Preparation method and application of hydrogel in-situ coated Au (at) Pt nano-enzyme flexible SERS (Surface Enhanced Raman Scattering) substrate

Abstract

The invention belongs to the technical field of Raman spectroscopy, and particularly discloses a preparation method and application of a hydrogel in-situ coated Au-Pt nano-enzyme flexible SERS (Surface Enhanced Raman Scattering) substrate, and the preparation method comprises the following steps: synthesis of concentrated gold nanoparticles: stirring and heating a HAuCl4 aqueous solution to boiling, quickly adding sodium citrate, stirring and heating to obtain a concentrated gold nanoparticle solution; heating and refluxing the mixed solution for 10-60 minutes until the mixed solution becomes wine red; gradually cooling to room temperature while stirring to obtain concentrated gold nanoparticles; and preparation of the hydrogel in-situ coated Au-Pt nano-enzyme flexible SERS substrate: mixing the hydrogel and concentrated gold nanoparticles according to a volume ratio, then adding H2PtCl6 and ascorbic acid, and carrying out a reaction to obtain the hydrogel in-situ coated Au-Pt nano-enzyme flexible SERS substrate Au-Pt-Atar. According to the prepared Au-Pt-Agar, due to the fact that the Au-Pt-Agar effectively coats the nano particles, the influence of the external environment (oxygen, acid, alkali and the like) on the nano particles is prevented, the particles are evenly dispersed in the hydrogel, and the detection uniformity and stability of an SERS substrate are improved under the condition that the sensitivity is guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of Raman spectroscopy, and in particular relates to a preparation method and application of a hydrogel in-situ coated Au@Pt nanozyme flexible SERS substrate. Background Art

[0002] Glutathione (GSH) is the most common non-protein biothiol in living systems and plays a key role in eliminating free radicals, improving immunity, maintaining the body's proper physiological redox environment, and gene regulation in physiological processes. Abnormal glutathione levels are believed to be associated with many diseases, such as cancer, human immunodeficiency virus (HIV), diabetes, liver damage, and Alzheimer's disease. In order to diagnose related diseases, it is urgent to develop sensors that can simultaneously detect glutathione. Many methods have been developed to detect GSH, such as high-performance liquid chromatography (HPLC), mass spectrometry (MS), fluorescence spectroscopy, and electrochemical analysis. However, these methods have disadvantages such as long detection time, cumbersome sample pretreatment, and poor selectivity. Surface-enhanced Raman scattering (SERS) is a powerful analytical method with the characteristics of fast detection speed, high sensitivity, and non-destructiveness, and can even achieve trace analysis at the single-molecule level. The key challenge to obtaining reliable SERS measurements is to prepare a plasmonic SERS substrate with excellent performance. Nano-gold and silver are the most commonly used SERS active substrates. Although such SERS substrates have a good enhancement effect, they have some inherent defects: (1) The random distribution of metal nanoparticles and the irregular arrangement of the molecules to be tested on the nanoparticles lead to poor repeatability of SERS detection. (2) The bare metal nanoparticles are easily contaminated by the surrounding environment, causing the stability of the substrate to decrease, resulting in inaccurate test results. Therefore, there is an urgent need to develop SERS active substrates with high sensitivity, high repeatability, stability and uniformity to achieve reliable quantitative detection of small biological molecules.

[0003] Nanozymes are a class of artificial enzyme mimics that have the unique properties of nanomaterials, and their catalytic efficiency and enzymatic reaction kinetics are similar to those of natural enzymes. Compared with natural enzymes, nanozymes have obvious advantages such as low cost, easy storage, high and adjustable catalytic activity, high stability, and easy mass production. They have been applied in biological detection, environmental monitoring, disease diagnosis, and biomedical development. Au@Pt nanoparticles (NPs) have excellent enzyme mimicking activity due to their platinum shell and unique surface plasmon resonance characteristics, which has aroused great research interest among scientific researchers. However, the unique surface plasmon resonance characteristics of the gold core have not been widely utilized, and the practical problem of the light damping properties of Pt has hindered the combination of the enzyme mimicking properties of Au@PtNPs with their surface enhanced Raman scattering (SERS) activity.

