Preparation method and application of hydrogel in situ coated Au@Pt nanoszyme flexible SERS substrate

Abstract

The application belongs to the technical field of Raman spectrum, and specifically discloses a preparation method of a hydrogel in-situ coated Au@Pt nanometer enzyme flexible SERS substrate and application, wherein the preparation method comprises the following steps: concentrated gold nanoparticle synthesis: stirring and heating HAuCl4 aqueous solution to boiling, quickly adding sodium citrate, heating the mixed solution to reflux for 10-60 min until becoming wine red; then gradually cooling to room temperature under stirring to obtain concentrated gold nanoparticles; preparation of hydrogel in-situ coated Au@Pt nanometer enzyme flexible SERS substrate: mixing hydrogel and concentrated gold nanoparticles according to the volume ratio, then adding H2PtCl6 and ascorbic acid, and reacting to obtain the hydrogel in-situ coated Au@Pt nanometer enzyme flexible SERS substrate Au@Pt-Agar. The Au@Pt-Agar prepared by the application can effectively coat the nanoparticles to prevent the influence of external environment (oxygen, acid and alkali, etc.) on the nanoparticles, and the particles are uniformly dispersed in the hydrogel, so that the detection uniformity and stability of the SERS substrate are improved under the condition of ensuring the sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of Raman spectroscopy technology, and particularly relates to a method for preparing and applying a flexible SERS substrate with Au@Pt nanozyme in situ coated with hydrogel. Background Technology

[0002] Glutathione (GSH) is the most common non-protein biothiol in living systems, playing a crucial role in eliminating free radicals, enhancing immunity, maintaining an appropriate physiological redox environment, and regulating genes in physiological processes. Abnormal glutathione levels are believed to be associated with many diseases, such as cancer, HIV, diabetes, liver damage, and Alzheimer's disease. Therefore, there is an urgent need to develop sensors that simultaneously detect glutathione for diagnosing these diseases. Currently, many methods for detecting GSH have been developed, such as high-performance liquid chromatography (HPLC), mass spectrometry (MS), fluorescence spectroscopy, and electrochemical analysis. However, these methods suffer from drawbacks such as long detection times, cumbersome sample pretreatment, and poor selectivity. Surface-enhanced Raman scattering (SERS), on the other hand, is a powerful analytical method with advantages such as fast detection speed, high sensitivity, and non-destructive nature, and can even achieve trace analysis at the single-molecule level. A key challenge in obtaining reliable SERS measurements is the preparation of high-performance plasma SERS substrates. Nanoparticles of gold and silver are the most commonly used active substrates for SERS. 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 analytes on the nanoparticles lead to poor repeatability of SERS detection. (2) Bare metal nanoparticles are easily contaminated by the surrounding environment, causing a decrease in substrate stability and resulting in inaccurate test results. Therefore, it is urgent to develop SERS active substrates with high sensitivity, high repeatability, and stable uniformity to achieve reliable quantitative detection of small biological molecules.

[0003] Nanozymes are a class of artificially induced enzymes that possess the unique properties of nanomaterials, exhibiting catalytic efficiency and enzymatic reaction kinetics similar to natural enzymes. Compared to natural enzymes, nanozymes offer significant advantages such as low cost, ease of storage, high and tunable catalytic activity, high stability, and ease of mass production, leading to their application in fields such as biosensoring, environmental monitoring, disease diagnosis, and biomedical development. Au@Pt nanoparticles (NPs), due to their platinum shell and unique surface plasmon resonance characteristics, possess excellent enzyme-mimicking activity, attracting considerable research interest. However, the unique surface plasmon resonance characteristics of the gold core have not yet been widely utilized, and the practical limitations of Pt's photodamping properties hinder the integration of Au@PtNPs' enzyme-mimicking properties with their surface-enhanced Raman scattering (SERS) activity.

[0004] Therefore, it is essential to develop a novel method for preparing a flexible SERS substrate with in-situ hydrogel coating of Au@Pt nanozymes. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the existing technology and provide a method for preparing a flexible SERS substrate with Au@Pt nanozyme in situ encapsulated by hydrogel and its application. Based on the excellent peroxidase-like properties of Au@Pt-Agar and combined with the encapsulation of hydrogel, the prepared SERS substrate is selective, uniform and stable, can quickly detect GSH, and is low in cost and highly sensitive.

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

[0007] S1. Synthesis of concentrated gold nanoparticles: HAuCl4 aqueous solution was stirred and heated to boiling, sodium citrate was quickly added, and the mixed solution was heated under reflux for 10-60 min until it turned wine red; then it was gradually cooled to room temperature with stirring to obtain concentrated gold nanoparticles.

