Preparation method of double-network silver nanoparticle carboxymethyl fiber hydrogel and application of hydrogel in anti-cancer drug detection

Abstract

The invention discloses a preparation method of double-network silver nanoparticle carboxymethyl cellulose hydrogel, which comprises the following steps: firstly, adding a chemical covalent cross-linking agent, natural anion polysaccharide pectin molecules and a photoinitiator into a reaction monomer acrylamide solution, fully mixing, adding carboxymethyl cellulose silver nanoparticles and a quantitative chitosan solution, and fully mixing to obtain the double-network silver nanoparticle carboxymethyl cellulose hydrogel. After ultrasonic full mixing and ultraviolet curing, polyacrylamide polymer molecules and carboxyl groups in natural anion polysaccharide pectin molecules can form a single-network structure, and carboxymethyl cellulose silver nanoparticles can continue to be polymerized to synthesize a double-network composite hydrogel material, so that a three-dimensional SERS substrate is formed. According to the method, a traditional hydrogel preparation method is innovated, uniform silver nanoparticles can be modified on the surface of the hydrogel, and a foundation is laid for subsequent detection. The prepared protein / Ag NPs / CMC / CS / PAAM composite hydrogel can be used as an SERS (Surface Enhanced Raman Scattering) substrate material in the detection of an anti-cancer drug (5-FU).

Description

Technical Field

[0001] The present invention relates to the technical field of materials science, and particularly relates to a preparation method of a double-network silver nanoparticle carboxymethyl cellulose hydrogel and its application in the detection of anti-cancer drugs. Background Art

[0002] 5-Fluorouracil (5-FU) is a widely used antimetabolic drug, mainly used for the treatment of digestive tract cancers and other solid tumors; however, due to its toxic side effects and individual differences during the treatment process, the detection and dosage management of 5-FU become particularly important. The research on 5-FU mainly focuses on improving its efficacy, reducing toxicity, and solving environmental pollution and occupational exposure problems. In recent years, electrochemical detection methods have received attention due to their advantages such as low detection limit, good reproducibility, simple pretreatment, and convenient operation. In particular, the application of new materials in modifying electrodes has further improved the detection effect. Currently, the detection methods of 5-FU include chromatographic methods, electrochemical methods, etc. Electrochemical methods, especially differential pulse voltammetry (DPV) and square wave voltammetry (SWV), are widely used in the detection of 5-FU due to their high sensitivity, good selectivity, and high precision. In addition, the detection of 5-FU also involves considerations of drug pharmacokinetics to achieve personalized chemotherapy regimens. Although progress has been made in the detection technology of 5-FU, there are still some limitations. For example, although chromatographic methods have high accuracy, they have a long analysis time, complex pretreatment, and require the use of toxic organic solvents, etc.

[0003] Hydrogels are polymeric materials with a three-dimensional network structure that can absorb and retain large amounts of water without dissolving. They are typically composed of cross-linked polymer chains, which can be natural or synthetic. NanoSilver is metallic silver in its elemental form with a particle size in the nanometer range. The particle size of NanoSilver is usually in the range of 1 - 100 nanometers. Silver nanoparticles have attracted much attention due to their unique physicochemical, biological, and antibacterial properties, making them widely used in the medical, biopharmaceutical, environmental, and antibacterial fields. The properties of hydrogels enable them to have a wide range of applications in multiple fields. The combination of the two described in this article for medical and health research. Hydrogels usually have good biocompatibility and softness, but poor mechanical properties. Therefore, by introducing silver nanoparticles, not only can their antibacterial properties be improved, but also their mechanical properties can be enhanced. The combination of silver nanoparticles and hydrogels can prepare composite materials with multiple functions. For example, intelligent hydrogels sensitive to changes in external conditions such as temperature, pH, and ionic strength can be developed. Such composite materials have broad application prospects in the fields of medical and health, biosensors, drug delivery systems, wearable devices, etc. For example, they can be used to develop antibacterial dressings, intelligent response medical devices, etc. Although the combination of silver nanoparticles and hydrogels shows great potential, there are still some challenges, such as the dispersion and reproducibility of nanoparticles.

[0004] Therefore, the present invention provides a double-network silver nanoparticle carboxymethyl cellulose hydrogel and a preparation method thereof. As an SERS substrate material, it can achieve the efficient detection of 5-FU and has great application prospects in the field of cancer drug detection. Summary of the Invention

[0005] Aiming at the above problems, the purpose of the present invention is to provide a preparation method of a double-network silver nanoparticle carboxymethyl cellulose hydrogel and its application in the detection of anti-cancer drugs. The constructed pectin / Ag NPs / CMC / CS / PAAM hydrogel has a uniform nanostructure, strong stability, and excellent adsorption capacity, and can rapidly adsorb a large amount of plasmas or plasma precursors, thereby improving the sensitivity and stabilizing the surface-enhanced Raman spectroscopy signal. As an SERS substrate, it can be used for the efficient detection of anti-cancer drugs (5-FU).

