Glutathione-specific detection method based on surface-enhanced raman spectroscopy

Abstract

The present application relates to the technical field of biomolecule detection and spectral analysis, and discloses a glutathione specific detection method based on surface enhanced Raman spectroscopy. The present application provides a preparation method of a molecular imprinting polymer, a thiol specific azo type labeling reagent is selectively reacted with the thiol in the glutathione molecule to form a template molecule, and the molecular imprinting polymer is obtained through a free radical polymerization reaction. The glutathione specific detection method provided by the present application combines the specific enrichment ability of the molecular imprinting polymer with the high sensitivity signal amplification advantage of the SERS technology, the molecular imprinting polymer is used to purify and enrich the labeled glutathione in the sample in advance, the impurities are removed, and then the characteristic Raman signal of the azo group is amplified through the silver nanoparticle film SERS detection substrate, so that the effective detection of low concentration glutathione is realized, the detection limit of the method is low, the sensitivity is high, the signal stability is good, the repeatability is good, and the quantitative accuracy is high.

Description

Technical Field

[0001] This invention relates to the field of biomolecular detection and spectroscopic analysis technology, and in particular to a method for the specific detection of glutathione based on surface-enhanced Raman spectroscopy. Background Technology

[0002] Glutathione (GSH) is a key tripeptide biomolecule composed of glutamic acid, cysteine, and glycine. It plays a core role in cellular antioxidation, resisting oxidative stress, and maintaining intracellular redox homeostasis. Abnormal levels of glutathione are closely related to various diseases such as neurodegenerative diseases, tumors, and cardiovascular diseases. Therefore, developing a sensitive and highly selective method for detecting glutathione has important clinical and scientific research value.

[0003] Currently, glutathione detection mainly relies on high-performance liquid chromatography (HPLC), fluorescence analysis, and electrochemical detection methods. These methods generally suffer from drawbacks such as cumbersome sample pretreatment, high instrument purchase and operating costs, and insufficient selectivity, making them unsuitable for the rapid and accurate detection needs of complex biological systems. Surface-enhanced Raman spectroscopy (SERS), with its localized surface plasmon resonance effect, can significantly enhance the Raman signal and possesses ultra-high detection sensitivity, making it a preferred technique for small molecule detection. However, this technique faces a serious selectivity problem when detecting glutathione.

[0004] Molecularly imprinted polymers (MIPs) are a class of functional materials that form specific recognition sites through template molecules, exhibiting excellent molecular recognition capabilities. Co-conjugation with SERS can effectively improve selectivity. However, glutathione molecules are small and highly polar, making it difficult to construct stable and highly selective imprinted sites. Furthermore, its intramolecular thiol groups possess strong reducing and free radical scavenging abilities, which can easily quench the free radical reaction process during the preparation of MIPs using free radical polymerization methods. This affects the polymerization reaction and the formation of imprinted sites, leading to reduced recognition performance of the resulting MIPs. In addition, glutathione itself has a very small Raman scattering cross section, resulting in weak signals during direct SERS detection, and is easily interfered with by other thiol-containing or structurally similar molecules, further reducing selectivity and sensitivity. Existing co-conjugation techniques cannot simultaneously solve the core problems of recognition stability, polymerization interference, signal strength, and anti-interference ability. Therefore, there is an urgent need to develop a new SERS detection method for glutathione that combines high selectivity and high sensitivity. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of the present invention is to provide a method for preparing molecularly imprinted polymers.

[0006] The second objective of this invention is to provide a molecularly imprinted polymer.

[0007] A third objective of this invention is to provide applications of such molecularly imprinted polymers.

[0008] The fourth objective of this invention is to provide a method for the specific detection of glutathione based on surface-enhanced Raman spectroscopy.

[0009] The fifth objective of this invention is to provide an application of this glutathione-specific detection method based on surface-enhanced Raman spectroscopy.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a method for preparing a molecularly imprinted polymer, comprising the following steps: A thiol-specific azo labeling reagent is added to glutathione to obtain the template molecule; The template molecule is subjected to free radical polymerization in the presence of functional monomers, crosslinking agents, and initiators; After the reaction is complete, the template molecule is removed to obtain the molecularly imprinted polymer.

