Three-dimensional hydrogel SERS (Surface Enhanced Raman Scattering) substrate with porous structure as well as preparation method and application thereof

Abstract

The invention discloses a three-dimensional hydrogel SERS (Surface Enhanced Raman Scattering) substrate with a porous structure as well as a preparation method and application thereof, and belongs to the technical field of material science. The Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate is synthesized by utilizing a photocuring method, the prepared hydrogel has a 3D porous structure, the Ag NPs can be more uniformly distributed in the hydrogel substrate, and aggregation of the Ag NPs is effectively reduced to construct richer'hot spots'; the three-dimensional hydrogel SERS substrate shows good specificity when being used for detecting anti-thyroid hormone drugs methimazole and 2-thiouracil in blood plasma, drug molecules to be detected can be rapidly separated and enriched in the actual sample detection process, complex sample pretreatment is not needed, and the detection sensitivity is high. And the detection limits of methimazole and 2-thiouracil which are anti-thyroid hormone drugs in plasma can respectively reach 7.48 * 10 <-9 > M and 2.35 * 10 <-8 > M.

Description

Technical Field

[0001] The present invention belongs to the field of material science and technology, and in particular relates to a three-dimensional hydrogel SERS substrate with a porous structure and a preparation method and application thereof. Background Art

[0002] SERS (Surface-Enhanced Raman Spectroscopy) is a highly sensitive molecular detection and analysis technology based on Raman spectroscopy. It utilizes the enhancement effect of the rough surfaces of metals such as gold, silver, and copper to enhance the Raman scattering signal of molecules, thereby achieving efficient detection of low-concentration or trace samples, with single-molecule-level surface detection sensitivity. The analytes used for SERS detection only require simple pretreatment (in situ detection is possible), which can maintain the original state of the analytes, making it a non-destructive and non-destructive detection method. These characteristics make SERS an ideal technology for detecting trace molecules in chemical detection applications. Currently, SERS technology has been widely used in many fields, such as in the biomedical field for the detection of biological molecules, cell analysis, and disease diagnosis; in the field of environmental monitoring for the detection of pollutants such as microplastics and heavy metal ions in water and soil; in the field of food safety for the detection of pesticide residues and additives in food; and in the field of cultural relics protection for the analysis of the composition and structure of ancient cultural relics.

[0003] SERS substrates are key to achieving signal enhancement, including metal nanosol substrates, two-dimensional substrates, and three-dimensional substrates. The surface structure and size of these materials have a significant impact on the enhancement effect. Three-dimensional porous SERS substrate materials include hydrogels, aerogels, silica microspheres, porous carbon, and metal-organic frameworks (MOFs). The three-dimensional SERS platform constructed from these substrates has high porosity, large specific surface area, and molecular sieving effect, allowing for rapid penetration and enrichment of target analytes. Its three-dimensional space can reduce the aggregation of metal nanoparticles, facilitate the construction of richer "hot spots", and make the molecular distribution more uniform, thereby improving the sensitivity, uniformity, repeatability, and specificity of the SERS platform. Among them, hydrogel is a porous polymer material with a three-dimensional network structure formed by chemical bonds or physical interactions. It can absorb and retain large amounts of water while maintaining its own shape and structure. It has excellent mechanical properties, environmental responsiveness, and biocompatibility. Its preparation process is relatively simple and low-cost compared to other materials, and it has good application prospects in instant detection.

[0004] Thyroid hormones can increase the cellular oxidation rate, increase oxygen consumption, promote the catabolism of proteins, carbohydrates, and fats, help the body regulate metabolism, and promote growth and development. Hyperthyroidism is the result of uncontrolled excessive secretion of thyroid hormones. Antithyroid hormone drugs such as methimazole and 2-thiouracil have high antithyroid activity and have been used to treat hyperthyroidism. However, they may cause side effects such as teratogenicity, allergic reactions, impaired taste, pharyngitis, nephritis, cirrhosis, and skin vasculitis. The presence of antithyroid hormone drugs in patients' blood is harmful to human health. Therefore, rapid quantitative monitoring of residual levels of antithyroid hormone drugs in the blood is very important for the diagnosis and treatment of thyroid diseases.

[0005] Currently, many methods for determining the concentration of antithyroid hormone drugs have been reported, including high-performance liquid chromatography, electrochemical analysis, chemiluminescence, and mass spectrometry. For example, Chinese Patent Publication No. CN107525791A, filed on August 17, 2017, discloses a method for detecting methimazole. The method comprises mixing methimazole with a first AuNPs solution to obtain a mixed solution, irradiating the mixed solution with a first fluorescent N / SCQDs solution to form a first mixed solution with ultraviolet light to obtain a fluorescence spectrum of the first mixed solution, irradiating a second AuNPs solution with a second fluorescent N / SCQDs solution to form a second mixed solution with ultraviolet light to obtain a fluorescence spectrum of the second mixed solution, and then determining the methimazole content based on the fluorescence spectra of the first and second mixed solutions.