[0006] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0007] A preparation method of a double-network silver nanoparticle carboxymethyl cellulose hydrogel, comprising the following steps:

[0008] 1): Ultrasonically mix the pectin aqueous solution, acrylamide aqueous solution, photoinitiator, and cross-linking agent evenly under water bath heating to obtain a hydrogel precursor solution;

[0009] 2): Add anionic cellulose carboxymethyl cellulose to the Ag NPs solution to obtain the Ag NPs / CMC solution;

[0010] 3): After thoroughly mixing the Ag NPs / CMC solution with the hydrogel precursor solution, add the chitosan solution and mix evenly. Then, place it in a UV curing oven for polymerization reaction to form the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel;

[0011] 4): Place the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel in a vacuum drying oven for drying.

[0012] As a possible implementation, further, in step 1), a 2 wt% aqueous pectin solution, an 8.4 M acrylamide aqueous solution 、 a 0.084 M photoinitiator, and a 0.3 M crosslinking agent are ultrasonically mixed evenly under water bath heating;

[0013] The volume ratio of the pectin, acrylamide, photoinitiator, and crosslinking agent is 1:1:1:1;

[0014] The crosslinking agent is selected as N,N′-methylenebisacrylamide, and the photoinitiator is selected as Irgacure2959.

[0015] As a possible implementation, further, in step 2), 100 μL of 0.1 wt% anionic cellulose carboxymethyl cellulose is added to 200 μL of the concentrated Ag NPs solution;

[0016] The preparation method of the concentrated Ag NPs solution includes the following steps:

[0017] Dissolve 36 mg of AgNO3 in 200 mL of deionized water and boil it. Add 4 mL of a 1% sodium citrate solution;

[0018] Then, boil the reaction solution at room temperature for 1 h and let it cool naturally. Centrifuge the obtained Ag NPs and collect the precipitate. Then, redisperse it with deionized water and remove the excess sodium citrate in the solution;

[0019] Next, concentrate the obtained Ag NPs by 7 times (from 7 mL to 1 mL) to obtain the concentrated Ag NPs solution.

[0020] As a possible implementation, further, in step 3), after thoroughly mixing 300 μL of the concentrated Ag NPs / CMC solution with 500 μL of the hydrogel precursor solution, add 200 μL of a 0.0625 wt% chitosan solution and mix evenly.

[0021] As a possible implementation, further, the water bath heating temperature in step 1) is 70 °C, and the ultrasonic mixing time is 20 min;

[0022] In step 3), during the UV curing process, it is irradiated with a UV lamp with a wavelength of 365 nm for 500 s.

[0023] As a possible implementation, further, in step 4), the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel is placed in a vacuum drying oven at 0.9 kPa and 50 °C for drying for 30 min.

[0024] The present invention also provides a double-network silver nanoparticle carboxymethyl cellulose hydrogel, which is prepared by the above preparation method and is used as a substrate material in the SERS detection process and can be applied to the detection of anticancer drugs (5-FU).

[0025] As a possible implementation, further, the 5-FU detection steps include:

[0026] A): Cut the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel substrate into fragments with a size of 0.5×0.5 cm 2 fragments;

[0027] B): Immerse the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel fragments in a neutral to alkaline solution to adjust the pH of the hydrogel SERS substrate, then take them out and dry them, and then immerse the hydrogel SERS substrate in the test solution containing 5-FU;

[0028] C): Take out the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel fragments and dry them, and then perform SERS detection.

[0029] As a possible implementation, further, the soaking time in step C) is 60 min.

[0030] The present invention has the following beneficial effects compared with the prior art:

[0031] The present invention prepares a double-network silver nanoparticle carboxymethyl cellulose pectin / Ag NPs / CMC / CS / PAAM polyacrylamide hydrogel, which can adsorb a large amount of Ag in an aqueous solution +A certain amount of chitosan was introduced to enhance the toughness and hardness of the hydrogel. Further, a photoinitiator, 0.028 M Irgacure 2959, was introduced for photocuring reaction and photopolymerization reaction, which could effectively reduce the exposed area of silver nanoparticles therein and prevent the oxidation of silver nanoparticles. The gap adjustment of AgNPs in the hydrogel could change the SERS signal. The SERS performance of the pectin / AgNPs / CMC / CS / PAAM composite hydrogel was evaluated by detecting dye molecules and the signal molecule R6G, which showed high sensitivity (detectable to 10 -8 M) and good signal uniformity, with a relative standard deviation (RSD) as low as 2.309%. Moreover, using pectin / Ag NPs / CMC / CS / PAAM as the SERS substrate, the high-efficiency detection of the concentration of 5-fluorouracil in pharmaceutical preparations or biological samples could be achieved by SERS detection, showing great application prospects in the field of cancer drug detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0033] Figure 1 It is the preparation mechanism of the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel material substrate.