[0011] In some embodiments of the present invention, the molar ratio of the thiol-specific azo labeling reagent to glutathione is 1:(0.85-1.25).

[0012] In some preferred embodiments of the present invention, the molar ratio of the thiol-specific azo labeling reagent to glutathione is 1:(0.95-1.15).

[0013] In some embodiments of the present invention, the thiol-specific azo labeling reagent includes N-(4-phenylazophenyl)maleimide.

[0014] In some embodiments of the present invention, the addition reaction further includes the use of a solvent, wherein the ratio of the thiol-specific azo labeling reagent to the solvent is 1 mmol: (8-12) mL.

[0015] In some preferred embodiments of the present invention, the solvent is selected from at least one of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and N-methylpyrrolidone (NMP).

[0016] In some embodiments of the present invention, the addition reaction is carried out at a temperature of 40-60°C for a time of 1-3 hours.

[0017] In some preferred embodiments of the present invention, the addition reaction is carried out at a temperature of 45-55°C for a time of 1.5-2.5 h.

[0018] In some embodiments of the present invention, the molar ratio of the functional monomer, crosslinking agent, initiator and glutathione is (3.0-4.6): (12.0-18.3): (0.28-0.43): 1.

[0019] In some preferred embodiments of the present invention, the molar ratio of the functional monomer, crosslinking agent, initiator and glutathione is (3.4-4.2): (13.7-16.8): (0.31-0.39): 1.

[0020] In some embodiments of the present invention, the functional monomer includes at least one of 4-vinylimidazole, 1-vinylimidazole, and 4-vinylpyridine.

[0021] In some preferred embodiments of the present invention, the functional monomer is 4-vinylimidazole.

[0022] In some embodiments of the present invention, the crosslinking agent includes at least one of ethylene glycol dimethacrylate, divinylbenzene, and N,N'-methylenebisacrylamide.

[0023] In some preferred embodiments of the present invention, the crosslinking agent is ethylene glycol dimethacrylate (EGDMA).

[0024] In some embodiments of the present invention, the initiator includes at least one of azobisisobutyronitrile, benzoyl peroxide, and ammonium persulfate.

[0025] In some preferred embodiments of the present invention, the initiator is azobisisobutyronitrile.

[0026] In some embodiments of the present invention, the free radical polymerization reaction further includes the use of a solvent, wherein the ratio of the functional monomer to the solvent is 1 mmol: (6-9) mL.

[0027] In some embodiments of the present invention, the solvent is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, and acetonitrile.

[0028] In some embodiments of the present invention, the temperature of the free radical polymerization reaction is 48-72°C and the time is 20-30 h.

[0029] In some preferred embodiments of the present invention, the temperature of the free radical polymerization reaction is 54-66°C and the time is 22-26h.

[0030] In some embodiments of the present invention, the step of causing the template molecule to undergo a free radical polymerization reaction in the presence of a functional monomer, a crosslinking agent, and an initiator specifically includes: adding a functional monomer to the template molecule, mixing thoroughly, adding a crosslinking agent, an initiator, and a solvent, dissolving by ultrasonication, removing oxygen, and then carrying out a free radical polymerization reaction under closed conditions.

[0031] In some embodiments of the present invention, the ultrasonic dissolution time is 8-12 minutes.

[0032] In some embodiments of the present invention, the deoxygenation includes nitrogen bubbling for 25-35 minutes.

[0033] In some embodiments of the present invention, after the free radical polymerization reaction is completed, the process further includes crushing and sieving the obtained block polymer to obtain polymer particles.

[0034] In some embodiments of the present invention, the removal of template molecules includes elution with a solvent selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, and N-methylpyrrolidone.

[0035] In some embodiments of the present invention, after removing the template molecules, the process further includes washing with an alcohol solvent and drying to obtain the molecularly imprinted polymer.

[0036] A second aspect of the present invention provides a molecularly imprinted polymer, which is prepared by the method for preparing the molecularly imprinted polymer described in the first aspect of the present invention.

