Asymmetric AgNPs / ANFs-PAM hydrogel film as well as preparation method and application thereof

Abstract

According to the asymmetric AgNPs / ANFs-PAM hydrogel film, the side, close to a light source, of the upper layer of the hydrogel film is a smooth and compact skin layer, and the side, far away from the light source, of the bottom of the hydrogel film is of a folded structure; agNPs are embedded in the hydrogel film. The invention further discloses a preparation method and application of the hydrogel film. The hydrogel film is a silver nanoparticle / aramid nanofiber-polyacrylamide (AgNPs / ANFs-PAM) hydrogel film with an asymmetric structure, the side, close to a light source, of the upper layer of the hydrogel film is a smooth and compact skin layer, the side, far away from the light source, of the bottom of the hydrogel film is of a hill-shaped wrinkle structure, and the compact network structure and the porous structure form obvious gradient screening. A small-molecule target object can smoothly enter the hydrogel film to be combined with a hydrogen bond generated by an amido bond of ANFs in the SERS substrate, and a large-molecule target object is intercepted outside the SERS substrate.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of hydrogels, in particular to an asymmetric AgNPs / ANFs-PAM hydrogel film, a preparation method and application. BACKGROUND

[0002] As a high-sensitivity molecular spectroscopy detection technology, surface-enhanced Raman scattering (SERS) can significantly amplify the Raman signal emitted by the target molecules adsorbed on the metal surface through the localized surface plasmon resonance effect of noble metal nanostructures (such as gold, silver, copper nanoparticles), thereby realizing the detection of trace substances or even single molecules. This technology has unique advantages such as "fingerprint identification", high sensitivity, non-destructive, simple operation, and is combined with various functional materials (metal-organic frameworks, aerogels, hydrogels, etc.) to construct flexible or rigid substrates, and is applied to the fields of food safety, chemical analysis, material science, biomedical engineering, etc. Although great progress has been made in SERS technology, in the detection of complex environments such as biological and food samples, there are a large number of biological molecules and matrix background interference, making it difficult to detect SERS signals; traditional SERS detection mainly relies on bare gold nanoparticles (AuNPs) and bare silver nanoparticles (AgNPs) substrates, which have poor reproducibility and insufficient stability, seriously limiting their application in actual samples. Therefore, constructing a new type of SERS substrate with high sensitivity, excellent mechanical properties, good chemical stability, and efficient separation and concentration capacity has become a key research direction for promoting SERS from the laboratory to practical application.

[0003] Hydrogels, as a kind of polymer material with three-dimensional cross-linked network structure, have unique advantages in constructing SERS active substrate due to its high porosity, excellent swelling performance and rich surface functional groups (such as hydroxyl, carboxyl, amino, etc.). Its network structure not only provides a large number of "hot spot" domains, but also can efficiently capture target molecules through hydrophilic interaction, and exclude large molecular interference in complex matrix by molecular sieving effect, significantly simplifying the sample pretreatment process. In addition, the good biocompatibility and easy modification of hydrogels enable them to further couple aptamers or recognition groups, improving detection specificity. For example, Zhang et al. used immersion reduction to load AgNPs to prepare β-lactoglobulin amyloid fibril-polyvinyl alcohol-silver nanoparticle adsorptive hydrogel. By adjusting the content of polyvinyl alcohol, the pore size of the hydrogel was adjusted to allow small molecule targets to enter the hydrogel and specifically bind to the β-lactoglobulin amyloid fibril. By drying the conditions, the hydrogel shrinks and then aggregates AgNPs and target molecules, improving the SERS signal. This hydrogel has been used for specific adsorption and detection of metamitron and ibuprofen. In terms of SERS enhancement mechanism, the dynamic structure regulation ability of hydrogel substrate is particularly prominent. Wu et al. prepared poly(N-isopropylacrylamide) hydrogel and successfully loaded AuNPs. By using the temperature-sensitive properties of the hydrogel itself to stimulate the shrinkage of the hydrogel, the distance between AuNPs is shortened, and the SERS signal is enhanced. Traditional SERS hot spots are usually limited to zero-dimensional, one-dimensional or two-dimensional structures, and the SERS electromagnetic field enhancement encounters a bottleneck. Yang et al. reported a three-dimensional hot spot matrix constructed by simply evaporating a drop of silver citrate sol. The AgNPs dispersed in the 3D space of this droplet shrink the gap between the particles as the water evaporates, and are uniformly deposited on the surface of the silicon wafer. In this process, AgNPs interact through van der Waals forces and electrostatic repulsion, and are fixed in three-dimensional space to form hot spots with three-dimensional geometry.