[0034] Figure 2 It is the characterization diagram of the hydrogel material; among them, (A, B) are the TEM microstructures of Ag NPs and Ag NPs / CMC; (C, D) are the physical pictures of PAAM, pectin / CS / PAAM, Ag NPs / CMC / PAAM, and pectin / Ag NPs / CMC / CS / PAAM; (E) Zeta potential; (F) particle size analysis; (G) ultraviolet-visible absorption spectrum; (H) FITR spectrum.

[0035] Figure 3Characterization diagrams of freeze-dried hydrogels (cross-sections); among them, (A, B) are SEM diagrams of Ag NPs / PAAM freeze-dried hydrogels (cross-sections); (C) is the SEM diagram of pectin / Ag NPs / CS / PAAM freeze-dried hydrogels (cross-sections); (D) is the low-magnification view corresponding to pectin / Ag NPs / CMC / CS / PAAM freeze-dried hydrogels (cross-sections); (E, F) are the high-magnification views corresponding to pectin / Ag NPs / CMC / CS / PAAM freeze-dried hydrogels (cross-sections); (G) is the elemental distribution diagram of pectin / CS / PAAM freeze-dried hydrogels (cross-sections); (H) is the elemental distribution diagram of pectin / Ag NPs / CS / PAAM freeze-dried hydrogels (cross-sections); (I) is the elemental distribution diagram of pectin / Ag NPs / CMC / CS / PAAM freeze-dried hydrogels (cross-sections).

[0036] Figure 4 Characterization diagrams of freeze-dried hydrogels (surfaces); among them, (A) is the SEM diagram of Ag NPs / PAAM hydrogels (surfaces); (B, C) are the SEM diagrams of pectin / Ag NPs / CS / PAAM freeze-dried hydrogels (surfaces); (D, E) are the low-magnification views corresponding to pectin / Ag NPs / CMC / CS / PAAM freeze-dried hydrogels (surfaces); (F) is the high-magnification view corresponding to pectin / Ag NPs / CMC / CS / PAAM freeze-dried hydrogels (surfaces); (G) is the elemental distribution diagram of Ag NPs / PAAM freeze-dried hydrogels (surfaces); (H) is the elemental distribution diagram of pectin / Ag NPs / CS / PAAM freeze-dried hydrogels (surfaces); (I) is the elemental distribution diagram of pectin / Ag NPs / CMC / CS / PAAM freeze-dried hydrogels (surfaces).

[0037] Figure 5 XPS diagrams of freeze-dried hydrogels; among them, (A) are the full XPS spectra of pectin / CS / PAAM freeze-dried hydrogels, pectin / Ag NPs / CS / PAAM freeze-dried hydrogels, and pectin / Ag NPs / CMC / CS / PAAM freeze-dried hydrogels; (B) is the partial XPS spectrum of Ag 3d; (C) is the partial XPS spectrum of C 1s; (D) is the partial XPS spectrum of O 1s.

[0038] Figure 6 SERS spectra of 8 hydrogels; (A) are the SERS spectra of Ag NPs / PAAM, pectin / Ag NPs / PAAM, Ag NPs / CS / PAAM, and pectin / Ag NPs / CS / PAAM under the same conditions (10 -4SERS intensity of (M); (B) is the error bar of the SERS intensity of Ag NPs / PAAM, pectin / Ag NPs / PAAM, Ag NPs / CS / PAAM, pectin / Ag NPs / CS / PAAM at 1510 cm -1 ; (C) is the SERS intensity of Ag NPs / CMC / PAAM, pectin / Ag NPs / CMC / PAAM, Ag NPs / CMC / CS / PAAM, pectin / Ag NPs / CMC / CS / PAAM under the same condition (10 -4 M); (D) is the error bar of the SERS intensity of Ag NPs / CMC / PAAM, pectin / Ag NPs / CMC / PAAM, Ag NPs / CMC / CS / PAAM, pectin / Ag NPs / CMC / CS / PAAM at 1510 cm -1 .

[0039] Figure 7 is the SERS characterization of the composite hydrogel using rhodamine 6G as the probe molecule; among them, (A) is the signal intensity of different concentrations of R6G (10 -4 M - 10 -8 M); (B) is the linear curve of the peak intensity of R6G at 1510 cm -1 corresponding to the concentration; (C, D) are the Raman spectra and intensity distributions at 1510 cm -1 at 50 randomly selected points on the hydrogel in a 10×10 μm area.

[0040] Figure 8 is the SERS characterization of 5-fluorouracil solutions with different pH values; among them, (A) is the SERS signal of 10 -1 M 5-fluorouracil prepared from an aqueous solution by the hydrogel SERS substrate adjusted with different pH values; (B) is the error bar of the peak corresponding to the SERS signal of 10 -1 M 5-fluorouracil prepared from an aqueous solution by the hydrogel SERS substrate adjusted with different pH values at 783 cm -1 ; (C) is the SERS signal of 10 -1 M 5-fluorouracil prepared from a phosphate buffer solution (pH = 7.4) by the hydrogel SERS substrate adjusted with different pH values; (D) is the error bar of the peak corresponding to the SERS signal of 10 -1 M 5-fluorouracil prepared from a phosphate buffer solution (pH 7.4) by the hydrogel SERS substrate adjusted with different pH values at 783 cm -1 .