[0037] A third aspect of the present invention provides the application of the molecularly imprinted polymer described in the second aspect of the present invention in the selective detection of glutathione using surface-enhanced Raman spectroscopy.

[0038] A fourth aspect of the present invention provides a method for the specific detection of glutathione based on surface-enhanced Raman spectroscopy, comprising the following steps: The sample to be tested is reacted with a mercapto-specific azo labeling reagent to generate a labeled product; The molecularly imprinted polymer described in the second aspect of this invention is added to specifically adsorb the labeled product; The labeled product was eluted and enriched, and the supernatant was collected and contacted with a surface-enhanced Raman spectroscopy (SERS) substrate for Raman spectroscopy detection of glutathione.

[0039] In some embodiments of the present invention, the reaction of the test sample with a mercapto-specific azo labeling reagent specifically includes: mixing the test sample with a solution containing a mercapto-specific azo labeling reagent, and carrying out an addition reaction at 40-60°C for 1-3 hours.

[0040] In some embodiments of the present invention, the concentration of the mercapto-specific azo labeling reagent in the solution is 0.8-1.2 mmol / L, and the volume ratio of the sample to be tested to the solution containing the mercapto-specific azo labeling reagent is 1:(1.5-2.5).

[0041] In some embodiments of the present invention, the solvent in the solution containing the mercapto-specific azo labeling reagent is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, and N-methylpyrrolidone.

[0042] In some embodiments of the present invention, the addition of the molecularly imprinted polymer for specific adsorption of the labeled product specifically includes: adding a solvent and the molecularly imprinted polymer, incubating at room temperature for 25-35 minutes, and then performing specific adsorption of the labeled product.

[0043] In some preferred embodiments of the present invention, the solvent is selected from at least one of acetonitrile, methanol, ethanol, and ethyl acetate.

[0044] In some embodiments of the present invention, after the specific adsorption is completed, the process further includes centrifugation to collect the solid phase and washing.

[0045] In some embodiments of the present invention, the elution and enrichment of the labeled product specifically includes: after the specific adsorption is completed, solid phase is collected by solid-liquid separation, washed and desorbed by adding solvent for 8-12 min, and supernatant is collected by solid-liquid separation.

[0046] In some embodiments of the present invention, the washing reagent includes acetonitrile, and the washing is performed 2-3 times.

[0047] In some embodiments of the present invention, the volume ratio of the desorption solvent to the sample to be tested is 1:(0.8-1.2).

[0048] In some embodiments of the present invention, the desorption solvent is selected from at least one of dimethyl sulfoxide, N,N-dimethylformamide, and N-methylpyrrolidone.

[0049] In some embodiments of the present invention, the surface-enhanced Raman spectroscopy detection substrate comprises a silver nanoparticle film.

[0050] In some embodiments of the present invention, the silver nanoparticle film is prepared by a method comprising the following steps: Silver nitrate, polyvinylpyrrolidone, and water are mixed to form a solution; Ascorbic acid is dissolved in water to form an ascorbic acid aqueous solution, which is then mixed with the mixture to obtain a silver nanoparticle solution. The silver nanoparticle solution was vacuum filtered and deposited through a 0.20-0.25 μm filter membrane to obtain the silver nanoparticle membrane.

[0051] In some embodiments of the present invention, the ratio of silver nitrate, polyvinylpyrrolidone and water is 1g:(1.6-2.4)g:(460-690)mL.

[0052] In some embodiments of the present invention, the solid-liquid ratio of ascorbic acid to water is (1.1-1.7) g: 10 mL.

[0053] In some embodiments of the present invention, the volume ratio of the ascorbic acid aqueous solution to the mixture is 1:(40-60).

[0054] In some embodiments of the present invention, the Raman spectroscopy detection of glutathione includes detection at 1133 cm⁻¹ under 532 nm laser excitation. -1 The characteristic peak intensity of the azo group in the labeled product is compared with the standard curve to achieve semi-quantitative detection of glutathione.