[0008] S2. Preparation of a flexible SERS substrate with in-situ hydrogel-coated Au@Pt nanozyme: Hydrogel and concentrated gold nanoparticles were mixed in a volume ratio, and then H2PtCl6 and ascorbic acid were added to obtain a flexible SERS substrate with in-situ hydrogel-coated Au@Pt nanozyme, 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 conditions in step S2 are water bath heating 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 feeding 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 to ascorbic acid is (1-3):1, and the feeding ratio of Au and Pt is (2-11):1.

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

[0014] To achieve the above objectives, the third technical solution of the present invention is: the application of a flexible SERS substrate for in-situ encapsulation of Au@Pt nanozymes with hydrogel obtained by the above preparation method in the detection of GSH.

[0015] In a preferred embodiment of the present invention, the specific steps for applying the hydrogel-coated Au@Pt nanozyme flexible SERS substrate in the detection of GSH are as follows: the prepared hydrogel-coated Au@Pt nanozyme flexible SERS substrate is immersed in 100-1000 μL of equal volumes of TMB (3,3',5,5'-tetramethylbenzidine) and H2O2 mixed solutions of different concentrations, reacted at 37°C for 20-60 min, and then SERS detection is performed; the excitation wavelength is 633 nm, the integration time is 10 s, and the integration is performed once; a 1611 cm⁻¹ substrate is then used. -1 A relationship was established between the SERS peak intensity and TMB concentration, yielding a linear regression equation: y = 144725.03x + 929776.19, with a correlation coefficient R0. 2 The value is 0.9988, and the TMB measurement is used as a standard reference. Then, Au@Pt-Agar is incubated with the TMB corresponding to the strongest SERS peak and the hydrogen peroxide concentration. After reacting for 20-60 min, the corresponding SERS spectrum is measured. Then, different concentrations of GSH solution are added to measure the SERS intensity at 1611 cm⁻¹. -1 The relationship between the intensity difference at a given location and the GSH concentration was established, yielding a linear regression equation: y = 0.1687x + 5.36, with a correlation coefficient R0. 2 It is 0.9925; finally, the SERS peak was measured at 1611 cm⁻¹ before and after the sample was dropped onto the SERS substrate. -1 The concentration of the sample can be obtained by substituting the intensity difference at each point into the corresponding linear regression equation.

[0016] By employing the above technical solution, Au@Pt nanozymes, serving as a SERS-enhancing substrate, are uniformly loaded within a hydrogel, resulting in a flexible SERS substrate possessing high sensitivity, high repeatability, and uniform stability. Based on redox reactions, Au@Pt-Agar placed in TMB and hydrogen peroxide solutions catalyzes the formation of a blue oxide from TMB, exhibiting a strong SERS signal. However, due to the reducing properties of GSH, the blue oxide reverts back to TMB, weakening the SERS signal. The strongest peak in its intrinsic SERS spectrum is 1611 cm⁻¹. -1 The intensity difference is used to detect GSH concentration.

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

[0018] 1. The Au@Pt-Agar prepared by this invention effectively encapsulates the nanoparticles, preventing the influence of the external environment (oxygen, acid and alkali, etc.) on the nanoparticles. The particles are uniformly dispersed inside the hydrogel, which improves the detection uniformity and stability of the SERS substrate while ensuring sensitivity.

[0019] 2. The Au@Pt nanozyme involved in this invention can catalyze the TMB reaction, exhibiting strong environmental stability and powerful catalytic activity. The bimetallic component provides additional tunability to the surface and significantly enhances catalytic activity. Compared with other metal-based materials, it demonstrates higher catalytic efficiency. Attached Figure Description

[0020] Figure 1 (a) is a physical image of the Au@Pt-Agar synthesized in situ according to the present invention, and (b) is a transmission electron microscope image of Au@Pt nanozymes with different shells in the hydrogel synthesized in situ.

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

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

[0023] Figure 4 (a) is the SERS spectrum of Au@Pt-Agar prepared in this invention for detecting different concentrations of TMB, and (b) is the UV spectrum of Au@Pt-Agar for detecting different concentrations of TMB.