[0041] Figure 9SERS characterization of 5-fluorouracil solutions with different concentrations; (A) SERS characterization of 5-fluorouracil solutions with different concentrations (10 -1 M-10 -6 (M) is the signal intensity of 5-fluorouracil; (B) is the signal intensity of 5-fluorouracil at 783 cm -1 The nonlinear fitting curve of the peak intensity and concentration at 783 cm -1 The peak intensity at is plotted against the error bars corresponding to the concentration.

[0042] Figure 10 This is the SERS characterization of the actual spiking experiment of the composite hydrogel material with anti-cancer drugs; where (A) is a normal human plasma sample; (B) is a plasma sample from a colon cancer patient. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0044] See attached Figure 1 As shown, the present invention provides a method for preparing a double-network silver nanoparticle carboxymethyl fiber hydrogel. First, a chemical covalent crosslinker (MBA), a natural anionic polysaccharide pectin molecule (pectin), and a photoinitiator (Irgacure2959) are added to a reaction monomer acrylamide (AM) solution, and after sufficient mixing, carboxymethyl cellulose silver nanoparticles (Ag NPs / CMC) and a quantitative chitosan (CS) solution are added, and after ultrasonic sufficient mixing, ultraviolet (UV) curing can form a single network structure between polyacrylamide polymer molecules and carboxyl groups in natural anionic polysaccharide pectin molecules, and carboxymethyl cellulose silver nanoparticles can continue to polymerize to synthesize a double-network composite hydrogel material, thereby forming a three-dimensional SERS substrate. This method has made innovations to the traditional hydrogel preparation method, and can modify uniform silver nanoparticles on the surface of the hydrogel, laying a foundation for subsequent detection.

[0045] Example 1: Preparation of pectin / Ag NPs / CMC / CS / PAAM hydrogel

[0046] The preparation method of pectin / Ag NPs / CMC / CS / PAAM hydrogel specifically comprises the following steps:

[0047] First step: First, mix 2 mL of 2 wt% pectin evenly with 2 mL of 8.4 M acrylamide. Subsequently, add 2 mL of 0.084 M Irgacure 2959 photoinitiator and 2 mL of 0.3 M N,N′-methylenebisacrylamide (MBA) crosslinker to it (volume ratio is 1:1:1:1, total volume is 8 mL); after ultrasonic stirring in a water bath at 70 °C for 20 min, a uniform hydrogel precursor solution is obtained.

[0048] Second step: Add 100 μL of 0.1 wt% anionic cellulose carboxymethyl cellulose to 200 μL of concentrated Ag NPs solution to obtain a concentrated Ag NPs / CMC solution.

[0049] Third step: After fully mixing the above 500 μL of hydrogel precursor solution with 300 μL of concentrated Ag NPs / CMC solution, add 200 μL of 0.0625 wt% chitosan solution; after mixing evenly, put it into a UV curing furnace (irradiate under a UV lamp with a wavelength of 365 nm for 500 s) for polymerization reaction to form a pectin / Ag NPs / CMC / CS / PAAM composite hydrogel.

[0050] Fourth step: Put the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel formed after the polymerization reaction in the UV curing furnace into a vacuum drying oven at 0.09 kPa and 50 °C for drying for 30 min.

[0051] Among them, the preparation method of the concentrated Ag NPs solution includes the following steps:

[0052] Dissolve AgNO3 (36 mg) in 200 mL of deionized water and boil. Add a solution of 1% sodium citrate (4 mL); then, boil the reaction solution at room temperature for 1 h and then cool it naturally. Centrifuge the obtained Ag NPs at a speed of 8000 rpm for 10 min, collect the precipitate, and then redisperse it with deionized water to remove the excessive sodium citrate in the solution. Concentrate the Ag NPs by 7 times (7 mL is concentrated to 1 mL) to obtain the concentrated Ag NPs for standby.

[0053] Example 2: Characterization of the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel material

[0054] Use TEM, ultraviolet-visible absorption spectroscopy, DLS, and Zeta potential analysis to analyze the microstructure and particle size of Ag NPs and Ag NPs / CMC;

[0055] The cross-sections and surfaces of Ag NPs / PAAM hydrogels, pectin / Ag NPs / CS / PAAM hydrogels, and pectin / Ag NPs / CMC / CS / PAAM hydrogel materials were characterized using a scanning electron microscope (SEM, Sigma 300, Oxford Xplore 30, Germany); to observe the microstructure of the composite hydrogels, the samples were freeze-dried (temperature: -80 °C); after freeze-drying, the hydrogel samples were observed using SEM at an acceleration voltage of 3.0 kV; the functional groups on the surface of the hydrogels were qualitatively analyzed using attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR, Nicolet 5700, Thermo, USA.); X-ray photoelectron spectroscopy (XPS, ESCALAB Xi+, Thermo Fisher Scientific, USA) was used for elemental analysis.