[0055] In some embodiments of the present invention, the standard curve is a linear fitting curve of the Raman characteristic peak intensity versus concentration of glutathione labeled with a thiol-specific azo labeling reagent as a standard.

[0056] In some embodiments of the present invention, the concentration of the standard is 1 × 10⁻⁶. -6 mol / L-1×10 -3 mol / L.

[0057] The fifth aspect of the present invention provides the application of the glutathione-specific detection method based on surface-enhanced Raman spectroscopy described in the fourth aspect of the present invention in the detection of glutathione content in serum, plasma or urine samples.

[0058] Compared with the prior art, the beneficial effects of the present invention are: 1) The method for preparing molecularly imprinted polymers provided by this invention is simple in steps. The thiol-specific azo labeling reagent used can quickly and selectively react with the thiol groups in glutathione molecules, thereby improving the recognition characteristics of the target molecule. Using the labeled glutathione as a template to construct molecularly imprinted polymers can effectively improve the structural stability and selectivity of the recognition sites. 2) The molecularly imprinted polymer provided by this invention has a high selective adsorption capacity for labeled glutathione, which can effectively eliminate the interference of cysteine, homocysteine ​​and other impurities in complex biological matrices such as serum and plasma, and solve the problem that the detection of glutathione in traditional methods is easily affected by matrix effects. 3) The glutathione-specific detection method based on surface-enhanced Raman spectroscopy provided by this invention combines the specific enrichment capability of molecularly imprinted polymers with the high-sensitivity signal amplification advantage of SERS technology. The molecularly imprinted polymers pre-purify and enrich the labeled glutathione in the sample, removing interference from impurities. Then, the characteristic Raman signal of the azo group is amplified using a silver nanoparticle membrane SERS detection substrate, achieving effective detection of low-concentration glutathione. The method's detection limit is as low as 1×10⁻⁶. -8 With a concentration of mol / L, its sensitivity is far superior to traditional Raman spectroscopy detection methods. It also boasts good signal stability, repeatability, and quantitative accuracy, making it effective for analyzing glutathione content in complex biological samples such as serum, plasma, and urine. Attached Figure Description

[0059] Figure 1 The 1H NMR spectrum of N-(4-phenylazophenyl)maleimide in Example 1; Figure 2 This is the positive ion mode ESI mass spectrum of the dimethyl sulfoxide solution of the template molecule in Example 1; Figure 3 This is a static adsorption diagram of the molecularly imprinted polymer for N-(4-phenylazophenyl)maleimide and the template molecule in Example 1; Figure 4 Raman spectra of template molecules at different concentrations in Example 2; Figure 5 This is the standard curve of the Raman signal intensity of the template molecule in Example 2; Figure 6 This is a comparison chart of selective Raman intensities tested using the method in Example 2; Figure 7 This is a confirmation graph of the detection limit of the method in Example 2; Figure 8 This is a graph showing the repeatability verification of the method in Example 2; Figure 9 This is a 14-day Raman signal stability graph from Example 2; Figure 10 Raman signal image of the fetal bovine serum sample glutathione content test in Application Example 1; Figure 11 The diagram shows the principle of molecularly imprinted polymer preparation (a) and the glutathione detection process (b) of this invention. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to specific accompanying drawings and embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0061] Example 1 This embodiment prepares a molecularly imprinted polymer: 1. Synthesis of N-(4-phenylazophenyl)maleimide, the synthetic route is shown below:

[0062] The synthesis steps are as follows: 1) Dissolve 15 mmol of 4-phenylazoaniline in 60 mL of ethyl acetate, add 18 mmol of maleic anhydride under stirring, and react at 60 °C for 8 h to generate the corresponding maleamide intermediate yellow precipitate. Filter and collect the precipitate, and wash the intermediate with ethyl acetate. 2) The intermediate was then transferred to 30 mL of tetrahydrofuran, and 30 mL of acetic anhydride and 2 g of anhydrous sodium acetate were added. The reaction was continued for 6 h under reflux at 80 °C to carry out the dehydration cyclization reaction. 3) After the reaction is complete, the reaction solution is cooled to room temperature and poured into 200 mL of ice water to precipitate the solid. After filtration, washing and drying, crude N-(4-phenylazophenyl)maleimide solid product is obtained. 4) The crude N-(4-phenylazophenyl)maleimide was recrystallized with anhydrous ethanol to further improve its purity and obtain pure N-(4-phenylazophenyl)maleimide.