[0004] As can be seen from the above, dispersing AgNPs in hydrogel to form a three-dimensional hot spot for SERS substrate, using hydrogel to realize the integration of separation, enrichment and detection of target analyte can effectively improve the detection sensitivity and SERS signal reproducibility. SUMMARY

[0005] To achieve the above-mentioned effect, the application aims to provide an asymmetric AgNPs / ANFs-PAM hydrogel film, a preparation method and application thereof, the hydrogel film is a silver nanoparticle / aramid nanofiber-polyacrylamide (AgNPs / ANFs-PAM) hydrogel film with an asymmetric structure, the upper layer of the hydrogel film is a smooth and dense skin layer on the near light source side, and the bottom far light source side is a "hill-shaped" wrinkle structure, the dense network structure and the porous structure form an obvious "gradient screening", so that small molecule target objects can smoothly enter the hydrogel film and be combined with the amide bond of the ANFs in the SERS substrate through hydrogen bonding, and the macromolecular target objects are intercepted outside the SERS substrate.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0007] The application discloses an asymmetric AgNPs / ANFs-PAM hydrogel film, wherein the upper layer of the hydrogel film is a smooth and dense skin layer on the near light source side, and the bottom far light source side is a wrinkle structure.

[0008] The hydrogel film embeds AgNPs.

[0009] Preferably, the wrinkle structure is hill-shaped.

[0010] Preferably, the AgNPs are embedded in the ANFs modified three-dimensional network porous hydrogel film.

[0011] The application further discloses a preparation method of the asymmetric AgNPs / ANFs-PAM hydrogel film, comprising the following steps:

[0012] a. uniformly dispersing ANFs in an AgNPs solution with an average diameter of 55 nm in a container;

[0013] b. adding acrylamide and polyethylene glycol diacrylate into the container in sequence and stirring;

[0014] c. stopping stirring, vacuumizing the container, and then adding phenyl bisoxidized phosphine and uniformly mixing to obtain a prepolymer solution;

[0015] d. injecting the prepolymer solution into a silicone rubber mold, covering a quartz glass plate on the opening of the silicone rubber mold, vertically irradiating the prepolymer solution with 365 nm ultraviolet light through the quartz glass plate to polymerize the prepolymer solution and form a hydrogel film with a smooth top layer and a hill-shaped wrinkle structure at the bottom;

[0016] e. after demolding, soaking the hydrogel film in secondary water to wash away unreacted substances.

[0017] Preferably, in steps a-c, the ratio of AgNPs solution, acrylamide, polyethylene glycol diacrylate, and phenyl bisoxidophosphine is 5.00 mL, 2.50 g, 75.0 μL, and 200 μL, and the concentration of phenyl bisoxidophosphine is 16.6 mg / mL.

[0018] Preferably, in step b, the stirring time is 2 h, and in step d, the ultraviolet irradiation time is 8 min.

[0019] Preferably, the preparation method of the AgNPs solution is as follows:

[0020] In the secondary water, 36.0 g / L of silver nitrate is added, a condenser tube is placed in an oil bath, and after magnetic stirring for 20 min, 1.00% of sodium citrate is added, and the reaction is stopped after the solution changes from yellow to silver gray after continuous heating and stirring.

[0021] Preferably, in the preparation of the AgNPs solution, the ratio of secondary water, silver nitrate, and sodium citrate is 200 mL, 1.00 mL, and 4.00 mL.

[0022] The application further discloses an application of the asymmetric AgNPs / ANFs-PAM hydrogel film, and the hydrogel film is used as a substrate of a SERS sensor, and the thickness of the substrate is 2 mm and the diameter is 5.0 mm.

[0023] Further, the SERS sensor is applied to detection and analysis of thiabendazole residues in vegetables and fruits.

[0024] The asymmetric AgNPs / ANFs-PAM hydrogel film has the following characteristics:

[0025] 1. The introduction of ANFs induces the hydrogel film to spontaneously form an asymmetric structure with a smooth upper layer and a "hill-shaped" bottom, and the ANFs have a large number of amide bonds, which provide abundant adsorption sites for target molecules;

[0026] 2. The hydrogel film is semi-shrunk by drying treatment, and when adsorption occurs, the hydrogel film swells rapidly, generating a negative pressure to quickly make the target molecules enter the hydrogel film, and form stable hydrogen bonds with the amide bonds in the ANFs, thereby significantly improving the enrichment performance on the target molecules;

[0027] 3. The dense network in the upper layer and the porous network structure of the hydrogel film together construct a gradient screening structure, which enriches small molecule target objects while reducing the interference of large molecule target objects, thereby significantly improving the selectivity of the detection system;

[0028] 4、Hydrogel films shrink in drying, the "hilly" structure can effectively squeeze AgNPs to the area with higher curvature to form stable 3D SERS "hot spots" with high density and strong enhancement ability, and in the shrinking process, the volume of hydrogel is reduced, the concentration of target molecules and AgNPs nanoparticles in the same area is increased, and a double enhancement signal effect is produced. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The figure is a schematic diagram of the preparation process of the asymmetric AgNPs / ANFs-PAM hydrogel film disclosed in the application.