[0004] Therefore, it is very necessary to develop a new method for preparing Au@Pt nanozyme flexible SERS substrate by in situ hydrogel coating. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide a preparation method and application of a hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate. Based on the excellent peroxidase-like properties of Au@Pt-Agar and combined with the hydrogel encapsulation, the prepared SERS substrate is selective, uniform and stable, can quickly detect GSH, has low cost and high sensitivity.

[0006] In order to achieve the above objectives, one of the technical solutions of the present invention is: a method for preparing a hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate, the specific steps are as follows:

[0007] S1. Synthesis of concentrated gold nanoparticles: Stir and heat the HAuCl4 aqueous solution to boiling, quickly add sodium citrate, and heat the mixed solution under reflux for 10-60 minutes until it turns wine red; then gradually cool to room temperature under stirring to obtain concentrated gold nanoparticles;

[0008] S2. Preparation of a flexible SERS substrate with Au@Pt nanozyme in situ coated by hydrogel: Mix the hydrogel and concentrated gold nanoparticles in a volume ratio, then add H2PtCl6 and ascorbic acid to react to obtain a flexible SERS substrate with Au@Pt nanozyme in situ coated by hydrogel, Au@Pt-Agar.

[0009] In a preferred embodiment of the present invention, the mass fraction of HAuCl4 in step S1 is 0.1wt%-1wt%, and the mass fraction of sodium citrate is 0.5wt%-5wt%.

[0010] In a preferred embodiment of the present invention, the reaction condition in step S2 is heating in a water bath at 50°C-100°C.

[0011] In a preferred embodiment of the present invention, the concentration of H2PtCl6 in step S2 is 1-10 mM, and the concentration of ascorbic acid is 2-20 mM.

[0012] In a preferred embodiment of the present invention, in step S2, the feed ratio of Au and Pt is controlled by adding different volumes of H2PtCl6 and ascorbic acid to obtain Au@P with different shell thicknesses; the volume ratio of H2PtCl6 and ascorbic acid is (1-3):1, and the feed ratio of Au and Pt is (2-11):1.

[0013] In order to achieve the above objectives, the second technical solution of the present invention is: a hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate obtained by the above preparation method.

[0014] In order to achieve the above objectives, the third technical solution of the present invention is: an application of a flexible SERS substrate in which the hydrogel in situ-coated Au@Pt nanozyme is obtained by the above preparation method in the detection of GSH.

[0015] In a preferred embodiment of the present invention, the specific steps of applying the hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate in detecting GSH are as follows: the prepared hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate is immersed in parallel in 100-1000 μL of a mixed solution of TMB (3,3',5,5'-tetramethylbenzidine) and H2O2 of different concentrations in equal volumes, reacted in a 37°C constant temperature box for 20-60 minutes, and then SERS detection is performed; the excitation wavelength used is 633 nm, the integration time is 10 s, and the number of integrations is 1; the 1611 cm -1 The relationship between the SERS peak intensity at and TMB concentration was established, and the linear regression equation y = 144725.03x + 929776.19 was obtained, and the correlation coefficient R 2 is 0.9988, and TMB is used as a standard reference. Then, Au@Pt-Agar is incubated with TMB and hydrogen peroxide concentrations corresponding to the strongest SERS peak. The corresponding SERS spectra are measured after 20-60 min of reaction. Then, GSH solutions of different concentrations are added to measure the SERS intensity. -1 The intensity difference at the point was related to the GSH concentration to obtain a linear regression equation y = 0.1687x + 5.36, and the correlation coefficient R 2 is 0.9925; Finally, the SERS peak was measured at 1611cm before and after the sample was dropped on the SERS substrate. -1 Substituting the intensity difference at into the corresponding linear regression equation, the concentration of the sample to be tested can be obtained.

[0016] By adopting the above technical solution: Au@Pt nanozyme as SERS enhancement substrate is uniformly loaded inside the hydrogel, and a SERS flexible substrate with high sensitivity, high repeatability and uniform stability is obtained. Based on the redox reaction, Au@Pt-Agar placed in TMB and hydrogen peroxide solution catalyzes TMB to obtain its blue oxide, which has a strong SERS signal. Due to the reducibility of GSH, the blue oxide turns back to TMB, and the SERS signal weakens. According to the strongest peak 1611cm in its intrinsic SERS spectrum, -1 The intensity difference is used to detect the GSH concentration.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The Au@Pt-Agar prepared by the present invention prevents the influence of the external environment (oxygen, acid and alkali, etc.) on the nanoparticles due to its effective coating of the nanoparticles. The particles are evenly dispersed inside the hydrogel, which improves the detection uniformity and stability of the SERS substrate while ensuring the sensitivity.