[0006] The European patent application number US20220390460A1, filed on August 19, 2022, discloses a methimazole adsorbent sample slide using graphene as a carrier modified with a dendrimer to allow controlled attachment of silver nanoparticles to its branches. The prepared graphene connected to silver nanoparticles (GD-Ag) stabilized by dendrimers was used as a SERS substrate for methimazole detection. However, these methods have limitations such as complex instrumentation, time-consuming procedures, relatively laborious sample pretreatment, and the need for trained technicians.

[0007] In summary, by combining SERS detection technology, a three-dimensional porous substrate material with good stability, uniformity and reproducibility has been developed, which enables the quantitative detection of trace antithyroid hormone drug molecules with a lower cost, simpler, faster and more sensitive analytical method. It can provide a reliable technical platform for the rapid detection of antithyroid drugs and has application potential in assisting the treatment of clinical thyroid diseases. Summary of the Invention

[0008] To solve the problems existing in the prior art, the present invention provides a three-dimensional hydrogel SERS substrate with a porous structure, as well as its preparation method and application. The prepared hydrogel has a 3D porous structure, which can make Ag NPs more evenly distributed in the hydrogel substrate and effectively reduce the aggregation of Ag NPs to construct richer "hot spots", showing good specificity in the detection of antithyroid hormone drugs methimazole and 2-thiouracil in plasma.

[0009] The technical solutions of the present invention are as follows:

[0010] One of the purposes of the present invention is to provide a three-dimensional hydrogel SERS substrate with a porous structure, which is composed of the following raw materials in parts by weight: 1 to 3 parts of silver nitrate, 1 to 3 parts of sodium citrate, 1 to 5 parts of N-isopropylacrylamide, 8 to 12 parts of polyethylene glycol diacrylate, 16 to 20 parts of 1-hydroxycyclohexyl phenyl ketone, and 2 to 6 parts of cellulose.

[0011] A second object of the present invention is to provide a method for preparing a porous three-dimensional hydrogel SERS substrate. The three-dimensional hydrogel SERS substrate is synthesized by a photocuring method. First, a nanosilver colloidal solution is prepared, and silver nanoparticles (Ag NPs) are dispersed and stabilized in a hydrogel network using cellulose. Then, N-isopropylacrylamide (NIPAM), a crosslinker (PEGDA), and a photoinitiator are added, mixed uniformly, and then the three-dimensional hydrogel SERS substrate is synthesized under ultraviolet light curing conditions. It is named Ag NPs / C-CNF / PNIPAM.

[0012] Further, the following steps are included:

[0013] S1. Prepare silver nanoparticles by a hydrothermal method: weigh silver nitrate and add it to deionized water. Heat and stir until boiling. Then add 1% sodium citrate solution by mass. Continue heating and reacting until the solution turns gray-green. Cool to room temperature and concentrate by centrifugation to obtain a silver nanoparticle colloidal solution.

[0014] S2. Weigh cellulose powder and dissolve it in deionized water, and dilute it to obtain a cellulose solution;

[0015] S3, weighing N-isopropylacrylamide (NIPAM) and dissolving it in the cellulose solution prepared in S2 to obtain a monomer solution;

[0016] S4, using ethanol as a solvent, preparing a photoinitiator containing 1 to 5 wt% of 1-hydroxycyclohexyl phenyl ketone;

[0017] S5, respectively measuring the nanosilver colloid solution, the monomer solution, the crosslinking agent polyethylene glycol diacrylate PEGDA and the photoinitiator, and uniformly mixing them to obtain a prepolymer;

[0018] S6. Drop the prepolymer prepared in S5 into a polytetrafluoroethylene template and use a UV curing box for cross-linking to prepare a three-dimensional hydrogel. After drying, the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate is obtained.

[0019] Furthermore, the volume ratio of deionized water to sodium citrate solution in S1 is 20:1; the heating and stirring speed is 500-550 rpm, and the heating reaction is continued for 1-1.3 hours.

[0020] Furthermore, the particle size of the nanosilver particles in S1 is 40 to 60 nm; and the concentration ratio of the nanosilver colloidal solution is 15 to 50 times.

[0021] Furthermore, the cellulose in S2 is any one of carboxymethyl cellulose, hydroxyethyl cellulose, mechanically ground nanofiber, sulfonated cellulose, carboxylated cellulose, and bacterial cellulose; and the cellulose concentration is 0.1% to 1%.

[0022] Furthermore, the weight ratio of N-isopropylacrylamide, nanosilver colloid solution, polyethylene glycol diacrylate and 1-hydroxycyclohexyl phenyl ketone used in preparing the prepolymer in S5 is 15-35:15-30:1-5:1-3.

[0023] Furthermore, the UV curing condition in S6 is crosslinking for 1 to 3 minutes under UV light with a wavelength of 365 nm and a power of 70 to 100%.

[0024] The third object of the present invention is to provide a three-dimensional hydrogel SERS substrate with a porous structure for use in methimazole (MMI) SERS detection in plasma.

[0025] Furthermore, the detection limit was 7.48×10 -9 M.

[0026] A fourth object of the present invention is to provide a three-dimensional hydrogel SERS substrate with a porous structure for use in SERS detection of 2-thiouracil (2-TU) in plasma.