[0063] Figure 1 The figure shows the 1H NMR spectrum of N-(4-phenylazophenyl)maleimide from Example 1. The 1H NMR data in the figure are as follows: 1 ¹H NMR (600MHz, DMSO-d6) δ: 8.04–7.98 (m, 2H, A), 7.94–7.90 (m, 2H, B), 7.65–7.56 (m, 5H, C), 7.24 (s, 2H, D). In the figure, A corresponds to the ortho-hydrogen signal on the para-substituted benzene ring; B corresponds to another set of symmetrical hydrogen signals on the same benzene ring; C corresponds to the hydrogen signal on the benzene ring on the other side of the azo structure; and D corresponds to the olefin hydrogen signal in the maleimide structure. Figure 1 It can be seen that the structure of N-(4-phenylazophenyl)maleimide is correct, and the labeling reagent was successfully synthesized.

[0064] 2. Preparation of molecularly imprinted polymers, the steps are as follows: 1) Dissolve 1.05 mmol of glutathione in 10 mL of dimethyl sulfoxide, add 1 mmol of N-(4-phenylazophenyl)maleimide, and react at 50 °C for 2 h to allow the maleimide group to undergo a Michael addition reaction with the thiol group in the glutathione molecule to obtain a dimethyl sulfoxide solution of the template molecule. The synthetic route for the template molecule is shown below:

[0065] Figure 2 This is the positive ion mode ESI mass spectrum of the dimethyl sulfoxide solution of the template molecule in Example 1, obtained from... Figure 2 It can be seen that the measured molecular ion peak of the target compound is close to the theoretical molecular weight (584.18+H). + The results (=585.18) are consistent with those of N-(4-phenylazophenyl)maleimide-labeled glutathione, indicating that the template molecule N-(4-phenylazophenyl)maleimide-labeled glutathione was successfully obtained.

[0066] 2) Add the dimethyl sulfoxide solution of the template molecule to the reaction vessel, add 4 mmol of 4-vinylimidazole as the functional monomer, and mix thoroughly; then add 16 mmol of ethylene glycol dimethacrylate as a crosslinking agent, add 60 mg of azobisisobutyronitrile as an initiator, add 30 mL of dimethyl sulfoxide, sonicate for 10 min to dissolve thoroughly, bubble with nitrogen for 30 min, seal the reaction vessel, and carry out free radical polymerization at 60 °C for 24 h; 3) After polymerization, a block polymer is obtained. The polymer particles are ground and sieved. Then, the polymer is eluted with dimethyl sulfoxide until the template molecules are no longer detectable in the supernatant. Finally, the polymer is washed three times with ethanol and dried under vacuum to obtain the molecularly imprinted polymer.

[0067] Figure 3 This is a static adsorption diagram of the molecularly imprinted polymer for N-(4-phenylazophenyl)maleimide and the template molecule in Example 1. The horizontal axis represents the concentration in the solution at equilibrium, and the vertical axis represents the amount of compound molecules adsorbed by the polymer at the corresponding equilibrium concentration. The maximum static adsorption amount (Q) of the molecularly imprinted polymer for N-(4-phenylazophenyl)maleimide and the template molecule was obtained by curve fitting using the Langmuir equation. max The concentrations were 0.65 μmol / g and 44.57 μmol / g, respectively, demonstrating that the molecularly imprinted polymer exhibits high specificity for the N-(4-phenylazophenyl)maleimide-labeled glutathione (template molecule).