[0056] 1) The microstructure of silver nanoparticles was studied using TEM and particle size potential characterization, as shown in Figure 2 A, B, E, F, and G. It was found that the particle sizes of the silver colloidal particles prepared in this experiment mostly ranged from 40 to 80 nm; the ultraviolet-visible absorption spectra showed the absorption characteristics of silver nanoparticles (Ag NPs) and the complex of silver nanoparticles and carboxymethyl cellulose (CMC) (Ag NPs / CMC). Both samples showed obvious absorption peaks in the wavelength range of about 410 - 420 nm, which is related to the surface plasmon resonance (SPR) phenomenon of silver nanoparticles. The absorption peak of Ag NPs / CMC was slightly higher than that of pure Ag NPs, indicating that the presence of CMC may enhance the light absorption ability of silver nanoparticles, probably due to the modulation of the surface plasmon resonance of silver nanoparticles by CMC. This enhanced light absorption property is of great significance for the development of new optical materials and sensors, and thus increases the experimental value of the subsequent composite hydrogels.

[0057] 2) The chemical structures of the four hydrogels were characterized using FITR spectroscopy. As shown in Figure 2 H, there were obvious vibration peaks near 3195 cm-1, which were caused by the N-H stretching vibration in PAAM; there were two obvious vibration peaks near 1655 cm-1 and 1607 cm-1, which were caused by the C=O vibration and N-H bending vibration of the amide. These two types of peaks existed in all four hydrogel composites, which could prove the chemical structure in polyacrylamide; for the two hydrogel materials modified with silver nanoparticles later: the two obvious peaks at 838 cm-1 and 607 cm-1 were related to the stretching vibration of the metal-oxygen (Ag-O) metal-ligand bond, thus proving the intervention of silver nanomaterials.

[0058] 3) The Ag NPs / PAAM hydrogel, pectin / Ag NPs / CS / PAAM hydrogel, and pectin / Ag NPs / CMC / CS / PAAM hydrogel materials were characterized by SEM to observe and analyze the cross-sectional morphology and microstructure of the hydrogels, as Figure 3 shown. It can be obtained from the SEM image analysis that the distribution in the pectin / Ag NPs / CMC / CS / PAAM hydrogel is more uniform and ordered, while in the Ag NPs / PAAM hydrogel, it shows irregular shapes and random distribution; from the elemental distribution map of the freeze-dried hydrogel (cross-section), it can be concluded that the latter two hydrogels have stronger silver adsorption than the pure Ag NPs / PAAM hydrogel, thus proving the feasibility of the solution of the present invention. At the same time, it is not difficult to find that the solidified structure of the substrate material prepared by the present invention cannot penetrate into the deeper part of the hydrogel.

[0059] 4) The Ag NPs / PAAM hydrogel, pectin / Ag NPs / CS / PAAM hydrogel, and pectin / Ag NPs / CMC / CS / PAAM hydrogel materials were characterized by SEM to further observe and analyze the surface morphology and microstructure of the hydrogels, as Figure 4 shown. It can be known from the SEM image that the average pore size of the pectin / Ag NPs / CMC / CS / PAAM hydrogel is about 100 - 200 nm, which is smaller than that of the pure Ag NPs / PAAM hydrogel; the silver nanoparticles are more uniformly and orderly distributed in the pectin / Ag NPs / CMC / CS / PAAM hydrogel and only form a three-dimensional double network system, while in the Ag NPs / PAAM hydrogel, they show irregular shapes and random distribution; from the elemental distribution map of the freeze-dried hydrogel (cross-section), it can be concluded that the latter two hydrogels have stronger silver adsorption than the pure Ag NPs / PAAM hydrogel, and the O content with the addition of carboxymethyl cellulose is further increased, thus also proving the feasibility of the solution of the present invention, increasing the distribution density of Ag NPs, resulting in a relatively large increase in plasma, which is beneficial to improving the SERS activity.

[0060] 5) The pectin / CS / PAAM hydrogel, pectin / Ag NPs / CS / PAAM hydrogel, and pectin / Ag NPs / CMC / CS / PAAM hydrogel materials were characterized by XPS to further observe and analyze the composition of the surface elements of the hydrogels, as Figure 5As shown, by comparing the XPS spectra of pectin / CS / PAAM hydrogel, pectin / Ag NPs / CS / PAAM hydrogel, and pectin / Ag NPs / CMC / CS / PAAM hydrogel, two different peaks were detected in the Ag 3d and Ag 4p energy level orbits of pectin / Ag NPs / CS / PAAM and pectin / Ag NPs / CMC / CS / PAAM hydrogels, and these peaks are attributed to the loading of Ag NPs on the hydrogel. As Figure 5 shown in B, there are two prominent peaks in the Ag 3d orbit, which are identified as Ag3d3 / 2 and Ag3d5 / 2 respectively; there are 2 peaks in the C 1s spectrum, and the corresponding binding energies are 284.08 and 286.78 respectively, which are assigned to C-C and O-C=O of pectin / Ag NPs / CMC / CS / PAAM hydrogel ( Figure 5 C). The Ag atoms of pectin / Ag NPs / CMC / CS / PAAM hydrogel can coordinate with O-C=O, thereby stabilizing Ag NPs.