[0019] 2. The Au@Pt nanozyme involved in the present invention can catalyze the TMB reaction, has strong environmental stability and strong catalytic activity. The bimetallic component can provide additional tunability to the surface and significantly improve the catalytic activity. Compared with other metal-based materials, it has higher catalytic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 (a) is a physical picture of Au@Pt-Agar synthesized in situ according to the present invention, and (b) is a transmission electron microscopy picture of Au@Pt nanozymes with different shell layers in the in situ synthesized hydrogel;

[0021] Figure 2 (a) is a transmission electron micrograph F200 of the Au@Pt nanozyme in the hydrogel synthesized in situ according to the present invention, and (b) is a scanning electron micrograph of the hydrogel SERS substrate loaded with Au@Pt nanozyme;

[0022] Figure 3 (a) is a line graph showing the SERS peak intensity of Au@Pt-Agar prepared by the present invention with different Pt shell thicknesses, and (b) is a uniformity test of 20 randomly selected points on Au@Pt-Agar;

[0023] Figure 4 (a) is a SERS spectrum of Au@Pt-Agar prepared by the present invention detecting different concentrations of TMB, (b) is a UV spectrum of Au@Pt-Agar detecting different concentrations of TMB;

[0024] Figure 5 (a) is a graph showing the SERS peak intensity and TMB concentration of Au@Pt-Agar prepared in the present invention detecting different concentrations of TMB; (b) is a graph showing the SERS peak intensity of the oxide obtained by Au@Pt-Agar catalyzing TMB at 1611 cm -1 The linear relationship between the peak intensity at and the logarithm of TMB concentration;

[0025] Figure 6 (a) is a graph showing the SERS peak intensity difference between Au@Pt-Agar prepared in the present invention and GSH concentration when detecting different concentrations of GSH; (b) is a graph showing the difference in SERS peak intensity at 1611 cm after oxTMB generated by Au@Pt-Agar catalytic oxidation and then reduced by GSH.-1 Linear relationship between the logarithm of the peak intensity and the logarithm of the GSH concentration.

[0026] Figure 7 (a) is the SERS spectra of different concentrations of GSH in the serum samples in Example 7, and (b) is the SERS spectra of GSH in serum at 1611 cm -1 The linear relationship between the logarithm of the peak intensity difference and the logarithm of its concentration. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0028] A method for preparing a hydrogel in-situ coated Au@Pt nanozyme flexible SERS substrate, the specific steps are as follows:

[0029] S1. Synthesis of concentrated gold nanoparticles: Stir and heat the HAuCl4 aqueous solution to boiling, quickly add sodium citrate, and heat the mixed solution under reflux for 10-60 minutes until it turns wine red; then gradually cool to room temperature under stirring to obtain concentrated gold nanoparticles;

[0030] S2. Preparation of a flexible SERS substrate with Au@Pt nanozyme in situ coated by hydrogel: Mix the hydrogel and concentrated gold nanoparticles in a volume ratio, then add H2PtCl6 and ascorbic acid to react to obtain a flexible SERS substrate with Au@Pt nanozyme in situ coated by hydrogel.

[0031] In the step S1, the mass fraction of HAuCl4 is 0.1wt%-1wt%, and the mass fraction of sodium citrate is 0.5wt%-5wt%.

[0032] The reaction condition in step S2 is heating in a water bath at 50°C-100°C.

[0033] In step S2, the concentration of H2PtCl6 is 1-10 mM, and the concentration of ascorbic acid is 2-20 mM.

[0034] In step S2, the feed ratio of Au and Pt is controlled by adding different volumes of H2PtCl6 and ascorbic acid to obtain Au@Pt with different shell thicknesses; the volume ratio of H2PtCl6 and ascorbic acid is (1-3):1, and the feed ratio of Au and Pt is (2-11):1.

[0035] A flexible SERS substrate in situ coated with Au@Pt nanozyme by hydrogel obtained by the preparation method.

[0036] A flexible SERS substrate in which the hydrogel in situ coated Au@Pt nanozyme is obtained by the above preparation method is used for detecting GSH.