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

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

[0026] Figure 7 (a) SERS spectra of different concentrations of GSH in serum samples from Example 7; (b) SERS spectra of GSH in serum at 1611 cm⁻¹. -1 A graph showing the linear relationship between the logarithm of the peak intensity difference and the logarithm of its concentration. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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 scope of protection of the present invention. The embodiments described in this invention are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0028] A method for preparing a flexible SERS substrate with Au@Pt nanozyme in situ encapsulated by hydrogel, the specific steps of which are as follows:

[0029] S1. Synthesis of concentrated gold nanoparticles: HAuCl4 aqueous solution was stirred and heated to boiling, sodium citrate was quickly added, and the mixed solution was heated under reflux for 10-60 min until it turned wine red; then it was gradually cooled to room temperature with stirring to obtain concentrated gold nanoparticles.

[0030] S2. Preparation of a flexible SERS substrate with in-situ hydrogel-coated Au@Pt nanozyme: Hydrogel and concentrated gold nanoparticles were mixed in a volume ratio, and then H2PtCl6 and ascorbic acid were added to react and obtain a flexible SERS substrate with in-situ hydrogel-coated Au@Pt nanozyme.

[0031] In 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 conditions in step S2 are water bath heating at 50℃-100℃.

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

[0035] A flexible SERS substrate with in-situ encapsulated Au@Pt nanozymes by a hydrogel prepared using the above method.

[0036] Application of a flexible SERS substrate for in-situ encapsulation of Au@Pt nanozymes with hydrogel obtained by the above preparation method in the detection of GSH.

[0037] The specific steps for applying the hydrogel-coated Au@Pt nanozyme flexible SERS substrate in GSH detection are as follows: The prepared hydrogel-coated Au@Pt nanozyme flexible SERS substrate is immersed in 100-1000 μL of equal volumes of TMB (3,3',5,5'-tetramethylbenzidine) and H2O2 mixed solutions of different concentrations, and reacted at 37℃ for 20-60 min before SERS detection; the excitation wavelength is 633 nm, the integration time is 10 s, and the integration is performed once; a 1611 cm⁻¹ substrate is then used. -1 A relationship was established between the SERS peak intensity and TMB concentration, yielding a linear regression equation: y = 144725.03x + 929776.19, with a correlation coefficient R0. 2 The value is 0.9988, and the TMB measurement is used as a standard reference. Then, Au@Pt-Agar is incubated with the TMB corresponding to the strongest SERS peak and the hydrogen peroxide concentration. After reacting for 20-60 min, the corresponding SERS spectrum is measured. Then, different concentrations of GSH solution are added to measure the SERS intensity at 1611 cm⁻¹. -1 The relationship between the intensity difference at a given location and the GSH concentration was established, yielding a linear regression equation: y = 0.1687x + 5.36, with a correlation coefficient R0. 2 It is 0.9925; finally, the SERS peak was measured at 1611 cm⁻¹ before and after the sample was dropped onto the SERS substrate. -1 The concentration of the sample can be obtained by substituting the intensity difference at each point into the corresponding linear regression equation.

[0038] Example 1

[0039] A flexible SERS substrate with Au@Pt nanozyme in situ encapsulated by a hydrogel was prepared by the following method, with specific steps 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. The mixed solution was heated under reflux for 30 min until the solution turned wine red. After gradually cooling to room temperature with stirring, concentrated gold nanoparticles were obtained.

[0041] S2. Preparation of hydrogel-coated Au@Pt nanozyme flexible SERS substrate: 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 mixture, ensuring that the volume ratio of H2PtCl6 to AA was 2:1, resulting in 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). The actual images are shown below. Figure 1 As shown in a.

[0042] Example 2

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

[0044] The Au@Pt nanoparticles with different shells synthesized in situ in Example 1 were characterized by TEM, such as... Figure 1 As shown in b, the in-situ synthesized Au@Pt nanoparticles exhibit uniform size with a particle size of approximately 45 nm. The Au is coated with Pt nanoclusters. By controlling the feed ratio of Au to Pt and varying the volumes of H₂PtCl₆ and AA added, the thickness of the Pt shell changes accordingly. Next, the Au@Pt nanoparticles within the in-situ synthesized hydrogel are characterized using transmission electron microscopy (TEM) F200. Figure 2 As shown in figure a, the presence of gold and platinum nanoparticles within the hydrogel was confirmed, with platinum successfully adhering to the surface of the gold particles. Finally, SEM characterization of Au@Pt-Agar revealed that the hydrogel exhibits a loose, porous, layered structure, with nanoparticles uniformly dispersed within the hydrogel's pores. This uniformly distributed SERS substrate contributes to improved reproducibility of SERS detection.