[0030] Figure 2 The figure is the UV-Vis absorption spectrum of AgNPs (a), the XRD (b), the XPS full spectrum (c) and the fine spectrum (d) of the AgNPs / ANFs-PAM hydrogel film.

[0031] Figure 3 The figure is the TEM (a) and the particle size distribution diagram (b) of AgNPs, the SEM of the AgNPs / ANFs-PAM hydrogel film before shrinking (c) and after shrinking (d).

[0032] Figure 4 The figure is the pore volume-pore size distribution curve (a) and the N2 adsorption-desorption isotherm (b) of the AgNPs / ANFs-PAM hydrogel film, the swelling rate (c) of the hydrogel film and the images before shrinking and after shrinking (d).

[0033] Figure 5 The figure is the SERS spectrum (a) and the SERS intensity diagram (b) of thiuram in the hydrogel film with different AgNPs concentrations, the SERS spectrum (c) and the SERS intensity diagram (d) of thiuram in the hydrogel film with different PEGDA contents, the tensile stress-strain curve (e), the toughness (f) and the Young's modulus (g) and the swelling rate (f) of the hydrogel film with different PEGDA contents.

[0034] Figure 6 The figure is the confocal microscope image of the bottom of the AgNPs-PAM hydrogel film (a) and the bottom of the AgNPs / ANFs-PAM hydrogel film (b), the SEM of the section of the AgNPs / ANFs-PAM hydrogel film after freeze-drying (c), the static contact angle of the bottom of the AgNPs-PAM hydrogel film (d) and the bottom of the AgNPs / ANFs-PAM hydrogel film (e), and the linear fitting diagram of the methylene blue concentration and the absorbance (f).

[0035] Figure 7SERS spectra (a) and SERS intensity (b) of thiram in different ANFs content of hydrogel films; SEM of the inner (c) and lower (d) layer of AgNPs / ANFs-PAM hydrogel films with 0.300 wt% ANFs; SERS spectra (e) of thiram in the upper and lower layer of AgNPs / ANFs-PAM hydrogel films; backscattered SEM of the upper (f) and lower (g) layer of naturally dried AgNPs / ANFs-PAM hydrogel films; SERS intensity (h) of thiram in AgNPs / ANFs-PAM hydrogel films with different thickness.

[0036] Figure 8 Effects of drying temperature (a), adsorption time (b), pre-drying time (c) and drying time (d) on the SERS performance of AgNPs / ANFs-PAM hydrogel films.

[0037] Figure 9 SERS spectra (a) of tetramethylthiuram disulfide (thiram), tetrabenzylthiuram disulfide and tetraethylthiuram disulfide; SERS spectra (b) of thiram at different positions on the same piece of hydrogel film; SERS intensity of thiram in different batches (c) and the same batch (d) of hydrogel films; SERS intensity of thiram at different times in the same batch (e); effects of different interferents on the detection of thiram by AgNPs / ANFs-PAM hydrogel films (f).

[0038] Figure 10 HPLC chart (a) and linear fitting chart (b) of thiram standard solution; HPLC chart of celery sample (c) and pear sample (d). DETAILED DESCRIPTION

[0039] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings.

[0040] Example 1

[0041] This example discloses an asymmetric AgNPs / ANFs-PAM hydrogel film, the upper layer of which is a smooth and dense skin layer near the light source side, and the bottom layer is a wrinkled structure far from the light source side; wherein:

[0042] The hydrogel film embeds AgNPs; the wrinkled structure is a hill-like structure.

[0043] This kind of asymmetric AgNPs / ANFs-PAM hydrogel film embeds AgNPs in the ANFs modified three-dimensional network porous hydrogel film, which significantly improves the SERS detection performance of the hydrogel film. It has the following innovative features:

[0044] (1) The introduction of ANFs induces the hydrogel film to spontaneously form an asymmetric structure with a smooth upper layer and a "hilly" bottom. The presence of a large number of amide bonds in ANFs provides abundant adsorption sites for target molecules.

[0045] (2) The hydrogel film is semi-shrunk by drying treatment. During adsorption, the hydrogel film swells rapidly, generating a negative pressure that allows target molecules to quickly enter the hydrogel film and form stable hydrogen bonds with the amide bonds in ANFs, thereby significantly improving the enrichment performance for target molecules.

[0046] (3) The dense network in the upper layer and the porous network structure of the hydrogel film together construct a gradient screening structure that enriches small molecule target substances while reducing the interference of large molecule target substances, thereby significantly improving the selectivity of the detection system.

[0047] (4) During the drying and shrinking of the hydrogel film, the "hilly" structure effectively squeezes AgNPs to gather in areas with higher curvature, forming stable 3D SERS "hot spots" with high density and strong enhancement capability. Moreover, during the shrinking process, the volume of the hydrogel decreases, and the concentration of target molecules and AgNPs nanoparticles in the same area increases simultaneously, resulting in a double enhancement signal effect. Experimental results show that the unique asymmetric structure of the hydrogel film exhibits excellent sensing performance in the field of food safety detection, with high sensitivity, high selectivity, and good reproducibility for detecting pesticide residues of thiram in vegetables and fruits. It provides a new technical solution for on-site rapid detection.