[0037] The specific steps of applying the hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate in detecting GSH are as follows: the prepared hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate is immersed in parallel in 100-1000 μL of a mixed solution of TMB (3,3',5,5'-tetramethylbenzidine) and H2O2 of different concentrations in equal volumes, reacted in a 37°C constant temperature box for 20-60 minutes, and then SERS detection is performed; the excitation wavelength used is 633 nm, the integration time is 10 s, and the number of integrations is 1; the 1611 cm -1 The relationship between the SERS peak intensity at and TMB concentration was established, and the linear regression equation y = 144725.03x + 929776.19 was obtained, and the correlation coefficient R 2 is 0.9988, and TMB is used as a standard reference. Then, Au@Pt-Agar is incubated with TMB and hydrogen peroxide concentrations corresponding to the strongest SERS peak. The corresponding SERS spectra are measured after 20-60 min of reaction. Then, GSH solutions of different concentrations are added to measure the SERS intensity. -1 The intensity difference at the point was related to the GSH concentration to obtain a linear regression equation y = 0.1687x + 5.36, and the correlation coefficient R 2 is 0.9925; Finally, the SERS peak was measured at 1611cm before and after the sample was dropped on the SERS substrate. -1 Substituting the intensity difference at into the corresponding linear regression equation, the concentration of the sample to be tested can be obtained.

[0038] Example 1

[0039] A hydrogel in-situ coated Au@Pt nanozyme flexible SERS substrate is prepared by the following method, and the specific steps are as follows:

[0040] S1. Synthesis of concentrated Au: 200 mL of 0.825 wt% HAuCl4 aqueous solution was heated to boiling under magnetic stirring, and 8.0 mL of 1 wt% sodium citrate aqueous solution was quickly added, and the mixed solution was heated to reflux for 30 min until the solution turned into wine red; after gradually cooling to room temperature under stirring, concentrated gold nanoparticles were obtained;

[0041] S2. Preparation of flexible SERS substrates with Au@Pt nanozyme coated in situ by hydrogel: 8 ml of hydrogel and 4 ml of concentrated gold nanoparticles were mixed; then, different volumes of 2 mM H2PtCl6 and 20 mM ascorbic acid were added to the mixed solution to ensure that the volume ratio of H2PtCl6:AA was 2:1, and hydrogel-coated Au@Pt nanozyme flexible SERS substrates Au@Pt-Agar with different Pt shell thicknesses (0.7 nm, 1.4 nm, 3.5 nm, 6.8 nm) were obtained. The actual picture is as follows Figure 1 As shown in a.

[0042] Example 2

[0043] Characterization of a hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate:

[0044] The Au@Pt nanoparticles in the Au@Pt-Agar with different shell layers synthesized in situ in Example 1 were characterized by TEM. Figure 1 As shown in Figure b, the size of the Au@Pt nanoparticles synthesized in situ is uniform, with a particle size of about 45 nm. The Au outer layer is coated with Pt nanoclusters. As the volume of H2PtCl6 and AA is added and the feed ratio of Au and Pt is controlled, the thickness of the Pt shell changes accordingly. Next, the Au@Pt nanoparticles in the in situ synthesized hydrogel were characterized by transmission electron microscopy F200, as shown in Figure 2 Figure 2 As shown in a, it is confirmed that the nanoparticles in the hydrogel contain gold and platinum, and platinum is successfully attached to the surface of the gold particles. Finally, Au@Pt-Agar was characterized by SEM. The hydrogel showed a loose and porous layered structure, and the nanoparticles were evenly dispersed in the pore structure of the hydrogel. This uniformly distributed SERS substrate helps to improve the repeatability of SERS detection.

[0045] Example 3

[0046] Evaluation of the optimal Pt shell thickness of the hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate:

[0047] Au@Pt with different shell thicknesses in the hydrogel, such as 0.7 nm, 1.4 nm, 2.8 nm, 3.5 nm, 6.8 nm and a blank control group, were prepared in situ by Example 1, and incubated for 20 min in the original concentration of TMB+H2O2 and ten-fold diluted TMB+H2O2, respectively, while keeping the other conditions the same, and then SERS detection was performed to draw a line graph of the SERS peak intensity with different Pt shell thicknesses, as shown in FIG. Figure 3 As shown in a. It can be seen from the figure that Au@Pt-Agar with a shell thickness of 0.7nm has the best comprehensive performance.