[0045] Example 3

[0046] Evaluation of the optimal Pt shell thickness for in-situ encapsulation of Au@Pt nanozymes on a flexible SERS substrate using hydrogel:

[0047] Au@Pt with different shell thicknesses in the hydrogel were prepared in situ according to Example 1, such as 0.7 nm, 1.4 nm, 2.8 nm, 3.5 nm, 6.8 nm, and a blank control group. These were incubated for 20 min in the original concentration of TMB+H2O2 and TMB+H2O2 diluted tenfold, respectively, while maintaining all other conditions the same. SERS detection was then performed, and a line graph of the SERS peak intensity versus different Pt shell thicknesses was plotted. Figure 3 As shown in figure a, the Au@Pt-Agar with a shell thickness of 0.7 nm exhibits the best overall performance.

[0048] Example 4

[0049] Reproducibility evaluation of hydrogel in-situ encapsulation of Au@Pt nanozymes on flexible SERS substrates:

[0050] To demonstrate the reproducibility of the prepared Au@Pt-Agar for SERS measurements, 20 locations were randomly selected on the same Au@Pt nanozyme hydrogel SERS substrate for SERS measurements. The results are as follows: Figure 3 As shown in b, the strongest signal is located at 1611 cm⁻¹. -1 The spectral peak at [value missing] was selected to evaluate the reproducibility of the SERS substrate. The relative standard deviation (RSD) of the Raman signal was 2.472%, which meets the requirement of RSD less than 20%. These data indicate that the hydrogel SERS substrate loaded with Au@Pt nanozymes exhibits good reproducibility and homogeneity, and can be used for accurate detection of glutathione in real samples.

[0051] Example 5

[0052] SERS measurements of different concentrations of TMB:

[0053] First, prepare TMB standard solutions of different concentrations. For example... Figure 4 As shown in figure a, this invention measured the oxides obtained by catalyzing different concentrations of TMB on an Au@Pt nanozyme hydrogel SERS substrate at 1611 cm⁻¹. -1 Peak intensity. Experimental results show that the SERS signal intensity of TMB oxide gradually increases with increasing TMB concentration, such as... Figure 5 As shown in figure a, the detection limit of this method for TMB is 6.24 × 10⁻⁶. -11 M, compared to the detection limit of 6.24 × 10 for TMB by ultraviolet spectroscopy. -5 M( Figure 4 b) It has very high sensitivity. (The 1611cm) -1 A correlation was established between the SERS peak intensity and the logarithm of the TMB concentration, revealing a strong linear relationship between the two. Figure 5 b) The linear regression equation is y = 144725.03x + 929776.19, and the correlation coefficient R0 is... 2 It is 0.9988. As a standard reference, the lower the detection limit of TMB, the stronger its ability to detect GSH.

[0054] Example 6

[0055] SERS measurements of different concentrations of GSH:

[0056] First, GSH standard solutions of different concentrations were prepared. Different concentrations of GSH were then added dropwise to the Au@Pt nanozyme hydrogel SERS substrate. After reacting for 3 minutes, the SERS peaks before and after the addition of GSH solutions were measured, and the SERS peak at 1611 cm⁻¹ was determined. -1The difference at the point is calculated, and a curve is plotted using this peak intensity difference versus GSH concentration, such as... Figure 6 As shown in figure a. This invention measured different concentrations of GSH on an Au@Pt nanozyme hydrogel SERS substrate at a depth of 1611 cm⁻¹. -1 The peak intensity difference was used to establish a linear relationship between the logarithm of the difference and the logarithm of the GSH concentration. Figure 6 b) The linear regression equation is y = 0.1687x + 5.36, and the correlation coefficient R0 is... 2 It reached 0.9925.

[0057] Example 7

[0058] SERS measurement of GSH in serum samples:

[0059] Serum collected from the hospital was centrifuged and diluted to obtain a serum solution diluted tenfold. This solution was then used to serially dilute GSH standard solution. Equal volumes of GSH at different concentrations were then dropped onto an Au@Pt-Agar surface and allowed to stand for 3 minutes. The SERS peak intensities before and after GSH addition were measured, and the SERS peak difference was calculated. The peak intensity was then measured at 1611 cm⁻¹. -1 Plot a curve of the peak intensity difference versus GSH concentration, as shown below. Figure 7 As shown in a. Then, 1611cm -1 A linear relationship was established between the logarithm of the SERS peak intensity difference 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 R0 is... 2 It reached 0.9968.