[0048] Example 2

[0049] Based on Example 1, this embodiment discloses a preparation method of the asymmetric AgNPs / ANFs-PAM hydrogel film, as shown in Figure 1 , specifically comprising:

[0050] 1. Synthesis of AgNPs

[0051] Silver nanoparticles (AgNPs) were synthesized based on the previously reported Rong method. A three-necked flask was charged with 200 mL of double-distilled water, 1.00 mL of 36.0 g / L silver nitrate, and placed in a 135°C oil bath. A condenser tube was placed, and magnetic stirring was performed for 20 min. Then, 4.00 mL of freshly prepared 1.00% sodium citrate was added to reduce the silver nitrate. The solution turned from yellow to silver gray after 1 h of continuous heating and stirring, and the reaction was stopped. After the solution cooled and aged, AgNPs with an average diameter of 55 nm were prepared for use.

[0052] 2. Preparation of asymmetric AgNPs / ANFs-PAM hydrogel film

[0053] In a 50 mL beaker, 5.00 mL of prepared AgNPs solution was added, first, ANFs were uniformly dispersed in the AgNPs solution, then 2.50 g of acrylamide (AM) and 75.0 μL of polyethylene glycol diacrylate (PEGDA) were added in turn, stirred for 2 h, vacuumized to remove oxygen, and finally 200 μL of 16.6 mg / mL phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO) was added and mixed. The prepared prepolymer solution was quickly filled into a silicone rubber circular hole mold with a thickness of 2.0 mm and a diameter of 4.0 cm, covered with a quartz glass plate, and a 365 nm ultraviolet light was vertically irradiated from above the mold for 8 min to polymerize and form a hydrogel film with a smooth surface and a "hill-shaped" wrinkle at the bottom. After demolding, it was soaked in secondary water to wash away the unreacted substances. A custom-made mold was used to cut the hydrogel film into uniform circular hydrogel film pieces with a thickness of 2.0 mm and a diameter of 5.0 mm for use. Unless otherwise specified, the following experiments used the bottom "hill-shaped" wrinkle surface as the SERS detection substrate.

[0054] Example 3

[0055] On the basis of Examples 1 and 2, this example discloses the characterization of the asymmetric AgNPs / ANFs-PAM hydrogel film, as follows:

[0056] To verify that the AgNPs were successfully loaded on the hydrogel film and their state of existence in the hydrogel film, the AgNPs / ANFs-PAM hydrogel film was subjected to freeze-drying dehydration treatment at -50 °C for 48-72 h, and then the hydrogel film was characterized in various ways. The results of the polycrystalline X-ray diffraction (XRD) analysis are shown in FIG. 8B, where four obvious diffraction peaks appear at 2θ of 38.2°, 44.4°, 64.7°, and 77.7°, corresponding to the (111), (200), (220), and (311) crystal planes of face-centered cubic (fcc) metal silver nanoparticles, respectively, consistent with the standard card (PDF #87-0719). The (111) crystal plane is a strong diffraction peak, indicating that the prepared silver nanoparticles have good crystallinity and have been successfully loaded in the hydrogel film network. Figure 2 b, where the characteristic peaks of C 1s, N 1s, O 1s, and Ag 3d are visible in the full spectrum Figure 2 c, and the fine spectrum Figure 2 d shows the binding energies of Ag 3d 5 / 2 ) and 372.94 eV (Ag 3d3 / 2 A pair of spin-orbit double peaks can be clearly observed at this location, with a binding energy difference of approximately 6.0 eV between the two peaks. This is similar to that of metallic silver (Ag). The results perfectly match the standard values, indicating that silver exists in the hydrogel membrane as zero-valent nanoparticles. These systematic characterization results fully demonstrate that silver nanoparticles have been successfully and stably embedded in the AgNPs / ANFs-PAM hydrogel membrane network structure.

