Raman instrument with the flexibility of the hydrogel SERS substrate, which allows people to directly detect the pH value of sweat, H2O2 and glucose concentration in sweat through a simple "stick-and-peel" method, with convenient, reliable and rapid detection capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the detection and use of the present invention;

[0027] Figure 2 It is an overview of the fabrication and application concepts of the 3D hydrogel SERS-based wearable sweat, pH, and glucose sensors of the present invention;

[0028] Figure 3 (a) TEM image of the gap between gold nanopetals; (b) Actual image of the 3D hydrogel SERS substrate@3-MPBA mold before freeze-drying; (c) SEM image of the 3D hydrogel SERS substrate after freeze-drying;

[0029] Figure 4 It is the pH Raman map and intensity fitting map of the standard system 3D hydrogel SERS substrate @4-MBA detection;

[0030] Figure 5 It is the Raman map and intensity fitting diagram of H2O2 detected by the standard system 3D hydrogel SERS substrate @3-MPBA;

[0031] Figure 6 This is the standard system 3D hydrogel SERS substrate @3-MPBA detection glucose Raman map and intensity fitting map;

[0032] Figure 7 The sweat system 3D hydrogel SERS substrate @4-MBA detects pH Raman intensity and fitting graph, and the 3D hydrogel SERS substrate @3-MPBA detects glucose Raman intensity and fitting graph. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0034] The detection method of the present invention can significantly improve the repeatability and stability of sweat analysis by encapsulating probe molecules (4-MBN) in nanoscale gaps to form gold nanoflower-like nanotags with internal standards, and then combining them with agarose hydrogel to prepare 3D flexible SERS substrates. The gold nanopetal gap particles modified with probe molecules are uniformly dispersed in the porous structure of the hydrogel, showing that the ratio-based 3D hydrogel SERS substrate has convenient, reliable and rapid detection capabilities, and has important application potential in analyzing pH and glucose in human sweat.

[0035] A method for preparing a 3D hydrogel SERS substrate comprises the following steps:

[0036] S1: First, synthesize gold nanoclusters, then generate gold cores from the gold nanoclusters, and use 4-MBN to modify the gold cores to prepare gold core@4-MBN particles;

[0037] S2: On the basis of the gold core@4-MBN particles prepared in step S1, gold nanopetal-like structures are further grown to form gap structures, and then 4-MBA or 3-MPBA probe molecules are modified to prepare gold nanopetal gap particles@4-MBA or gold nanopetal gap particles@3-MPBA with internal standard function;

[0038] S3: The gold nanopetal gap particles @4-MBA or 3-MPBA prepared in step S2 are mixed with the hydrogel and freeze-dried to prepare a 3D hydrogel SERS substrate @4-MBA or a 3D hydrogel SERS substrate @3-MPBA.

[0039] In step S1, the gold core @ 4-MBN particles are prepared by the following method: first, a gold nanocluster solution is prepared, chloroauric acid and NaBH4 solution are added to a CTAC (tri(hexadecyl)methylammonium chloride) solution, ultrasonic treatment is performed until fully mixed, and then the mixture is allowed to stand for 0.5-1.5 hours to generate a gold nanocluster solution; then, chloroauric acid solution, ascorbic acid (AA) solution and gold nanocluster solution are added to the CTAC solution to obtain gold nanospheres; after the gold nanospheres are synthesized, 4-MBN solution is added for modification, ultrasonication is performed at room temperature, and the gold core @ 4-MBN particles are prepared.

[0040] The molar ratio of the CTAC solution, chloroauric acid and NaBH4 solution for generating the gold nanocluster solution is 100-2000:1-20:5-30.

[0041] The molar ratio of the CTAC solution, chloroauric acid, ascorbic acid and gold nanocluster solution for preparing the gold nanospheres is 100-2000:1-20:10-200, and the ratio of the gold nanospheres and the 4-MBN solution for modifying the gold nanospheres is 1-100 μL:0.1-5 mmol.

[0042] The ultrasonic time is 5-30 minutes, and centrifugal washing is performed after the ultrasonication, and the number of washing times is 1-3 times.