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

[0061] This study prepared Au@Pt-Agar with excellent peroxidase-like activity, capable of catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) under H2O2 conditions. Under acidic conditions, GSH with reducing ability can inhibit the oxidation of TMB, leading to 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, combined with hydrogel encapsulation, a selective, uniform, and stable SERS substrate was prepared, enabling rapid detection of GSH at low cost and with high sensitivity. Furthermore, comparisons were made with naked-eye analysis and UV-Vis spectroscopy; this method outperformed previous methods in both sensitivity and detection range.

[0062] In summary, the in-situ preparation method of the Au@Pt nanozyme hydrogel SERS substrate involved in this invention is simple and efficient, and the SERS substrate is uniform, reducing interference from other substances on the detection of target analytes. Furthermore, the dense porous structure of the hydrogel surface can prevent the passage of interfering substances, allowing only target small molecules to enter, avoiding complex and time-consuming sample pretreatment steps, and exhibiting a certain enrichment effect, enabling 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, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting GSH by using a hydrogel to in-situ coat Au@Pt nanoszyme flexible SERS substrate, characterized in that, comprises the following steps: the prepared hydrogel in situ coated Au@Pt nanoscale enzyme flexible SERS substrate respectively parallel immersion in 100-1000 μL equal volume of different concentrations of TMB (3,3',5,5'-tetramethylbenzidine) and H2O2 mixed solution, in 37℃ incubator reaction 20-60 min after SERS detection; the excitation wavelength used is 633 nm, the integral time is 10 s, the integral number is 1 times; the SERS peak intensity at 1611 cm -1 The relationship between the concentration of TMB is established, and the linear regression equation y=144725.03x+929776.19 is obtained, the correlation coefficient R 2 is 0.9988, and the determination of TMB is used as a standard reference; the strongest SERS peak corresponding to the concentration of TMB and hydrogen peroxide is used to incubate Au@Pt-Agar, and the corresponding SERS spectrum is determined after reaction for 20-60 min, and then different concentrations of GSH solution are added to determine the SERS intensity, and the intensity difference at 1611 cm -1 The relationship between the concentration of GSH is established, and the linear regression equation y=0.1687x+5.36 is obtained, and the correlation coefficient R 2 is 0.9925; the intensity difference of the SERS spectrum peak at 1611 cm -1 before and after adding the sample on the SERS substrate is determined, and the concentration of the sample to be measured can be obtained by substituting the corresponding linear regression equation. The preparation method of the hydrogel in-situ coated Au@Pt nanoszyme flexible SERS substrate comprises the following steps: S1, concentrated gold nanoparticles synthesis: the aqueous solution of HAuCl4 is heated to boiling with stirring, sodium citrate is quickly added, the mixed solution is heated to reflux for 10-60 min until it becomes wine red; then gradually cool to room temperature with stirring to obtain concentrated gold nanoparticles; S2, preparation of hydrogel in-situ coated Au@Pt nanoszyme flexible SERS substrate: mix the hydrogel and concentrated gold nanoparticles according to the volume ratio, then add H2PtCl6 and ascorbic acid, and react to obtain the hydrogel in-situ coated Au@Pt nanoszyme flexible SERS substrate Au@Pt-Agar.

2. The method for detecting GSH by using the hydrogel to in situ coat the Au@Pt nanoszyme flexible SERS substrate according to claim 1, wherein, The mass fraction of HAuCl4 in step S1 is 0.1wt%-1wt%.

3. The method for detecting GSH by using the hydrogel to in situ coat the Au@Pt nanoszyme flexible SERS substrate according to claim 1, wherein, The mass fraction of sodium citrate in step S1 is 0.5wt%-5wt%.

4. The method for detecting GSH by using the hydrogel to in situ coat the Au@Pt nanoszyme flexible SERS substrate according to claim 1, wherein, The reaction condition in step S2 is 50℃-100℃ water bath heating.

5. The method for detecting GSH by using the hydrogel to in situ coat the Au@Pt nanoszyme flexible SERS substrate according to claim 1, wherein, The concentration of H2PtCl6 in step S2 is 1-10mM.

6. The method for detecting GSH by using the hydrogel to in situ coat the Au@Pt nanoszyme flexible SERS substrate according to claim 1, wherein, The concentration of ascorbic acid in step S2 is 2-20mM.

7. The method for detecting GSH by using the hydrogel to in situ coat the Au@Pt nanoszyme flexible SERS substrate according to claim 1, wherein, The volume ratio of H2PtCl6 to ascorbic acid in step S2 is (1-3):1, and the feeding ratio of Au to Pt is (2-11):1.

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