[0057] To investigate the morphology and size distribution of AgNPs in AgNPs / ANFs-PAM hydrogel membranes, the swollen hydrogel membranes were quenched with liquid nitrogen and then rapidly ground into powder. The powder was dispersed in secondary water, sonicated for 20 min, and then 10 μL of the suspension was dropped onto a copper grid. After drying, transmission electron microscopy (TEM) analysis was performed. Figure 3 a) It can be clearly seen that AgNPs have a clear outline, appearing as slightly irregular, approximately spherical polygons, with particle sizes mainly concentrated in the 50-60 nm range. Figure 3 b). To further observe the microstructure of the hydrogel membrane, the hydrogel membrane was freeze-dried, quenched with liquid nitrogen, and sputter-coated with gold before scanning electron microscopy (SEM) analysis. Before shrinkage ( Figure 3 c) It exhibits a continuous porous structure with interconnected pores of a wide size distribution and smooth pore walls. This structure facilitates the containment of large amounts of water and solution in a swollen state, providing ample storage space and diffusion channels for the target material. After natural drying and water loss, the original pores of the hydrogel membrane close, resulting in significant shrinkage. Figure 3 d). Furthermore, to investigate the pore structure of the hydrogel membrane, the membrane was freeze-dried after swelling equilibrium, and N2 adsorption-desorption tests were performed. The hydrogel membrane was analyzed using a fully automated specific surface area and microporous / mesoporous physical adsorption analyzer. The degassing procedure involved raising the sample temperature to 120 °C at room temperature, holding it for 12 h, and then cooling it to room temperature before measuring again. Figure 4 As shown in Figure a, the pore size distribution curve exhibits a distinct peak in the 0-20 nm range, and a relatively flat, broad curve in the 20-160 nm range, indicating that the material is predominantly mesoporous, with a small number of widely distributed macropores. N2 adsorption-desorption isotherms ( Figure 4 (b) The material exhibits a typical Type IV curve and an H1 hysteresis loop, further confirming the formation of highly uniform and well-structured mesopores within the material. BET calculations show an average pore size of 25.7 nm and a specific surface area of ​​0.669 m². 2 / g. This hydrogel membrane possesses an abundant mesoporous structure and a small number of macropores. The macroporous network endows the hydrogel membrane with excellent swelling properties and serves as the main transport channel, ensuring rapid diffusion and efficient enrichment of target molecules within the hydrogel membrane. The abundant mesoporous structure provides the hydrogel membrane with active surfaces and molecular sieving functions, facilitating the selective enrichment and uniform distribution of target analytes. This rational configuration of pore structure lays a solid structural foundation for achieving highly selective, reproducible, and sensitive SERS detection.

[0058] To quantify the mass and volume changes of hydrogel membranes during swelling and shrinkage, the swelling properties of the hydrogel membranes were tested. Uniform AgNPs / ANFs-PAM hydrogel membrane samples were prepared using a 5.0 mm diameter circular mold and dried at 50°C. The samples were weighed every 5 minutes. ), until completely dried to obtain dry weight ( ), calculate the swelling ratio of the hydrogel membrane using Formula 1: (1)

[0059] The results are as follows Figure 4 As shown in cd, with the increase of drying time, the mass of the hydrogel membrane gradually decreases due to water loss, and the swelling rate decreases accordingly. Until the hydrogel membrane is completely dry, the volume shrinks significantly by about 5 times. This process not only achieves efficient enrichment of trace concentration target substances through the concentration effect, but also reduces the spacing of the embedded AgNPs, forming a dense local enhanced electric field, thereby significantly improving the SERS detection sensitivity.

[0060] Example 4

[0061] Based on Examples 1 and 2, this example discloses that the hydrogel membrane is used as a substrate for a SERS sensor. The substrate has a thickness of 2 mm and a diameter of 5.0 mm.

[0062] SERS performance optimization of this asymmetric AgNPs / ANFs-PAM hydrogel membrane

[0063] Using 100 μg / L thiram as the probe molecule, the effects of AgNPs concentration, PEGDA dosage, ANF dosage and hydrogel membrane thickness on the SERS performance of the hydrogel membrane were investigated.

[0064] The effect of AgNP concentration (0.880-3.52 g / L) on SERS signal was investigated while keeping the PEGDA content at 1.00%, the ANF content at 0.0900 wt%, and the film thickness at 2.0 mm. Figure 5As shown in ab, the SERS signal gradually strengthens with increasing AgNP concentration. When the AgNP concentration is 3.52 g / L, the double characteristic peak of thiram (1380 cm⁻¹) is observed. -1 When the concentration of AgNPs exceeds 3.52 g / L, the high number of "hot spots" leads to nanoparticle aggregation, resulting in a decrease in the SERS enhancement effect. With other conditions unchanged, the AgNPs concentration was kept at 3.52 g / L, and the PEGDA content was adjusted (0.120%-2.50%). Experiments showed that the SERS signal of the hydrogel membrane was optimal when the PEGDA content was 1.50%. Figure 5 To visually evaluate the effect of PEGDA content on the hydrogel membrane network structure, this study conducted uniaxial tensile tests on dumbbell-shaped samples with a width of 2.00 mm using an electronic universal testing machine (Sansi, UTM5504, China) at room temperature and a tensile speed of 50.0 mm / min. Tensile strain... Calculate according to Formula 2:

[0065] (2)

[0066] In the formula, For the displacement of the crossbeam, This represents the initial length of the hydrogel film. Stress. Calculate according to formula 3:

[0067] = (1+ε)(3)