[0027] Furthermore, the detection limit was 2.35×10 -8 M.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention discloses for the first time a porous structured Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate, stabilizes silver nanoparticles (Ag NPs) by cellulose, and introduces N-isopropylacrylamide (NIPAM) to construct a three-dimensional porous network structure, thereby achieving dual optimization of material design. Cellulose not only effectively inhibits the aggregation of Ag NPs, but also, through its hydrophilicity and high dispersibility, evenly distributes the nanoparticles in the hydrogel, enriches the hydrogel "hot spots", and significantly enhances the Raman signal. In addition, the three-dimensional porous structure has both molecular sieve effect and enrichment function, which can selectively adsorb small molecule drugs while blocking large molecular interferences in plasma, solving the problems of poor uniformity and insufficient stability of traditional SERS substrates. This design integrates the synergistic effect of materials science and nanotechnology, providing a new technology platform for high-sensitivity detection of anti-thyroid hormone drugs.

[0030] 2. The Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate disclosed in this invention has a 3D porous structure and exhibits excellent practical value in drug detection. Its three-dimensional porous structure can quickly separate and enrich trace drug molecules, and can directly detect plasma samples without complex pretreatment. Experiments show that the detection limit of the hydrogel SERS substrate for methimazole (MMI) and 2-thiouracil (2-TU) is as low as 7.48×10 -9 M and 2.35×10 -8 M, with high sensitivity, uniformity, reproducibility, and specificity, accurately identifying target molecules even in the presence of high concentrations of interfering substances. Combined with machine learning algorithms (PCA-LDA-SVM, PCA-LDA-RF, and PCA-LDA-MLP), the model achieved 100% classification accuracy for both drugs, with an AUC value of 1. This highlights the potential of combining spectroscopy with artificial intelligence in sample testing and provides new insights into the search for rapid, sensitive, and reliable drug detection tools.

[0031] 3. The present invention provides a method for preparing the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate, whose controllability and environmental protection characteristics ensure the feasibility of large-scale preparation of the three-dimensional hydrogel SERS substrate. The Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate is synthesized by ultraviolet curing, which is simple and low-cost. By systematically optimizing the Ag NPs concentration multiple, cellulose type and content, the uniformity and stability of the SERS signal are significantly improved. During the preparation process, the present invention not only enhances the colloidal stability of Ag NPs through the introduction of cellulose, but also gives the hydrogel excellent mechanical properties and environmental responsiveness. In addition, cellulose, as a renewable biomaterial, has both low Raman background and biocompatibility, which further reduces detection interference and expands application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the preparation principle of the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate of the present invention;

[0033] Figure 2 Characterization of the silver nanoparticles prepared in Example 1 of the present invention, including (A) UV-Vis image of Ag NPs; (B) DLS spectrum of Ag NPs; (C) TEM image of Ag NPs; (D) Zeta potential of Ag NPs, C-CNFs, and Ag NPs / C-CNFs;

[0034] Figure 3 SEM images and elemental analysis of the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate prepared in Example 1 of the present invention, where (A) under freeze-drying conditions, scale bar: 10 μm; (B) 5 μm; (C) 2 μm; (D) under oven-dried conditions, scale bar: 10 μm; (E) 5 μm; (F) 2 μm; (G) all elements; (H) EDS elemental mapping of C, N, O, and Ag; (I) EDS spectrum of the dried hydrogel;

[0035] Figure 4 Spectral characterization of the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate prepared in Example 1 of the present invention, including (A) ATR-FTIR spectrum; (B) XRD spectrum; (C) full XPS spectrum; (D) Ag 3d; (E) C 1s; (F) N 1s; (G) O 1s XPS spectrum;

[0036] Figure 5 The Raman signal sensitivity test results of the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate prepared in Example 1 of the present invention for detecting R6G, wherein (A) is composed of 10 -5 M to 10 -9 SERS spectrum of MR6G; (B) 1514 cm -1 Linear regression curve of SERS signal intensity at and R6G concentration;

[0037] Figure 6 The results of the Raman signal uniformity test of R6G detected by the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate prepared in Example 1 of the present invention are shown in Figure 1, where (A) AgNPs / PNIPAM detected 10 -5 SERS spectrum of MR6G; (B) 1514 cm -1SERS signal intensity distribution at (C) AgNPs / C-CNF / PNIPAM detection 10 -5 SERS spectrum of MR6G; (D) 1514 cm -1 SERS signal intensity distribution at ;

[0038] Figure 7 The stability test results of Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate prepared in Example 1 of the present invention are shown in Figure 1. (A) 5 batches were prepared and tested for 10 -5 SERS spectrum of M R6G;

[0039] (B)1514cm -1 (C) Preservation for 50 days and detection of 10 -5 SERS spectrum of MR6G; (D) 1514 cm -1 SERS signal intensity at ;

[0040] Figure 8 This is the detection test of MMI by Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate prepared in Example 1 of the present invention, wherein the detection concentration (A) is from 10 -4 M to 10 -8 SERS spectrum of M MMI; (B) 1365 cm -1 Linear regression curve of SERS signal intensity and concentration at 1365 cm; (C) SERS spectra of different interfering substances; (D) -1 SERS signal intensity at 1365 cm; (E) SERS spectra of MMI in different plasma samples; (F) 1365 cm -1 SERS signal intensity and recovery rate at