[0043] In step S2, the gold nanopetal gap particles @4-MBA or gold nanopetal gap particles @3-MPBA are prepared by the following method: the gold core @4-MBN particles prepared in step (1) are added to a mixture of CTAC solution, ascorbic acid and HAuCl4 to grow gold nanopetals to form a gap structure, and then 4-MBA or 3-MPBA solution is added to react for 0.5-1.5h, centrifuged and washed, and finally resuspended in CTAC solution for washing to prepare gold nanopetal gap particles @4-MBA or gold nanopetal gap particles @3-MPBA with internal standard function.

[0044] The molar ratio of the CTAC solution, AA, HAuCl4, 4-MBA or 3-MPBA solution is 10-1000: 1-20: 0.1-10: 10-500.

[0045] The concentration ratio of the CTAC solution for washing to the CTAC solution for growing the gold nano-petal-shaped gap structure is 0.01-1:1-50, and the volume ratio of the two is 1:1.

[0046] In the step S3, the 3D hydrogel SERS substrate @4-MBA or 3D hydrogel SERS substrate @3-MPBA is prepared by the following method: the concentrated solution of gold nanopetal gap particles @4-MBA or gold nanopetal gap particles @3-MPBA concentrated 5-50 times is mixed with an agarose solution with a concentration of 0.1-5wt% in a volume ratio of 1:1-1:5, poured into a mold, and then freeze-dried for 3-30 hours to obtain a new 3D hydrogel SERS substrate @4-MBA or 3D hydrogel SERS substrate @3-MPBA.

[0047] A 3D hydrogel SERS substrate prepared by a preparation method of a 3D hydrogel SERS substrate.

[0048] A 3D hydrogel SERS substrate was used to detect sweat pH as well as H2O2 and glucose in sweat.

[0049] The method for detecting the pH of sweat using the 3D hydrogel SERS substrate is as follows: the 3D hydrogel SERS substrate@4-MBA is placed on a glass slide, 10-1000 μL of sweat sample is then added to the substrate, and a portable Raman spectrometer is used to collect spectra.

[0050] The method for detecting H2O2 and glucose in sweat by using the 3D hydrogel SERS substrate is as follows: immersing the 3D hydrogel SERS substrate @3-MPBA in 10-1000 μL sweat sample, incubating it at an incubation temperature of 25-45° C. for 5-60 min, then placing it on a microscope slide, and using a portable Raman spectrometer to collect spectra.

[0051] Figure 1 This is a schematic diagram of the detection principle of the present invention. After the 3D hydrogel SERS substrate @4-MBA collects sweat, the portable Raman spectroscopy is used to directly collect signals. After the 3D hydrogel SERS substrate @3-MPBA is mixed with glucose oxidase and then sweat is collected, the portable Raman spectroscopy is used to directly collect signals.

[0052] Figure 2 This paper provides a conceptual overview of the fabrication and application of the 3D hydrogel SERS-based wearable sweat, pH, and glucose sensors of the present invention.

[0053] The pH tests in the following examples used artificial sweat samples with large pH differences; the samples used in glucose detection were actual sweat samples.

[0054] Example 1

[0055] A novel 3D hydrogel SERS substrate is prepared by the following method, which specifically includes the following steps:

[0056] (1) Synthesis of gold core@4-MBN: First, a gold cluster solution was prepared. 5 μmol chloroauric acid and 5 μmol NaBH4 solution were added to 10 mL 200 mM CTAC solution, respectively. The mixture was sonicated until fully mixed, and then the mixture was allowed to stand for 1 h to generate a gold nanocluster solution. Then, 5 μmol chloroauric acid solution, 10 μmol ascorbic acid (AA) solution, and 50 μL gold nanocluster solution were added to 10 mL 0.1 M CTAC solution, respectively, to obtain gold nanospheres. After the gold nanospheres were synthesized, 2.5 mmol 4-MBN solution was added for modification. The mixture was sonicated at room temperature for 20 min, washed three times, and resuspended in the original volume of 0.1 M CTAC solution to prepare gold core@4-MBN particles.

[0057] (2) Synthesis of gold nanopetal gap particles @4-MBA: The gold core @4-MBN solution was added to a 10 mL mixture of 0.1 M CTAC solution, 5 mM ascorbic acid and 5 mM HAuCl4 to grow gold nanopetals to form gap structures. Then 25 mL of 10 mM 4-MBA solution was added to the solution in the previous step, allowed to react for 1 hour, centrifuged and washed, and finally resuspended in the original volume of 0.1 M CTAC solution to prepare gold nanopetal gap particles @4-MBA with internal standard function, such as Figure 3 (a) shown.