[0061] Example 3: Sensitivity and uniformity of the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel material substrate

[0062] To estimate the application of the constructed SERS platform, the sensitivity and uniformity of the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel material substrate were studied in this example. Rhodamine 6G was used as the probe molecule in the experiment to perform SERS characterization on the substrate.

[0063] 1) Drop 10 -4 M R6G dye solution on 8 kinds of composite hydrogels such as pectin / Ag NPs / CMC / CS / PAAM (0.5×0.5 cm 2 ), then dry the hydrogel in the oven for 3 hours, and then perform SERS detection to explore the SERS intensity of different hydrogels, as Figure 6 shown. It can be found that the SERS intensity of pectin / Ag NPs / CMC / CS / PAAM hydrogel is the strongest after modifying R6G.

[0064] 2) Prepare R6G standard solutions with different concentrations (10 -4 mol / L, 10 -5 mol / L, 10 -6 mol / L, 10 - 7 mol / L, 10 -8mol / L). The pectin / Ag NPs / CMC / CS / PAAM composite hydrogel substrate was cut into pieces with a size of 0.5×0.5 cm. 2 The fragments were then immersed in 500 μL of R6G with different concentrations for 60 min. After SERS detection, the working curve was established and the linear curve was fitted.

[0065] The SERS spectra were obtained by a portable Raman analyzer (RMS1000, Ruhai Optoelectronics, China) equipped with 785 nm. For each SERS measurement, the laser source power used was: 20 mW and the integration time was 5 s.

[0066] like Figure 7 A and B show that the SERS signal of R6G decreases gradually with the decrease of concentration, among which the 1510 cm -1 The peak correlation coefficient reached 0.98418. -8 M, 1510cm -1 The peak at the location can still be identified. In addition to high sensitivity, the hydrogel SERS platform also has excellent uniformity. In order to eliminate the randomness of the sample area, 50 locations were randomly selected from the hydrogel SERS platform to collect SERS signals. The experiment found that there was no obvious SERS signal fluctuation in any characteristic Raman peak of the 50 spectra collected ( Figure 7 C, D), RSD at 1510 cm -1 It is only 2.309% at 40°C, indicating that the hydrogel SERS platform has good sensitivity and substrate uniformity, and is worthy of detection and application in different fields.

[0067] Example 4: Reproducibility of pectin / Ag NPs / CMC / CS / PAAM composite hydrogel material substrate

[0068] For practical applications, the reproducibility of hydrogels is also very important. This example records the SERS spectra of pectin / Ag NPs / CMC / CS / PAAM hydrogels (25 sites on each substrate) from five different batches: the RSD value of the hydrogel SERS platform is only 0.66%, and the pectin / Ag NPs / CMC / CS / PAAM hydrogel matrix has excellent batch-to-batch reproducibility.

[0069] The discussion in Examples 3 and 4 demonstrates that the pectin / Ag NPs / CMC / CS / PAAM hydrogel SERS substrate has excellent performance, including high sensitivity, uniformity and excellent reproducibility.

[0070] Example 5: SRES detection of 5-fluorouracil standard solution with optimal pH

[0071] 5-Fluorouracil (5-FU) is a widely used antimetabolic drug, mainly used for the treatment of digestive tract cancers and other solid tumors. 5-FU exerts its antitumor effect by affecting the nucleic acid synthesis and metabolism of cells. Specifically, it can inhibit thymidylate synthase, thereby inhibiting DNA synthesis and, to a certain extent, RNA synthesis. However, it is not difficult to find that its detection is greatly affected by the pH acid-base environment, and its behavior at different pH values has a significant impact on its efficacy and bioavailability. Therefore, the Raman and surface-enhanced Raman scattering (SERS) spectra of 5-FU are recorded and discussed at different pH values to explore its chemical forms and behaviors in different environments (such as Figure 8 shown).

[0072] In the pH-exploratory Raman study, different pH values (10 -3 M NaOH (pH = 9.05), 5 × 10 -4 M NaOH (pH = 8.18), 10 -4 M NaOH (pH = 7.12) and 10 -3 M HCl (pH = 4.02)) were obtained by preparing aqueous solutions of different concentrations of HCl and NaOH. The pectin / Ag NPs / CMC / CS / PAAM composite hydrogel substrate was cut into fragments with a size of 0.5 × 0.5 cm 2 as the hydrogel SERS substrate, and then immersed in 500 μL of 5-fluorouracil (5-FU) with different concentrations for 60 minutes. The substrate was taken out and dried for SERS detection to explore the performance of the hydrogel SERS substrate at the optimal pH.