[0041] Figure 9 This is the detection test of 2-TU by Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate prepared in Example 1 of the present invention, wherein the detection concentration (A) is from 10 -3.5 M to 10 -7.5 SERS spectrum of M 2-TU; (B) 923 cm -1 Linear regression curve of SERS signal intensity and concentration at 923 cm; (C) SERS spectra of different interfering substances; (D) -1 SERS signal intensity at 923 cm; (E) SERS spectra of 2-TU in different plasma samples; (F) -1 SERS signal intensity and recovery rate at . DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with preferred embodiments. The endpoints of the ranges and any values ​​disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0043] Unless otherwise specified, the experimental methods in the following examples are conventional methods and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0045] The main reagents and manufacturers in the following examples are as follows:

[0046]

[0047] The main instrument models and production places in the following examples are as follows:

[0048]

[0049]

[0050] Example 1

[0051] This embodiment provides a method for preparing an Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate, comprising the following steps:

[0052] S1. Prepare silver nanoparticles by a hydrothermal method: weigh 0.017 g of silver nitrate and add it to 100 mL of deionized water. Heat and stir at 500 rpm until boiling. Add 5 mL of 1% sodium citrate solution and continue heating for 1 h until the solution turns gray-green. Cool to room temperature and concentrate by centrifugation to obtain a silver nanoparticle colloidal solution.

[0053] S2. Weigh 1 g of carboxymethyl cellulose and dissolve it in 100 mL of deionized water, and dilute it to obtain a 1% carboxymethyl cellulose solution.

[0054] S3, weigh 0.57 g of N-isopropylacrylamide (NIPAM) and dissolve it in 5 mL of the carboxymethyl cellulose solution prepared in S2 to obtain a 1 M monomer solution;

[0055] S4. Weigh 0.09 g of 1-hydroxycyclohexyl phenyl ketone and dissolve it in 4 mL of ethanol to prepare a photoinitiator with a concentration of 2.25 wt %;

[0056] S5, respectively measuring 50 μL of monomer solution, 50 μL of nanosilver colloid solution, 5 μL of cross-linking agent polyethylene glycol diacrylate, and 2 μL of photoinitiator, and evenly mixing them to obtain a prepolymer;

[0057] S6. The prepolymer prepared in S5 was dropped into a polytetrafluoroethylene template, and a three-dimensional hydrogel was prepared by crosslinking for 1 minute in a UV curing box under UV light with a wavelength of 365 nm and a power of 90%. After drying at 50°C for 1 hour, the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate was obtained.

[0058] Example 2

[0059] This embodiment provides a method for preparing an Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate, comprising the following steps:

[0060] S1. Prepare silver nanoparticles by a hydrothermal method: weigh 0.017 g of silver nitrate and add it to 100 mL of deionized water. Heat and stir at 500 rpm until boiling. Add 5 mL of 1% sodium citrate solution and continue heating for 1 h until the solution turns gray-green. Cool to room temperature and concentrate by centrifugation to obtain a silver nanoparticle colloidal solution.

[0061] S2. Weigh 0.5 g of hydroxyethyl cellulose and dissolve it in 100 mL of deionized water, and dilute it to obtain a 0.5% hydroxyethyl cellulose solution.

[0062] S3, weighing 0.57 g of N-isopropylacrylamide (NIPAM) and dissolving it in 5 mL of the cellulose solution prepared in S2 to obtain a 1 M monomer solution;

[0063] S4, weighing 0.1 g of 1-hydroxycyclohexyl phenyl ketone and dissolving it in 4 mL of ethanol to prepare a photoinitiator with a concentration of 2.5 wt %;

[0064] S5, respectively measuring 50 μL of monomer solution, 40 μL of nanosilver colloid solution, 5 μL of cross-linking agent polyethylene glycol diacrylate, and 3 μL of photoinitiator, and evenly mixing them to obtain a prepolymer;

[0065] S6. The prepolymer prepared in S5 was dropped into a polytetrafluoroethylene template, and a three-dimensional hydrogel was prepared by crosslinking for 1.5 minutes under ultraviolet light with a wavelength of 365 nm and a power of 80% using a UV curing box. After drying at 50°C for 1 hour, the AgNPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate was obtained.

[0066] Example 3

[0067] This embodiment provides a method for preparing an Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate, comprising the following steps:

[0068] S1. Prepare silver nanoparticles by a hydrothermal method: weigh 0.017 g of silver nitrate and add it to 100 mL of deionized water. Heat and stir the mixture at 550 rpm until boiling. Add 5 mL of a 1% sodium citrate solution and continue heating for 1.3 h until the solution turns gray-green. Cool to room temperature and concentrate by centrifugation to obtain a silver nanoparticle colloidal solution.