[0058] (3) Preparation of 3D hydrogel SERS substrate @4-MBA: The gold nanopetal gap particles @4-MBA were concentrated 10 times and mixed with 1% wt agarose solution in a ratio of 1:2, poured into a mold, and then freeze-dried for 3 hours to obtain a novel 3D hydrogel SERS substrate @4-MBA, such as Figure 3 (b) and Figure 3 (c) as shown.

[0059] Example 2

[0060] A novel 3D hydrogel SERS substrate is prepared by the same method as in Example 1, except that the 4-MBA solution in step (2) is replaced by a 3-MPBA solution to obtain gold nanopetal gap particles @3-MPBA, and finally a 3D hydrogel SERS substrate @3-MPBA.

[0061] Example 3

[0062] The 3D hydrogel SERS substrate @4-MBA prepared in Example 1 was used for pH value determination. The specific method included: placing the 3D hydrogel SERS substrate @4-MBA on a glass slide, then adding 100 μL of PB buffer with different pH levels of 5.5-8 to the substrate, and then using a portable Raman spectrometer to collect spectra, such as Figure 4 As shown, it shows a good linear detection curve. Figure 4 This is a fitting diagram of Raman intensity and pH. Combined with the Raman intensity fitting diagram, the corresponding pH value can be found according to the Raman spectrum intensity.

[0063] Example 4

[0064] The 3D hydrogel SERS substrate @3-MPBA prepared in Example 2 was used for H2O2 and glucose detection: For H2O2 detection in the standard system, the 3D hydrogel SERS substrate @3-MPBA was immersed in 200 μL aqueous solution samples containing different concentrations of H2O2, such as Figure 5 As shown, the linear dynamic range of the assay is from 0.01 to 5 mM, R 2 is 0.9947. For glucose detection in the standard system, the substrate was immersed in a mixture of 200 μL aqueous solution samples containing different concentrations of glucose and 20 ul 10 mg / mL GOx (glucose oxidase) solution and incubated at 30 ° C for 30 min. The hydrogel was then placed on a microscope slide and the spectrum was collected using a portable Raman spectrometer. The detection linear range was 0.1 mM to 10 mM, such as Figure 6 shown.

[0065] like Figure 5 and 6 As shown, a fitting graph of Raman intensity and H2O2 and glucose concentration has been made. Combined with the Raman intensity fitting graph, the corresponding H2O2 and glucose concentrations can be found according to the Raman spectrum intensity.

[0066] Example 5

[0067] The 3D hydrogel SERS substrate @4-MBA prepared in Example 1 was used to measure the pH value in artificial sweat. The specific method included: placing the 3D hydrogel SERS substrate @4-MBA on a glass slide, then adding 100 μL of artificial sweat samples with different pH levels of 5.5-8 to the substrate, and then using a portable Raman spectrometer to collect spectra, such as Figure 7 As shown in a, the pH of the artificial sweat samples was highly correlated with the standard curve, and the pH test results of four sweat samples (pH 5.5, 6, 6.5, and 7.4) showed that the repeated measurement errors were 0.945%, 1.13%, 0.108%, and 0.486%, indicating that the pH detection accuracy was excellent.

[0068] The 3D hydrogel SERS substrate @3-MPBA prepared in Example 2 was used for actual glucose detection in sweat: For glucose detection in sweat, a sweat sample was first taken from a volunteer, and then the substrate was immersed in a mixture of 200 μL sweat sample containing different concentrations of glucose and 20 ul 10 mg / mL GOx (glucose oxidase) solution, and incubated at 30°C for 30 min. The hydrogel was then placed on a microscope slide and a portable Raman spectrometer was used for spectrum acquisition. Figure 7 As shown in b, the linear range of detection is 0.5 to 10 mM, and the linear correlation coefficient R 2 The linear regression equation for glucose detection is (y=0.028x±0.024), and the detection limit of glucose is 0.25 mM. These results show that the 3D hydrogel SERS chip provides convenient, reliable and rapid detection capabilities, and this detection platform has great potential in analyzing pH and glucose in sweat.