[0068] Example 2 In this embodiment, the molecularly imprinted polymer prepared in Example 1 is applied to the selective detection of glutathione using surface-enhanced Raman spectroscopy: 1. Preparation of a surface-enhanced Raman spectroscopy detection substrate: 1) Dissolve 0.425g of silver nitrate and 0.85g of polyvinylpyrrolidone in 245mL of deionized water to obtain a mixture; 2) Dissolve 0.7g of ascorbic acid in 5mL of water to obtain an ascorbic acid aqueous solution; 3) Add ascorbic acid aqueous solution to the mixture and stir for 15 minutes to change the color of the solution from dark brown to clay color, thus obtaining silver nanoparticle solution; 4) A dense silver nanoparticle film was deposited by vacuum filtration of 50 mL of silver nanoparticle solution through a 0.22 μm filter membrane. The film was then washed with 50 mL of deionized water, and the filter membrane was removed and dried in an oven at 50 °C to obtain a surface-enhanced Raman spectroscopy detection substrate.

[0069] 2. Draw the standard curve: A dimethyl sulfoxide solution of the template molecule (N-(4-phenylazophenyl)maleimide-labeled glutathione) was prepared according to the method in Example 1. 10 μL of a 1×10⁻⁶ solution was taken from each solution. -6 mol / L, 1×10 -5 mol / L, 1×10 -4 mol / L and 1×10 -3 A dimethyl sulfoxide solution of template molecules in mol / L was dropped onto a surface-enhanced Raman spectroscopy detection substrate, and the detection was performed using a Raman spectrometer with a laser wavelength of 532 nm, a power of 10 mW, and an integration time of 2 s.

[0070] Figure 4 The images shown are Raman spectra of template molecules at different concentrations in Example 2. Figure 4 It can be seen that the Raman intensity is positively correlated with the concentration of template molecules, indicating that the SERS signal of N-(4-phenylazophenyl)maleimide-labeled glutathione is strong, the peak position is stable, and the concentration dependence is good, making it suitable for quantitative detection.

[0071] Figure 5 The standard curve for the Raman signal intensity of the template molecule in Example 2 is shown at 1133 cm⁻¹. -1 The characteristic peak intensity of the azo group in the template molecule was plotted on the ordinate, and the negative logarithm (-log(c)) of the glutathione concentration in the template molecule was plotted on the abscissa. The data showed that at 1×10 -6 mol / L-1×10 -3 Within the mol / L range, the linear relationship is good (y=-3136x+19315, R). 2 =0.9814), this quantitative relationship can be used to calculate the glutathione concentration in unknown samples, achieving semi-quantitative detection.

[0072] 3. Detection of glutathione: 1) Mix 1 mL of the sample to be tested with 2 mL of 1 mol / L N-(4-phenylazophenyl)maleimide dimethyl sulfoxide solution and react at 50 °C for 30 min to generate the labeled product; 2) Add 7 mL of acetonitrile and 30 mg of the molecularly imprinted polymer prepared in Example 1, and incubate at room temperature for 30 min to allow the labeled product to be selectively recognized and enriched by the molecularly imprinted polymer; 3) Centrifuge to remove the supernatant, wash the polymer twice with acetonitrile, add 1 mL of dimethyl sulfoxide to elute and enrich the labeled product, and centrifuge to collect the supernatant after 10 min; 4) Add 10 μL of supernatant to the silver nanoparticle membrane SERS detection substrate and perform Raman spectroscopy. The laser wavelength is 532 nm, the power is 10 mW, the integration time is 2 s, and the value is recorded at 1133 cm⁻¹. -1 The characteristic peak intensity of the azo group in the labeled product was used to perform semi-quantitative detection of glutathione using a standard curve.

[0073] To verify the selectivity of this detection method, following the above-described detection steps for glutathione, the test samples containing glutathione were replaced with test samples containing glutamic acid, lysine, alanine, glycine, tryptophan, N-acetylcysteine, and cysteine, respectively, for testing. Figure 6 This is a comparison chart of the selective Raman intensity tests performed using the method in Example 2. Figure 6 It can be seen that, compared with substances such as glutamic acid, lysine, and alanine under the same conditions, the 1133 cm⁻¹ -1 At the peak intensity, only the glutathione signal was significant, indicating that the detection method has good selectivity for glutathione.