[0069] S2, weighing 10 mL of 2% mechanically ground nanofibers, dissolving them in 10 mL of deionized water, and diluting them to obtain a 1% mechanically ground nanocellulose solution;

[0070] S3, weighing 0.57 g of N-isopropylacrylamide (NIPAM) and dissolving it in 5 mL of the cellulose solution prepared in S2 to obtain a 1 M monomer solution;

[0071] S4, weighing 0.15 g of 1-hydroxycyclohexyl phenyl ketone and dissolving it in 4 mL of ethanol to prepare a photoinitiator with a concentration of 3.75 wt %;

[0072] S5, respectively measuring 50 μL of monomer solution, 45 μL of nanosilver colloid solution, 5 μL of cross-linking agent polyethylene glycol diacrylate, and 2 μL of photoinitiator, and evenly mixing them to obtain a prepolymer;

[0073] S6. The prepolymer prepared in S5 was dropped into a polytetrafluoroethylene template, and a three-dimensional hydrogel was prepared by crosslinking for 2 minutes under ultraviolet light with a wavelength of 365 nm and a power of 80% in a UV curing box. After drying at 50°C for 1 hour, the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate was obtained.

[0074] Example 4

[0075] This embodiment provides a method for preparing an Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate, comprising the following steps:

[0076] S1. Prepare silver nanoparticles by a hydrothermal method: weigh 0.017 g of silver nitrate and add it to 100 mL of deionized water. Heat and stir at 500 rpm until boiling. Add 5 mL of 1% sodium citrate solution and continue heating for 1.2 h until the solution turns gray-green. Cool to room temperature and concentrate by centrifugation to obtain a silver nanoparticle colloidal solution.

[0077] S2. Weigh 10 mL of 1% sulfonated cellulose and dissolve it in 40 mL of deionized water. Dilute to obtain a 0.2% sulfonated cellulose solution.

[0078] S3, weighing 0.57 g of N-isopropylacrylamide (NIPAM) and dissolving it in 5 mL of the cellulose solution prepared in S2 to obtain a 1 M monomer solution;

[0079] S4. Weigh 0.05 g of 1-hydroxycyclohexyl phenyl ketone and dissolve it in 4 mL of ethanol to prepare a photoinitiator with a concentration of 1.25 wt %;

[0080] S5, respectively measuring 50 μL of monomer solution, 45 μL of nanosilver colloid solution, 5 μL of cross-linking agent polyethylene glycol diacrylate, and 4 μL of photoinitiator, and evenly mixing them to obtain a prepolymer;

[0081] S6. The prepolymer prepared in S5 was dropped into a polytetrafluoroethylene template, and a three-dimensional hydrogel was prepared by crosslinking for 3 minutes under ultraviolet light with a wavelength of 365 nm and a power of 70% using a UV curing box. After drying at 50°C for 1.5 hours, the AgNPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate was obtained.

[0082] Example 5

[0083] This embodiment provides a method for preparing an Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate, comprising the following steps:

[0084] S1. Prepare silver nanoparticles by a hydrothermal method: weigh 0.017 g of silver nitrate and add it to 100 mL of deionized water. Heat and stir the mixture at 525 rpm until boiling. Add 5 mL of a 1% sodium citrate solution and continue heating for 1 h until the solution turns gray-green. Cool to room temperature and concentrate by centrifugation to obtain a silver nanoparticle colloidal solution.

[0085] S2. Weigh 10 mL of 6% carboxylated cellulose and dissolve it in 50 mL of deionized water, and dilute it to obtain a 1% carboxylated cellulose solution.

[0086] S3, weighing 0.57 g of N-isopropylacrylamide (NIPAM) and dissolving it in 5 mL of the cellulose solution prepared in S2 to obtain a 1 M monomer solution;

[0087] S4. Weigh 0.16 g of 1-hydroxycyclohexyl phenyl ketone and dissolve it in 4 mL of ethanol to prepare a photoinitiator with a concentration of 4 wt %;

[0088] S5, respectively measuring 50 μL of monomer solution, 40 μL of nanosilver colloid solution, 6 μL of crosslinker polyethylene glycol diacrylate, and 2 μL of photoinitiator, and evenly mixing them to obtain a prepolymer;

[0089] S6. The prepolymer prepared in S5 was dropped into a polytetrafluoroethylene template, and a three-dimensional hydrogel was prepared by crosslinking for 1 minute in a UV curing box under UV light with a wavelength of 365 nm and a power of 100%. After drying at 50°C for 1.5 hours, the AgNPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate was obtained.

[0090] Example 6

[0091] This embodiment provides a method for preparing an Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate, comprising the following steps:

[0092] S1. Prepare silver nanoparticles by a hydrothermal method: weigh 0.017 g of silver nitrate and add it to 100 mL of deionized water. Heat and stir at 500 rpm until boiling. Add 5 mL of 1% sodium citrate solution and continue heating for 1.2 h until the solution turns gray-green. Cool to room temperature and concentrate by centrifugation to obtain a silver nanoparticle colloidal solution.

[0093] S2. Weigh 10 mL of 1% bacterial cellulose and dissolve it in 10 mL of deionized water, and dilute it to obtain a 0.5% bacterial cellulose solution.