[0068] in, It is the load divided by the initial cross-sectional area. The toughness of the hydrogel membrane, which measures its ability to absorb energy during fracture, is calculated using the integral stress-strain curve and Equation 4: (4) This represents the fracture strain of the hydrogel. Elastic modulus. Also known as Young's modulus, it reflects the hardness of a material during the elastic deformation stage. It is determined by the slope of the initial linear portion of the stress-strain curve and is calculated using formula 5:

[0069] (5)

[0070] The results are as follows Figure 5 As shown in the figure, with the increase of PEGDA content, the slope of the stress-strain curve gradually increases, the toughness decreases, and the Young's modulus increases, indicating that the stiffness and resistance to elastic deformation of the hydrogel film are continuously enhanced. Simultaneously, swelling performance tests were performed on it. Figure 5(h) The swelling ratio decreases with increasing PEGDA content. Combined with mechanical test results, the influence of PEGDA on the cross-linking degree of the hydrogel membrane is further verified. When the PEGDA content is low, the hydrogel network has a low degree of cross-linking, a loose structure, and more space to accommodate water, resulting in a high swelling ratio. Although it can adsorb more target analytes, its mechanical properties are poor and it cannot effectively support the formation of 3D hot spots by AgNPs. As the PEGDA content increases, the degree of cross-linking increases. Excessive PEGDA leads to a denser hydrogel membrane network structure, reducing the space that can accommodate water and decreasing the swelling ratio. In summary, the hydrogel membrane with 1.50% PEGDA has both excellent mechanical properties and swelling-shrinkage ability, effectively adsorbing target analytes while effectively controlling the spacing of AgNPs.

[0071] Subsequently, after optimizing the above parameters, the effect of ANFs composites on the SERS performance of the hydrogel membrane was investigated. The AgNPs-PAM hydrogel has a uniform structure and smooth surfaces on both sides. Figure 6 a) The AgNPs / ANFs-PAM hydrogel membrane formed a "hill-like" wrinkled structure. Figure 6 b). The introduction of ANFs alters the hydrogel's curing process. When ultraviolet light shines from above, a light intensity gradient effect occurs as the light penetrates the medium, resulting in stronger light on the upper layer and weaker light on the lower layer, triggering top-down layered gelation. Therefore, the AgNPs / ANFs-PAM hydrogel membrane exhibits a unique asymmetric structure: the upper layer near the light source is a smooth and dense skin, while the lower layer far from the light source forms a porous structure of varying sizes. Figure 6 c). The contact angle of the sample surface before and after ANFs composite was measured using a contact angle meter (SDC-350) at room temperature and pressure using ultrapure water (8.0 μL). The results showed that the contact angle of the AgNPs-PAM hydrogel membrane was 66.187° ( Figure 6 d), the contact angle of the ANFs-composite hydrogel membrane is 57.627° ( Figure 6 e), the contact angles of both sets of data were less than 90°. Notably, the contact angle of the ANFs-composite hydrogel membrane was smaller than that of the AgNPs-PAM hydrogel membrane. Furthermore, the effect of ANFs composites on the adsorption capacity of the AgNPs-PAM hydrogel membrane was investigated using a UV-Vis spectrophotometer. A linear fit was established between methylene blue concentration and UV-Vis absorption light ( Figure 6f), the initial concentration of methylene blue was 3.73 mg / L, and the adsorption capacity of AgNPs-PAM hydrogel membrane was 21.0 μg / g and the adsorption capacity of AgNPs / ANFs-PAM hydrogel membrane was 62.9 μg / g at the same adsorption time, which showed that the adsorption capacity of the hydrogel membrane after ANFs complexation increased by about 3 times. This is because ANFs in AgNPs / ANFs-PAM hydrogel membrane contain a large number of amide bonds, which can form stable hydrogen bonds with polar molecules such as -NH2 and -COOH, thereby improving the adsorption performance of the hydrogel membrane. The hydrogel membranes with different contents of ANFs were systematically studied, and with the increase of ANF content (0.0300-0.210wt%), the hydrogel membrane showed strong SERS signal for thiram, but when the content exceeded 0.0600wt%, the SERS signal showed a downward trend. Figure 7 a-b). This is mainly due to the fact that excessive ANFs cannot be uniformly dispersed, and accumulate in the interior of the hydrogel membrane, Figure 7 c) and the bottom Figure 7 d) causing pore blockage, reducing the active sites in the interior of the hydrogel membrane, and at the same time increasing the optical shielding effect of the incident laser, reducing the laser energy received by the surface of AgNPs, making it difficult to produce stronger SERS "hot spots", which restricts the SERS performance of the hydrogel membrane. When the content of ANFs is 0.0600wt%, the SERS signal of 100 μg / L thiram on the lower wrinkled surface of AgNPs / ANFs-PAM hydrogel membrane is higher than that on the upper smooth surface of AgNPs / ANFs-PAM hydrogel membrane. Figure 7 e). SEM test of naturally dried AgNPs / ANFs-PAM hydrogel membrane in backscattering mode, as shown in Figure 7 f and Figure 7 g, the upper surface contains almost no AgNPs, and the lower surface contains rich AgNPs. This is because at the initial stage of polymerization, AM and PEGDA on the upper layer polymerize rapidly, AM and PEGDA on the lower layer diffuse upward, causing AgNPs to sink unstably and be embedded in the lower layer of the hydrogel membrane. The above experimental results all prove that the excellent SERS performance of AgNPs / ANFs-PAM hydrogel membrane is mainly due to the formation of "hill-like" structure after ANFs complexation, which can increase the SERS active sites, and the formation of trapezoidal pore structure by the hydrogel. Finally, the thickness of the hydrogel membrane (1.0-3.0mm) was optimized, as shown in Figure 7h, the 2.0 mm thickness of hydrogel film showed strong SERS response. The AgNPs loading of hydrogel film was sufficient and the mass transfer efficiency was high under this thickness condition. The diffusion was blocked when the thickness was too thick (3.0 mm), and the active sites were insufficient when the thickness was too thin (1.0 mm). By optimizing the above parameters, the optimal conditions of the hydrogel film were determined as follows: the AgNPs concentration was 3.52 g / L, the PEGDA content was 1.50 %, the ANFs content was 0.0600 wt %, and the film thickness was 2.0 mm. In order to obtain the optimal SERS detection conditions of AgNPs / ANFs-PAM hydrogel film, the effects of drying temperature, adsorption time, pre-drying time, and drying time on SERS signal were studied. The experimental results showed that the SERS performance of the hydrogel film was best when the drying temperature was 55 ℃ (a), the adsorption time was 8 min (b), the pre-drying time was 1.5 min (c), and the drying time was 4 min (d). Figure 8 a), the adsorption time was 8 min ( Figure 8 b), the pre-drying time was 1.5 min ( Figure 8 c), and the drying time was 4 min ( Figure 8 d).