[0073] This experiment used the pectin / Ag NPs / CMC / CS / PAAM hydrogel as the SERS substrate to study the orientation and morphology of 5-FU during its adsorption; the experimental results showed that the SERS signal of the 5-FU solution prepared with phosphate buffer solution (pH = 7.4) was more excellent than that of the 5-FU solution prepared with aqueous solution; under alkaline pH conditions, the deprotonated form of 5-FU existed in the solution and adsorbed on the Ag surface perpendicular to the silver surface or with a direction not significantly inclined to the surface normal. The N3 deprotonated form seems to be the main isomer in the adsorbed state and is more likely to bind to the surface through the O7 atom; while at acidic pH values, the N3 deprotonated form was again confirmed to be the main chemisorbed species, adopting a similar orientation.

[0074] According to experimental findings, it can be inferred that the optimal pH value of 5-FU may be in the neutral to alkaline range because in this pH range, the deprotonated form of 5-FU exists stably in solution and can adsorb on the silver surface in a manner beneficial to its pharmacodynamic effect. All subsequent experiments used a 5×10 -4 M NaOH solution (pH = 8.18) as the environmental solution.

[0075] Example 6: SRES Detection of 5-Fluorouracil Standard Solution

[0076] Prepare 5-FU standard solutions with different concentrations (10 -1 mol / L, 10 - 2 mol / L, 10 -3 mol / L, 10 -4 mol / L, 10 -5 mol / L, 10 -6 mol / L) using a phosphate buffer solution (pH = 7.4). Cut the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel substrate into fragments with a size of 0.5×0.5 cm 2 . Immerse the hydrogel SERS substrate in the environmental solution with the optimal pH (pH = 8.18), take it out and dry it, then soak it in 500 μL of 5-fluorouracil (5-FU) with different concentrations for 60 minutes. Take out the substrate and dry it for SERS detection. After SERS detection, establish a working curve.

[0077] Raman detection was performed on 5-fluorouracil (5-FU) buffer solutions with different concentrations (10 -1 M - 10 -6 M). In the SERS detection, it was found that the Raman spectrum showed that the main peaks were located at 783 cm -1 (pyrimidine ring breathing mode), 1226 cm -1 (coupled ring mode), 1349 cm -1 (ring stretching mode), 1426 cm -1 (N-H in-plane deformation), and the peaks were located at 1673 cm -1 , 1650 cm -1 (both C=O stretching modes). The lowest detection limit concentration of 5-fluorouracil (5-FU) was as low as 10 -6 M, and the SERS signal intensity at 783 cm -1 had a non-linear relationship with the logarithmic concentration of 5-fluorouracil (5-FU) ( Figure 9 B).

[0078] Example 7: Detection of 5-Fluorouracil in the Blood of Cancer Patients

[0079] To evaluate the interference of real samples on the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel SERS chip, 5-fluorouracil (5-FU) in the blood of cancer patients was detected. In the actual detection, a recovery experiment was adopted. Using the blood samples provided by Fujian Provincial Hospital, a spiking experiment was carried out. The blood samples used in this experiment need to be centrifuged at a speed of 3000 rpm for 10 min, and the supernatant, that is, plasma, was extracted. Specifically, the blood samples were centrifuged at 3000 rpm for 10 minutes to obtain the supernatant, and the supernatant was stored in a refrigerator at 4 °C for further detection. Finally, the hydrogel SERS chip soaked in the solution with the optimal pH environment (pH = 8.18) was dried and then immersed in 500 μL of the supernatant for 60 min for subsequent SERS detection.

[0080] In this experiment, non-spiked experiments were carried out on the plasma samples of two normal people as the background Raman peaks for Raman detection. Subsequently, an equal volume of 10 -2 M and 10 -3 M 5-fluorouracil (5-FU) buffer solutions were added, and detection was carried out again according to the same method as above; subsequently, non-spiked experiments were carried out on the plasma samples of colon cancer patients as the background Raman peaks, and then the spiking experiment was carried out according to the same protocol. These two concentrations of 5-fluorouracil (5-FU) buffer solutions were added to the actual samples. After the measurement was completed, it was then used for the recovery rate test. Due to the error in the plasma environment, in the spiking experiment, the peak at 803 cm -1 was used for calibration. As Figure 10 shown, that is, the SERS characterization spectra of 6 types of plasma samples.

[0081] The recovery rates of 5-fluorouracil at different concentrations in the plasma samples of normal people were 99.97% - 124.28%, and the recovery rates of 5-fluorouracil (5-Fu) at different concentrations in the plasma samples of colon cancer patients were 90.83% - 104.68%, as shown in Table 1 below.

[0082] Table 1 Recovery experiment for detecting 5-fluorouracil in plasma samples using the hydrogel SERS platform

[0083]

[0084] In the above Examples 5 - 7, the SERS spectra were obtained by a portable Raman analyzer (RMS1000, China - Ruhai Optoelectronics) equipped with 785 nm; for each SERS measurement, the power of the laser source used was: 40 milliwatts, and the integration time was 5 seconds.