[0094] S3, weighing 0.57 g of N-isopropylacrylamide (NIPAM) and dissolving it in 5 mL of the cellulose solution prepared in S2 to obtain a 1 M monomer solution;

[0095] S4. Weigh 0.14 g of 1-hydroxycyclohexyl phenyl ketone and dissolve it in 4 mL of ethanol to prepare a photoinitiator with a concentration of 3.5 wt %;

[0096] S5, respectively measuring 50 μL of monomer solution, 40 μL of nanosilver colloid solution, 6 μL of crosslinker polyethylene glycol diacrylate, and 2 μL of photoinitiator, and evenly mixing them to obtain a prepolymer;

[0097] S6. The prepolymer prepared in S5 was dropped into a polytetrafluoroethylene template, and a three-dimensional hydrogel was prepared by crosslinking for 1.5 minutes under ultraviolet light with a wavelength of 365 nm and a power of 100% in a UV curing box. After drying at 50°C for 1.5 hours, the AgNPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate was obtained.

[0098] Performance Characterization

[0099] 1. Characterization of Ag NPs

[0100] The optical properties of Ag NPs, C-CNF and Ag NPs / C-CNF colloids were studied by UV-Vis. Figure 2As shown in Figure A. The Ag NPs sol has a broad optical absorption band at approximately 416 nm, generating strong plasmon excitation. The C-CNF does not exhibit a UV characteristic peak, while the maximum absorption peak of the Ag NPs / C-CNF appears at 420 nm. Through DLS testing, it can be approximately characterized that the particle size of the Ag NPs is mainly distributed around 50-60 nm, as shown in Figure 4. Figure 2 B. The micromorphology of AgNPs was characterized by TEM, as shown in Figure 2 As shown in Figure C, the Ag NPs have a spherical shape and a uniform size of about 50 nm. They have well-defined edges and are well distributed without obvious aggregation. Therefore, Ag NPs have a beneficial effect on the enhancement of SERS signals.

[0101] The stability of Ag NPs / C-CNF / PNIPAM hydrogel was evaluated by measuring the Zeta potential of Ag colloid solution and Ag / C-CNF colloid solution using nanoparticle size and Zeta potential analyzer. Figure 2 As shown in Figure D, the Zeta potential of Ag NPs is -19.00 mV, indicating that the Ag NPs solution is unstable. The Zeta potential of C-CNF is -43.43 mV, which has good stability. The Zeta potential of Ag / C-CNF is -38.94 mV, indicating that Ag NPs form a stable colloidal solution in the presence of C-CNF. This is because the stability of the colloid depends on the Zeta potential value. When the Zeta potential is greater than +30 mV or less than -30 mV, the colloid is considered to be stable.

[0102] Stable colloids can enable hydrogels to produce stable SERS signals. The test results show that C-CNF has a certain degree of regulatory effect on the SERS signal of Ag NPs / C-CNF / PNIPAM hydrogel, which has better stability.

[0103] 2. Morphological and structural characterization

[0104] The morphology and structure of the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate prepared in Example 1 were characterized. Figure 3 As shown in A to C. After freeze-drying, the hydrogel was observed by SEM. The surface was a porous structure in the range of 10-30 μm, and the pores were relatively uniform and orderly. Figure 3 As shown in D to F, the morphology of the hydrogel was observed by SEM after drying in an oven. It can be seen that the volume of the hydrogel shrank significantly after drying, and the pores were closed. Figure 3 G to H show that the EDS spectrum is as follows Figure 3As shown in Figure 1, it shows that Ag NPs were successfully added to the hydrogel PNIPAM, and the Ag NPs were evenly distributed on PNIPAM.

[0105] 3. Spectral characterization

[0106] The chemical structures of PNIPAM and Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel in Example 1 were characterized by FTIR. Figure 4 As shown in A. 3458cm -1 The broad absorbance peak at 2877 cm corresponds to the OH stretching vibration of PNIPAM hydrogel. -1 The absorption peak at 1720 cm is the CH stretching vibration. -1 The absorption peak at 1551 cm is mainly attributed to the C=O stretching vibration of the ester group. -1 and 1638cm -1 The two absorption peaks at NH=O represent the bending vibration of NH and the stretching vibration of NC=O, respectively. Compared with PNIPAM hydrogel, the spectrum of Ag NPs / PNIPAM hydrogel does not show any new vibration absorption peaks, and the absorption peak positions do not change much. This indicates that there is only physical interaction between PNIPAM and Ag NPs, and no new covalent bonds are formed. The loading of Ag NPs has almost no effect on PNIPAM hydrogel.

[0107] The structure and composition of the hydrogel were characterized by XRD patterns. Figure 4 As shown in Figure B, the prepared C-CNF / PNIPAM hydrogel is non-crystalline. For the synthesized Ag NPs-C-CNF / PNIPAM hydrogel, strong diffraction peaks are shown at 2θ = 38.3, 44.4, 64.7, and 77.4°, corresponding to the (111), (200), (220), and (311) crystal planes of the face-centered cubic Ag crystal, respectively. The high intensity of these diffraction peaks indicates the high crystallinity of the Ag NPs.