[0074] To verify the detection limit of this detection method, following the detection steps described above, Raman spectrometry was used to detect concentrations of 1×10⁻⁶. -6 mol / L, 1×10 -7 mol / L, 1×10 -8 A dimethyl sulfoxide solution of template molecules at a concentration of mol / L: Figure 7 This is the detection limit confirmation chart for the method in Example 2, provided by... Figure 7 It can be seen that the limit of detection (LOD) of this glutathione-specific detection method based on surface-enhanced Raman spectroscopy is 1×10⁻⁶. -8 The method has a high sensitivity and is suitable for detecting low-content samples.

[0075] To verify the repeatability of the detection method, following the detection steps described above, the same test sample (a dimethyl sulfoxide solution of the template molecule) was repeatedly irradiated 10 times using a Raman spectrometer. Figure 8 This is the repeatability verification graph for the method in Example 2, by... Figure 8 It can be seen that when the same sample (dimethyl sulfoxide solution of template molecules) is detected 10 times consecutively, the value is 1133 cm⁻¹. -1The peak intensity fluctuation was small, with an RSD of 10.95%, demonstrating good repeatability, high detection stability, and reliable data.

[0076] To verify the Raman signal stability of this detection method, the same sample (a dimethyl sulfoxide solution of the template molecule) was stored at room temperature for 14 days, and Raman spectrometry was performed daily under the same detection conditions. Figure 9 This is the 14-day Raman signal stability diagram from Example 2, provided by... Figure 9 It can be seen that after the sample was stored at room temperature for 14 days, the volume was 1133 cm. -1 The peak intensity showed no significant attenuation, the signal retention rate was high, and the RSD was 3.63%, proving that the labeled product and the SERS detection substrate had good stability, the sample could be stored for a short period of time, and the detection timeliness was strong.

[0077] Application Example 1 This application example uses a glutathione-specific detection method based on surface-enhanced Raman spectroscopy to determine the glutathione content in fetal bovine serum samples: 1) Mix 1 mL of commercially available fetal bovine serum sample with 2 mL of 1 mol / L N-(4-phenylazophenyl)maleimide dimethyl sulfoxide solution and react at 50 °C for 30 min to generate the labeled product; 2) Add 7 mL of acetonitrile and 30 mg of the molecularly imprinted polymer prepared in Example 1, and incubate at room temperature for 30 min to allow the labeled product to be selectively recognized and enriched by the molecularly imprinted polymer; 3) Centrifuge to remove the supernatant, wash the polymer twice with acetonitrile, add 1 mL of dimethyl sulfoxide to elute and enrich the labeled product, and centrifuge to collect the supernatant after 10 min; 4) Add 10 μL of the supernatant to the silver nanoparticle membrane SERS detection substrate prepared in Example 2, and perform detection using a Raman spectrometer. The laser wavelength is 532 nm, the power is 10 mW, the integration time is 2 s, and the value is recorded at 1133 cm⁻¹. -1 The characteristic peak intensity of the azo group in the labeled product was used to semi-quantitatively detect glutathione using the standard curve established in Example 2.

[0078] Figure 10 To obtain the Raman signal image of the fetal bovine serum sample glutathione content test in Example 1, based on 1133 cm⁻¹... -1 The concentration of glutathione in the commercially available fetal bovine serum sample was calculated to be 1 × 10⁻⁶ by substituting the characteristic peak intensity of the azo group in the labeled product into the standard curve. -5 The method, measured in mol / L, demonstrates its applicability for detecting glutathione in complex biological samples (serum), proving its strong practicality.