[0048] Example 4

[0049] Repeatability evaluation of Au@Pt nanozyme in situ coated on flexible SERS substrate by hydrogel:

[0050] In order to prove the detection repeatability of the prepared Au@Pt-Agar, the present invention randomly selected 20 positions on the same Au@Pt nanozyme hydrogel SERS substrate for SERS measurement. Figure 3 As shown in b, the strongest signal is located at 1611cm -1 The peak at was selected to evaluate the repeatability of the SERS substrate. The relative standard deviation (RSD) of the Raman signal was 2.472%, which met the requirement of RSD less than 20%. The above data show that the hydrogel SERS substrate loaded with Au@Pt nanozymes has good reproducibility and uniformity, and can be used to accurately detect glutathione in actual samples.

[0051] Example 5

[0052] SERS determination of different concentrations of TMB:

[0053] First, prepare TMB standard solutions of different concentrations. Figure 4 As shown in a, the present invention measured the oxides obtained by catalyzing different concentrations of TMB on the Au@Pt nanozyme hydrogel SERS substrate at 1611 cm -1 The experimental results show that the SERS signal intensity of TMB oxide gradually increases with the increase of TMB concentration, such as Figure 5 a. The detection limit of this method for TMB is 6.24×10 -11 M, compared with the detection limit of 6.24×10 -5 M( Figure 4 b) has a high sensitivity. -1 The SERS peak intensity at 100 nm was related to the logarithm of TMB concentration, and a good linear relationship was found between the two ( Figure 5 b) The linear regression equation is y = 144725.03x + 929776.19, and the correlation coefficient is R 2 The detection limit of TMB is 0.9988. The lower the detection limit of TMB as a standard reference, the stronger its ability to detect GSH is.

[0054] Example 6

[0055] SERS determination of different concentrations of GSH:

[0056] First, GSH standard solutions of different concentrations were prepared, and GSH of different concentrations was added to the Au@Pt nanozyme hydrogel SERS substrate for 3 min. The SERS peaks before and after the addition of GSH solution were measured to find the SERS peak at 1611 cm -1The peak intensity difference and GSH concentration are used to draw a curve, such as Figure 6 The Au@Pt nanozyme hydrogel SERS substrate was used to measure the GSH concentration at 1611 cm -1 The peak intensity difference at the 200 nm position was taken and its logarithm was used to establish a linear relationship with the logarithm of the GSH concentration ( Figure 6 b) The linear regression equation is y = 0.1687x + 5.36, and the correlation coefficient is R 2 Reached 0.9925.

[0057] Example 7

[0058] SERS determination of GSH in serum samples:

[0059] Serum taken from the hospital was centrifuged and diluted to obtain a ten-fold diluted serum solution, which was then used to gradually dilute the GSH standard solution. GSH of different concentrations and the same volume was dropped on the Au@Pt-Agar surface and allowed to stand for 3 minutes. The SERS peak intensity before and after the addition of GSH was measured, and the SERS peak difference was calculated. -1 The peak intensity difference at the 200 nm position is plotted against the GSH concentration. Figure 7 a. Then 1611cm -1 A linear relationship was established between the logarithm of the SERS peak intensity difference at and the logarithm of the GSH concentration ( Figure 7 b), the linear regression equation is y = 0.2336x + 5.2956, and the linear correlation coefficient is R 2 Reached 0.9968.

[0060] Au@Pt NPs with an Au:Pt feed ratio of 11:1 produced the highest Raman signal within 40 min, which was due to the strong electric field retained by the modified Pt and the gold core, which significantly accelerated the catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine. This study not only demonstrates the prospect of combining multiple functions of bimetallic nanomaterials, but also provides a reasonable guide for the design of high-performance nanozymes with potential biomedical applications.

[0061] In this study, Au@Pt-Agar with excellent peroxidase-like activity was prepared, which can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. Under acidic conditions, GSH with reducing ability can inhibit the oxidation of TMB, resulting in a reduction in the formation of blue oxygen TMB and a weakening of the SERS signal. Based on the excellent peroxidase-like properties of Au@Pt-Agar and combined with hydrogel encapsulation, a selective, uniform and stable SERS substrate was prepared, which can quickly detect GSH with low cost and high sensitivity. In addition, naked eye analysis and UV-vis spectrometer were compared, and this method is superior to previous methods in terms of sensitivity and detection range.