[0108] In order to further verify the elements on the surface, the XPS spectrum of Ag NPs / C-CNF / PNIPAM hydrogel was obtained by XPS analysis. Figure 4 As shown in Figure C, it can be clearly found that different peaks are detected on the Ag 3d and Ag 4p energy level orbitals of the Ag NPs / C-CNF / PNIPAM hydrogel, which are attributed to the loading of Ag NPs on the hydrogel. Figure 4 As shown in D, there are two prominent peaks of Ag 3d orbital at 373.9eV and 368.1eV, which are identified as Ag 3d 3 / 2 and Ag 3d 5 / 2The C 1s spectrum has two peaks with corresponding binding energies of 284.8 eV and 286.3 eV, which are assigned to CC and CO of Ag NPs / C-CNF / PNIPAM hydrogel, respectively. Figure 4 E). The N 1s spectrum fits a peak with a binding energy of 399.6 eV, which is related to the -CONH2 of PNIPAM ( Figure 4 F). The O 1s spectrum has one peak with a corresponding binding energy of 532.5 eV, which is assigned to the CC / C=O( Figure 4 G).

[0109] 4. SERS performance evaluation of Ag NPs / C-CNF / PNIPAM hydrogel

[0110] (1) Sensitivity

[0111] Different concentrations of R6G dye molecules were used to investigate the relationship between the SERS intensity of the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel described in Example 1 and the concentration of the tested substance. Figure 5 As shown in A and B, the Raman signal of R6G gradually increases with the increase of concentration, and the 1514 cm -1 The peak has a good linear relationship with the R6G concentration (correlation coefficient R 2 The detection limit of R6G was as low as 9.21×10 -10 M.

[0112] (2) Uniformity

[0113] 100 random Raman spectra of R6G were scanned on Ag NPs / PNIPAM hydrogel and Ag NPs / C-CNF / PNIPAM 3D hydrogel, respectively. Figure 6 As shown in A to D, it was found that the hydrogel without cellulose addition had a peak at 1514 cm -1 The RSD at 1514 cm was 10.07%, while the RSD at 1514 cm was 10.07%. -1 The RSD at is only 3.57%, indicating that the SERS spectrum of the cellulose-containing hydrogel of the present invention has better consistency, and the Ag NPs / C-CNF / PNIPAM hydrogel SERS substrate has good uniformity.

[0114] (3) Stability and reproducibility

[0115] The SERS signals of the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel described in Example 1 were recorded every ten days to evaluate the stability of the hydrogel. Figure 7As shown in A and B. With the increase of time, the SERS signal of the hydrogel showed a slight attenuation. After 50 days, the signal only decreased by 16.7%, which shows that the substrate can prevent the oxidation of Ag NPs and the hydrogel has good stability.

[0116] Records from five different batches of Ag NPs / C-CNF / PNIPAM hydrogels, from Figure 7 As can be clearly seen in Figures C and D, the SERS signals of the hydrogel SERS substrate are basically the same, indicating that the Ag NPs / C-CNF / PNIPAM hydrogel SERS substrate has excellent batch-to-batch reproducibility. These results show that the good performance of the hydrogel is suitable for detection in practical applications.

[0117] 5. MMI detection test

[0118] The Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate described in Example 1 of the present invention was used to measure the MMI standard solution from 10 -4 M to 10 -8 M was quantitatively tested, and the results were as follows Figure 8 As shown in A and B, the SERS intensity increases with the increase of MMI concentration, and the minimum detection concentration of MMI is as low as 7.48×10 -9 M. 1365cm -1 The SERS signal intensity at the position is linearly related to the logarithm of the MMI concentration, and the correlation coefficient (R 2 ) is 0.9965.

[0119] At the same time, potential interfering substances (KCl, K2CO3, NaCl, Na2SO4, GSH, L-glutamic acid, GLU and UA) were studied to evaluate the selectivity of this method for detecting MMI. Figure 8 As shown in C and D, even though the concentrations of other interfering substances are much higher than that of MMI, the -1 There is no characteristic peak of MMI at the position of , which only appears when MMI is present. The results show that the proposed method is suitable for the detection of MMI in actual samples.

[0120] In addition, Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate was used to detect MMI in spiked human plasma. Figure 8 As shown in E and F, the spiked concentration was 10 -4 M, 3.16×10 -6 M and 10 -6The recoveries of MMI were 101.1%, 99.8%, and 99.7% at 40 °C, 75 °C, and 100 °C, respectively. This indicates that the Ag NPs / C-CNF / PNIPAM hydrogel can selectively adsorb small molecules and block the entry of large molecules. Therefore, it can be applied to the detection of MMI in plasma.

[0121] 6. 2-TU detection test

[0122] Ag NPs / C-CNF / PNIPAM hydrogel SERS substrate was used to analyze the 2-TU standard solution from 10 -3.5 M to 10 -7.5 M was quantitatively tested, and the results were as follows Figure 9 As shown in A and B, the SERS intensity increases with the increase of 2-TU concentration, and the minimum detection concentration of 2-TU is as low as 2.35×10 -8 M. 923cm -1 The SERS signal intensity at the site is linearly related to the logarithm of the 2-TU concentration, and the correlation coefficient (R 2 ) is 0.9900.

[0123] Potential interfering substances (KCl, K2CO3, NaCl, Na2SO4, GSH, L-glutamic acid, GLU and UA) were studied to evaluate the selectivity of the method for detecting 2-TU. Figure 9 As shown in C and D, even though the concentrations of other interfering substances are much higher than that of 2-TU, the -1 The characteristic peak of 2-TU does not appear at the position of the sample, and it only appears when 2-TU is present. The results show that the proposed method is suitable for the detection of 2-TU in actual samples.