[0069] like Figure 7 As shown in Figs. 7a and 7b, fitting diagrams of Raman intensity and H2O2 and glucose concentration have been made respectively. Combined with the Raman intensity fitting diagram, the corresponding H2O2 and glucose concentrations can be found according to the Raman spectrum intensity.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a 3D hydrogel SERS substrate, characterized in that: The steps include: S1: First, synthesize gold nanoclusters, then generate gold cores from the gold nanoclusters, and use 4-MBN to modify the gold cores to prepare gold core@4-MBN particles; S2: On the basis of the gold core@4-MBN particles prepared in step S1, gold nanopetal-like structures are further grown to form gap structures, and then 4-MBA or 3-MPBA probe molecules are modified to prepare gold nanopetal gap particles@4-MBA or gold nanopetal gap particles@3-MPBA with internal standard function; S3: The gold nanopetal gap particles @4-MBA or 3-MPBA prepared in step S2 are mixed with the hydrogel and freeze-dried to prepare a 3D hydrogel SERS substrate @4-MBA or a 3D hydrogel SERS substrate @3-MPBA.

2. The preparation method according to claim 1, characterized in that In step S1, the gold core @ 4-MBN particles are prepared by the following method: adding chloroauric acid and NaBH4 solution to the CTAC solution, ultrasonically treating until fully mixed, and then allowing the mixture to stand for 0.5-1.5 hours to generate a gold nanocluster solution; then adding chloroauric acid solution, ascorbic acid solution and gold nanocluster solution to the CTAC solution to obtain gold nanospheres; after the gold nanospheres are synthesized, adding 4-MBN solution for modification, ultrasonicating at room temperature, washing and resuspending in the CTAC solution to prepare gold core @ 4-MBN particles.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the CTAC solution, chloroauric acid and NaBH4 solution for generating the gold nanocluster solution is 100-2000:1-20:5-30, the molar ratio of the CTAC solution, chloroauric acid, ascorbic acid and gold nanocluster solution for preparing the gold nanospheres is 100-2000:1-20:10-200, and the ratio of the gold nanospheres and the 4-MBN solution for modifying the gold nanospheres is 1-100 μL:0.1-5 mmol.

4. The preparation method according to claim 1, characterized in that: In step S2, the gold nanopetal gap particles @4-MBA or gold nanopetal gap particles @3-MPBA are prepared by the following method: the gold core @4-MBN particles prepared in step (1) are added to a mixture of CTAC solution, ascorbic acid and HAuCl4 to grow gold nanopetal-shaped gap structures, and then the probe molecule 4-MBA or 3-MPBA solution is added to react for 0.5-1.5 hours, centrifuged and washed, and finally resuspended in CTAC solution for washing to prepare gold nanopetal gap particles @4-MBA or gold nanopetal gap particles @3-MPBA with internal standard function.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the CTAC solution, ascorbic acid, HAuCl4, 4-MBA or 3-MPBA solution is 10-1000: 1-20: 0.1-10: 10-500, the concentration ratio of the CTAC solution for washing and the CTAC solution for growing gold nano-petal-shaped gap structures is 0.01-1: 1-50, and the volume ratio is 1:

1.

6. The preparation method according to claim 1, characterized in that: The 3D hydrogel SERS substrate @4-MBA / 3-MPBA in step S3 is prepared by the following method: a concentrated solution of gold nanopetal gap particles @4-MBA / 3-MPBA concentrated 5-50 times and an agarose solution with a concentration of 0.1-5wt% are mixed in a volume ratio of 1:1-1:5, poured into a mold, and then freeze-dried for 3-30 hours to obtain a new 3D hydrogel SERS substrate @4-MBA or 3D hydrogel SERS substrate @3-MPBA.

7. A 3D hydrogel SERS substrate prepared by the preparation method according to any one of claims 1 to 6.

8. An application of the 3D hydrogel SERS substrate as claimed in claim 7 in detecting sweat pH and H2O2 and glucose in sweat.

9. The use according to claim 8, characterized in that The method for detecting the pH of sweat is to place the 3D hydrogel SERS substrate @4-MBA on a glass slide, then add 10-1000 μL of sweat sample to the substrate, and use a portable Raman spectrometer to collect spectra.

10. The use according to claim 8, characterized in that The method for detecting H2O2 and glucose in sweat is to immerse the 3D hydrogel SERS substrate @3-MPBA in 10-1000 μL sweat sample, incubate it at an incubation temperature of 25-45°C for 5-60 minutes, then place it on a microscope slide, and use a portable Raman spectrometer to collect spectra.

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