[0085] The present invention uses a novel UV photopolymerization method to prepare a double-network pectin / Ag NPs / CMC / CS / PAAM hydrogel as a SERS substrate material for the rapid and efficient detection of the anticancer drug 5-fluorouracil (5-FU). The double-network pectin / Ag NPs / CMC / CS / PAAM composite hydrogel prepared in the present invention has carboxyl groups on the molecular chains of natural anionic polysaccharide pectin and a covalently crosslinked polyacrylamide network, which can efficiently modify silver nanoparticles, making the silver nanoparticles more uniform and dense. At the tips, edges of metal nanostructures or the gaps between nanoparticles, the electromagnetic field intensity will be significantly enhanced, thereby improving the uniformity and reproducibility of the substrate. At the same time, the hydrogel prepared by the solution of the present invention exhibits excellent performance in the detection of anticancer drugs, and the LOD can be controlled at 10 -6 M, showing great application prospects in the field of cancer drug detection.

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of a double-network silver nanoparticle carboxymethyl cellulose hydrogel, characterized in that, It includes the following steps: 1): Ultrasonically mix the pectin aqueous solution, acrylamide aqueous solution, photoinitiator, and crosslinker evenly under water bath heating to obtain a hydrogel precursor solution; 2): Add anionic cellulose carboxymethyl cellulose to the Ag NPs solution to obtain an Ag NPs / CMC solution; 3): After fully mixing the Ag NPs / CMC solution and the hydrogel precursor solution, add the chitosan solution and mix evenly, then put it into a UV curing furnace for polymerization reaction to form a pectin / Ag NPs / CMC / CS / PAAM composite hydrogel; 4): Dry the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel in a vacuum drying oven.

2. The preparation method of the double-network silver nanoparticle carboxymethyl cellulose hydrogel according to claim 1, wherein In step 1), 2 wt% aqueous pectin solution, 8.4 M acrylamide aqueous solution 、 0.084 M photoinitiator and 0.3 M crosslinking agent are ultrasonically mixed evenly under water bath heating; The volume ratio of the pectin, acrylamide, photoinitiator, and crosslinker is 1:1:1:1; The crosslinker is selected as N,N′-methylenebisacrylamide, and the photoinitiator is selected as Irgacure2959.

3. The preparation method of the double-network silver nanoparticle carboxymethyl cellulose hydrogel according to claim 1, characterized in that, In step 2), 100 μL of 0.1 wt% anionic cellulose carboxymethyl cellulose is added to 200 μL of the concentrated Ag NPs solution; The preparation method of the concentrated Ag NPs solution includes the following steps: Dissolve 36 mg of AgNO3 in 200 mL of deionized water and boil it, then add 4 mL of a 1% sodium citrate solution; Then, boil the reaction solution at room temperature for 1 h and then let it cool naturally. Centrifuge the obtained Ag NPs and collect the precipitate, then redisperse it with deionized water and remove the excessive sodium citrate in the solution; Next, concentrate the obtained Ag NPs by 7 times to obtain a concentrated Ag NPs solution.

4. The preparation method of the double-network silver nanoparticle carboxymethyl cellulose hydrogel according to claim 1, characterized in that, In step 3), after fully mixing 300 μL of the concentrated Ag NPs / CMC solution and 500 μL of the hydrogel precursor solution, add 200 μL of a 0.0625 wt% chitosan solution and mix evenly.

5. The preparation method of the double-network silver nanoparticle carboxymethyl cellulose hydrogel according to claim 1, wherein In step 1), the water bath heating temperature is 70 °C, and the ultrasonic mixing time is 20 min; In step 3), during the UV curing process, irradiate with a UV lamp with a wavelength of 365 nm for 500 s.

6. The preparation method of the double-network silver nanoparticle carboxymethyl cellulose hydrogel according to claim 1, wherein, In step 4), the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel is placed in a vacuum drying oven at 0.9 kPa and 50 °C for drying for 30 min.

7. A double-network silver nanoparticle carboxymethyl cellulose hydrogel, characterized in that It is prepared by the preparation method according to any one of claims 1 to 8.

8. Use of the double-network silver nanoparticle carboxymethyl cellulose hydrogel as described in claim 7 in the detection of anti-cancer drugs, characterized in that, The double-network silver nanoparticle carboxymethyl cellulose hydrogel is used as a substrate material in the SERS detection process, and the anticancer drug is 5-FU.

9. The application according to claim 8, wherein The 5-FU detection steps include: A): Cut the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel substrate into pieces with dimensions of 0.5×0.5 cm 2 fragments; B): Immerse the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel fragments in a neutral to alkaline solution to adjust the pH of the hydrogel SERS substrate, then take it out and dry it, and then immerse the hydrogel SERS substrate in the test solution containing 5-FU; C): Take out the pectin / Ag NPs / CMC / CS / PAAM composite hydrogel fragments, dry them, and perform SERS detection.

10. The application according to claim 9, characterized in that, In step C), the immersion time is 60 min.

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