[0124] In addition, Ag NPs / C-CNF / PNIPAM hydrogel SERS substrate was used to detect 2-TU in spiked human plasma. Figure 9 As shown in E and F, the spiked concentration was 10 -4 M, 10 -5 M and 10 -6 The recoveries of 2-TU were 96.8%, 100.8%, and 99.3% at 400 nm, respectively, indicating that the Ag NPs / C-CNF / PNIPAM hydrogel can selectively adsorb small molecules and block the entry of large molecules. Therefore, it can be applied to the detection of 2-TU in plasma.

[0125] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A three-dimensional hydrogel SERS substrate with a porous structure, characterized in that: The invention is composed of the following raw materials in parts by weight: 1 to 3 parts of silver nitrate, 1 to 3 parts of sodium citrate, 1 to 5 parts of N-isopropylacrylamide, 8 to 12 parts of polyethylene glycol diacrylate, 16 to 20 parts of 1-hydroxycyclohexyl phenyl ketone and 2 to 6 parts of cellulose.

2. A method for preparing the porous three-dimensional hydrogel SERS substrate according to claim 1, characterized in that: The three-dimensional hydrogel SERS substrate was synthesized by a photocuring method. First, a nanosilver colloidal solution was prepared, and silver nanoparticles (Ag NPs) were dispersed and stabilized in the hydrogel network using cellulose. N-isopropylacrylamide (NIPAM), a crosslinker (polyethylene glycol diacrylate) (PEGDA), and a photoinitiator were then added, mixed evenly, and then cured under ultraviolet light to synthesize the three-dimensional hydrogel SERS substrate, named Ag NPs / C-CNF / PNIPAM.

3. The method for preparing a porous three-dimensional hydrogel SERS substrate according to claim 2, characterized in that: The following steps are involved: S1. Prepare silver nanoparticles by a hydrothermal method: weigh silver nitrate and add it to deionized water. Heat and stir until boiling. Then add 1% sodium citrate solution by mass. Continue heating and reacting until the solution turns gray-green. Cool to room temperature and concentrate by centrifugation to obtain a silver nanoparticle colloidal solution. S2. Weigh cellulose powder and dissolve it in deionized water, and dilute it to obtain a cellulose solution; S3, weighing N-isopropylacrylamide (NIPAM) and dissolving it in the cellulose solution prepared in S2 to obtain a monomer solution; S4, using ethanol as a solvent, preparing a photoinitiator containing 1 to 5 wt% of 1-hydroxycyclohexyl phenyl ketone; S5, respectively measuring the nanosilver colloid solution, the monomer solution, the crosslinking agent polyethylene glycol diacrylate PEGDA and the photoinitiator, and uniformly mixing them to obtain a prepolymer; S6. Drop the prepolymer prepared in S5 into a polytetrafluoroethylene template and use a UV curing box for cross-linking to prepare a three-dimensional hydrogel. After drying, the Ag NPs / C-CNF / PNIPAM three-dimensional hydrogel SERS substrate is obtained.

4. The method for preparing a porous three-dimensional hydrogel SERS substrate according to claim 3, characterized in that: The volume ratio of deionized water to sodium citrate solution in S1 is 20:1; the heating and stirring speed is 500-550 rpm, and the heating reaction is continued for 1-1.3 hours.

5. The method for preparing a porous three-dimensional hydrogel SERS substrate according to claim 3, characterized in that: The particle size of the nano-silver particles in S1 is 40 to 60 nm; and the concentration ratio of the nano-silver colloid solution is 15 to 50 times.

6. The method for preparing a porous three-dimensional hydrogel SERS substrate according to claim 3, characterized in that: The cellulose in S2 is any one of carboxymethyl cellulose, hydroxyethyl cellulose, mechanically ground nanofiber, sulfonated cellulose, carboxylated cellulose, and bacterial cellulose; and the cellulose concentration is 0.1% to 1%.

7. The method for preparing a porous three-dimensional hydrogel SERS substrate according to claim 3, characterized in that: The weight ratio of N-isopropylacrylamide, nanosilver colloid solution, polyethylene glycol diacrylate and 1-hydroxycyclohexyl phenyl ketone used in preparing the prepolymer in S5 is 15-35:15-30:1-5:1-3.

8. The method for preparing a porous three-dimensional hydrogel SERS substrate according to claim 3, characterized in that: The UV curing conditions in S6 are crosslinking for 1 to 3 minutes under UV light with a wavelength of 365 nm and a power of 70 to 100%.

9. Use of a porous three-dimensional hydrogel SERS substrate prepared by the method according to any one of claims 2 to 8 in the detection of methimazole (MMI) SERS in plasma, characterized in that: The detection limit was 7.48×10 -9 M.

10. Use of a porous three-dimensional hydrogel SERS substrate prepared by the method according to any one of claims 2 to 8 in SERS detection of 2-thiouracil (2-TU) in plasma, characterized in that: The detection limit was 2.35×10 -8 M.

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