[0072] Example 5

[0073] On the basis of Example 4, this example discloses the use of the hydrogel film as a substrate for a SERS sensor for detecting thiuram in vegetables and fruits.

[0074] Thiuram (tetramethylthiuram disulfide) is a widely used dithiocarbamate fungicide in agriculture (seed treatment, fruit and vegetable sterilization), which can effectively prevent and control many diseases and is used for vegetable and fruit disease control (such as pear black star disease and celery early blight). Compared with new fungicides, thiuram is inexpensive and is the first choice of many farmers for fungicides. Due to its potential toxicity, eating vegetables and fruits with excessive thiuram residues can cause serious harm to human health, such as nausea, vomiting, diarrhea, and even cancer. Therefore, it is particularly important to detect the content of thiuram in vegetables and fruits.

[0075] In order to detect the residual thiuram in vegetables and fruits, the anti-interference performance of the AgNPs / ANFs-PAM hydrogel film constructed in this study was tested, including inorganic and organic interference substances that may coexist in vegetable and fruit samples such as vitamin B1, nicotinic acid, vitamin E, glutamic acid, ZnCl2, MgCl2, KCl, CaCl2, NaCl, and pectin. Figure 9 f, when 1.25 × 10 3 times of vitamin B1, nicotinic acid, vitamin E, glutamic acid, and pectin, 2.5 × 10 3 times of ZnCl2, MgCl2, KCl, CaCl2, NaCl, and all substances were mixed to explore the anti-interference ability of the hydrogel film to thiuram, the SERS signal of thiuram at 1380 cm -1The SERS signal intensity of the two samples did not change significantly and the RSD was less than 4.5 %, indicating that the hydrogel film had good anti-interference performance on the inorganic and organic substances that might coexist in the actual sample. To verify the detection ability of the established SERS sensing method in the actual sample, celery and pear samples were used as representatives to perform the standard addition recovery experiment of thiram.

[0076] The results are shown in Table 1. At three standard addition concentration levels (0.0750 mg / kg, 0.375 mg / kg and 0.750 mg / kg), the standard addition recovery rate of thiram in celery was 86.7%-91.3% and the RSD was 3.3%-4.8%; the standard addition recovery rate in pear was 87.7%-95.2% and the RSD was 1.0%-5.8%. The standard addition recovery rate of the two samples was 86.7%-95.2% and the RSD was less than 5.8%, which met the requirements of trace detection. The experimental results fully proved that the AgNPs / ANFs-PAM hydrogel film disclosed in the present application had excellent anti-matrix interference ability and good reproducibility, and could meet the precise detection of thiram in a complex environment.