[0062] In summary, the in-situ preparation method of the Au@Pt nanozyme hydrogel SERS substrate involved in the present invention is simple and efficient, the SERS substrate is uniform, and the interference of other substances on the detection of the target object is reduced. In addition, the dense pore structure on the surface of the hydrogel can exclude the passage of interfering substances and only allow the target small molecules to enter, avoiding complex and time-consuming sample pretreatment steps, and having a certain enrichment effect, realizing the rapid and direct detection of GSH in real samples.

[0063] 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 flexible SERS substrate with Au@Pt nanozyme in situ coated with hydrogel, characterized in that: The steps include: S1. Synthesis of concentrated gold nanoparticles: Stir and heat the HAuCl4 aqueous solution to boiling, quickly add sodium citrate, and heat the mixed solution under reflux for 10-60 minutes until it turns wine red; then gradually cool to room temperature under stirring to obtain concentrated gold nanoparticles; S2. Preparation of a flexible SERS substrate with Au@Pt nanozyme in situ coated by hydrogel: Mix the hydrogel and concentrated gold nanoparticles in a volume ratio, then add H2PtCl6 and ascorbic acid to react to obtain a flexible SERS substrate with Au@Pt nanozyme in situ coated by hydrogel, Au@Pt-Agar.

2. The method for preparing a flexible SERS substrate with Au@Pt nanozyme in situ coated with hydrogel according to claim 1, characterized in that: The mass fraction of HAuCl4 in step S1 is 0.1wt%-1wt%.

3. The method for preparing a flexible SERS substrate with Au@Pt nanozyme in situ coated with hydrogel according to claim 1, characterized in that: The mass fraction of sodium citrate in step S1 is 0.5wt%-5wt%.

4. The method for preparing a flexible SERS substrate with Au@Pt nanozyme in situ coated with hydrogel according to claim 1, characterized in that: The reaction condition in step S2 is heating in a water bath at 50°C-100°C.

5. The method for preparing a flexible SERS substrate with Au@Pt nanozyme in situ coated with hydrogel according to claim 1, characterized in that: The concentration of H2PtCl6 in step S2 is 1-10 mM.

6. The method for preparing a flexible SERS substrate with Au@Pt nanozyme in situ coated with hydrogel according to claim 1, characterized in that: The concentration of ascorbic acid in step S2 is 2-20 mM.

7. The method for preparing a flexible SERS substrate with Au@Pt nanozyme in situ coated with hydrogel according to claim 1, characterized in that: In step S2, the volume ratio of H2PtCl6 and ascorbic acid is (1-3):1, and the feed ratio of Au and Pt is (2-11):

1.

8. The hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate obtained by the preparation method of the hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate as described in any one of claims 1 to 7.

9. Use of the hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate as claimed in claim 8 in detecting GSH.

10. The use of the hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate in detecting GSH as claimed in claim 9, characterized in that: The method comprises the following steps: immersing the prepared hydrogel in situ coated Au@Pt nanozyme flexible SERS substrate in parallel in 100-1000 μL of a mixed solution of TMB (3,3',5,5'-tetramethylbenzidine) and H2O2 of different concentrations in equal volumes, reacting in a 37°C constant temperature box for 20-60 minutes, and then performing SERS detection; the excitation wavelength used is 633 nm, the integration time is 10 s, and the number of integrations is 1; the 1611 cm -1 The relationship between the SERS peak intensity at and TMB concentration was established, and the linear regression equation y = 144725.03x + 929776.19 was obtained, and the correlation coefficient R 2 is 0.9988, and TMB is used as a standard reference. Au@Pt-Agar is incubated with TMB and hydrogen peroxide concentrations corresponding to the strongest SERS peak. The corresponding SERS spectra are measured after 20-60 min of reaction. Then, GSH solutions of different concentrations are added to measure the SERS intensity. -1 The intensity difference at the point was related to the GSH concentration to obtain a linear regression equation y = 0.1687x + 5.36, and the correlation coefficient R 2 is 0.9925; the SERS peak measured before and after the sample was dropped on the SERS substrate was 1611cm -1 Substituting the intensity difference at into the corresponding linear regression equation, the concentration of the sample to be tested can be obtained.

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