[0079] Figure 11The diagrams (a) illustrating the preparation principle of the molecularly imprinted polymer and (b) the glutathione detection process of this invention are provided by [the relevant authority / organization]. Figure 11 As can be seen, the present invention first utilizes a mercapto-specific azo labeling reagent (such as N-(4-phenylazophenyl)maleimide) to undergo an addition reaction with the mercapto group in the glutathione molecule to form a Raman-active labeled glutathione; then, using the labeled glutathione as a template molecule, a molecularly imprinted polymer is prepared by free radical polymerization in the presence of a functional monomer (such as 4-vinylimidazole), a crosslinking agent (such as ethylene glycol dimethacrylate), and an initiator (such as azobisisobutyronitrile); and the template molecule is removed by elution to form imprinted holes with specific recognition capabilities. When this molecularly imprinted polymer is applied to the selective detection of glutathione using surface-enhanced Raman spectroscopy (SERS), the polymer is first brought into contact with the sample to selectively adsorb labeled glutathione. Unbound thiol-specific azo labeling reagents are then removed by centrifugation and washing. The polymer is then extracted with a solvent, releasing the labeled glutathione bound to the imprinted sites into the solution, and the supernatant is collected. Simultaneously, a silver nanoparticle solution is filtered through a membrane to deposit a dense nanoparticle film, which serves as the SERS detection substrate. The supernatant is then dropped onto the surface of the SERS substrate, and glutathione is detected by measuring its Raman characteristic signal. This invention improves the selectivity and sensitivity of glutathione detection by combining chemical labeling, molecular imprinting recognition, and SERS signal enhancement.

[0080] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing a molecularly imprinted polymer, characterized in that, Includes the following steps: A thiol-specific azo labeling reagent is added to glutathione to obtain the template molecule; The template molecule is subjected to free radical polymerization in the presence of functional monomers, crosslinking agents, and initiators; After the reaction is complete, the template molecule is removed to obtain the molecularly imprinted polymer. The thiol-specific azo labeling reagent is selected from N-(4-phenylazophenyl)maleimide; the functional monomer is selected from at least one of 4-vinylimidazolium, 1-vinylimidazolium, and 4-vinylpyridine; the molar ratio of the thiol-specific azo labeling reagent to glutathione is 1:(0.85-1.25); the addition reaction is a Michael addition reaction between the maleimide group and the thiol group in the glutathione molecule. The molar ratio of the functional monomer, crosslinking agent, initiator and glutathione is (3.0-4.6): (12.0-18.3): (0.28-0.43):

1.

2. The method for preparing the molecularly imprinted polymer according to claim 1, characterized in that, The addition reaction is carried out at a temperature of 40-60℃ for 1-3 hours.

3. The method for preparing the molecularly imprinted polymer according to claim 2, characterized in that, The free radical polymerization reaction is carried out at a temperature of 48-72℃ for 20-30 hours.

4. The method for preparing the molecularly imprinted polymer according to claim 3, characterized in that, The crosslinking agent includes at least one of ethylene glycol dimethacrylate, divinylbenzene, and N,N'-methylenebisacrylamide.

5. A molecularly imprinted polymer, characterized in that, It is prepared by the method for preparing molecularly imprinted polymers according to any one of claims 1-4.

6. The application of the molecularly imprinted polymer of claim 5 in the selective detection of glutathione using surface-enhanced Raman spectroscopy.

7. A method for the specific detection of glutathione based on surface-enhanced Raman spectroscopy, characterized in that, Includes the following steps: The sample to be tested is reacted with a mercapto-specific azo labeling reagent to generate a labeled product; The molecularly imprinted polymer of claim 5 is added to specifically adsorb the labeled product; The labeled product was eluted and enriched, and the supernatant was collected and contacted with a surface-enhanced Raman spectroscopy (SERS) substrate for Raman spectroscopy detection of glutathione.

8. The method for specific detection of glutathione based on surface-enhanced Raman spectroscopy according to claim 7, characterized in that, The surface-enhanced Raman spectroscopy detection substrate includes a silver nanoparticle film.

9. The method for specific detection of glutathione based on surface-enhanced Raman spectroscopy according to claim 7, characterized in that, The Raman spectroscopy detection of glutathione includes detection at 1133 cm⁻¹ under 532 nm laser excitation. -1 The characteristic peak intensity of the azo group in the labeled product is compared with the standard curve to achieve semi-quantitative detection of glutathione.

10. The application of the glutathione-specific detection method based on surface-enhanced Raman spectroscopy according to any one of claims 7-9 in the detection of glutathione content in serum, plasma or urine samples.

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