[0077] Table 1, SERS analysis method for determining thiram in celery and pear

[0078]

[0079] To verify the reliability of the SERS detection method of the AgNPs / ANFs-PAM hydrogel film, it was compared with the traditional high performance liquid chromatography (HPLC) method. The standard analysis method for detecting thiram by HPLC was established, and the experimental results are shown in Figure 10 a and Figure 10 b. The thiram standard solution showed a good linear relationship with the chromatographic peak area in the concentration range of 0.600-9.00 mg / L, the linear regression equation was y = 119.6x + 19.01, the correlation coefficient (R²) was 0.9981, and the method detection limit (LOD) was 152 μg / L. On this basis, the actual samples such as celery and pear were detected by HPLC, and the results are shown in Figure 10 c and Figure 10As shown in FIG. 1, no thiuram residual was found in the celery juice and pear juice samples, and the peak area of the sample spiked with 2.00 mg / kg thiuram standard solution was close to that of the 5.00 mg / L thiuram standard solution. Subsequently, the actual samples spiked with 1.00 mg / kg, 2.00 mg / kg and 3.00 mg / kg thiuram were detected by HPLC. Since the detection range of the SERS sensor developed in the present study for thiuram was lower than that of HPLC, the HPLC spiked solution was diluted 100 times before being detected by SERS. The results are shown in Table 2. The detection results of thiuram in celery and pear by the two methods were consistent, and the relative error was less than 7.7 %, verifying the accuracy and practical application potential of the developed SERS detection method. Compared with other related literature reports shown in Table 3, the AgNPs / ANFs-PAM hydrogel film SERS substrate disclosed in the present application has a low LOD and good stability after being placed for 90 days, and the overall analysis time is 14.5 min. The results show that the AgNPs / ANFs-PAM hydrogel film sensor disclosed in the present application has the characteristics of high sensitivity and rapid detection in the aspect of trace detection of SERS technology.

[0080] Table 2, Determination of thiuram in celery and pear by HPLC and SERS method

[0081]

[0082] Table 3, Comparison of different SERS substrates for detecting thiuram

[0083]

[0084] Of course, the present application can have other various embodiments, and those skilled in the art can make various corresponding changes and modifications according to the present application without departing from the spirit and essence of the present application, but these corresponding changes and modifications should all belong to the protection scope of the claims attached to the present application.

Claims

1. An asymmetric AgNPs / ANFs-PAM hydrogel membrane, characterized in that, The upper layer of the hydrogel film, near the light source, is a smooth and dense skin layer, while the bottom layer, far from the light source, has a wrinkled structure. AgNPs were embedded in a hydrogel membrane.

2. The asymmetric AgNPs / ANFs-PAM hydrogel membrane according to claim 1, characterized in that, The folded structure is hill-shaped.

3. The asymmetric AgNPs / ANFs-PAM hydrogel membrane according to claim 1 or 2, characterized in that, The AgNPs are embedded in a three-dimensional network porous hydrogel membrane modified with ANFs.

4. A method applicable to the preparation of the asymmetric AgNPs / ANFs-PAM hydrogel membrane as described in claim 3, characterized in that, Includes the following steps: a. Disperse ANFs uniformly in an AgNPs solution with an average diameter of 55 nm in a container; b. Add acrylamide and polyethylene glycol diacrylate to the container in sequence and stir; c. After stirring is stopped, the container is evacuated and then phenylphosphine dioxide is added and mixed well to obtain the prepolymer solution. d. Inject the prepolymer liquid into the silicone rubber mold, cover the opening of the silicone rubber mold with a quartz glass plate, and use 365 nm ultraviolet light to vertically irradiate the prepolymer liquid through the quartz glass plate to form a hydrogel film with a smooth top layer and hill-shaped wrinkles at the bottom. e. After demolding, soak the hydrogel membrane in secondary water to wash away any unreacted substances.

5. The preparation method according to claim 4, characterized in that, In step ac, the ratio of AgNPs solution, acrylamide, polyethylene glycol diacrylate, and phenylphosphine dioxide is 5.00 mL, 2.50 g, 75.0 μL, and 200 μL, respectively, and the concentration of phenylphosphine dioxide is 16.6 mg / mL.

6. The preparation method according to claim 5, characterized in that, In step b, the stirring time is 2 hours; in step d, the ultraviolet light irradiation time is 8 minutes.

7. The preparation method according to claim 4, characterized in that, The preparation method of the AgNPs solution is as follows: Add 36.0 g / L silver nitrate to secondary water, place it in an oil bath, place a condenser in it, stir magnetically for 20 min, then add 1.00% sodium citrate, continue heating and stirring until the solution changes from yellow to silvery-gray, then stop the reaction and allow the solution to cool and age.

8. The preparation method according to claim 7, characterized in that, In the preparation of the AgNPs solution, the ratio of deionized water, silver nitrate, and sodium citrate is 200 mL, 1.00 mL, and 4.00 mL, respectively.

9. The application of the asymmetric AgNPs / ANFs-PAM hydrogel membrane according to claim 3, characterized in that, The hydrogel membrane is used as the substrate for the SERS sensor, and the substrate has a thickness of 2 mm and a diameter of 5.0 mm.

10. The application according to claim 9, characterized in that, The SERS sensor is used for the detection and analysis of thiram residues in vegetables and fruits.

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