SERS (Surface Enhanced Raman Scattering) detection method based on spherical hydrogel metal nanoparticle composite gel beads
By constructing a SERS substrate of spherical hydrogel-metal nanoparticle composite beads, the problems of signal inhomogeneity and repeatability in complex samples were solved, achieving trace analysis with high sensitivity and high reliability, which is suitable for the detection of complex biological fluids and food samples.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
- Filing Date
- 2026-03-13
- Publication Date
- 2026-05-08
AI Technical Summary
Existing SERS detection technologies are susceptible to interference from high ionic strength, complex matrix composition, and proteins in complex biological fluids and food samples, leading to signal attenuation, uneven spatial distribution, and reduced repeatability, making it difficult to achieve highly sensitive and reliable trace analysis.
A SERS substrate material based on spherical hydrogel metal nanoparticle composite beads was constructed. By uniformly loading silver nanoparticles into a three-dimensional network, spherical sensing units with uniform macroscopic size and symmetrical microstructure were formed. The isotropic shrinkage characteristics of silver nanoparticles during the evaporation process were utilized to avoid the coffee ring effect and achieve signal uniformity and reproducibility.
It significantly improves the signal uniformity and batch-to-batch reproducibility of SERS detection, achieves femtomolar-level detection sensitivity in high-salt and high-protein environments, and reliably identifies target components in complex samples, making it suitable for highly reliable detection of complex real samples.
Smart Images
Figure CN121994776A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of surface-enhanced Raman spectroscopy (SERS) technology, specifically relating to a SERS detection method based on spherical hydrogel-metal nanoparticle composite beads. Background Technology
[0002] Surface-enhanced Raman scattering (SERS) is a highly sensitive detection technique that leverages the localized electromagnetic field enhancement effect generated by metallic nanostructures to achieve trace and even single-molecule level analysis. With its advantages such as strong molecular fingerprint recognition capabilities, good resistance to photobleaching, and non-destructive nature, SERS has shown significant application potential in the analysis of complex systems such as biological fluids, environmental water samples, and food safety.
[0003] The performance of SERS detection largely depends on the structural stability and interface controllability of the SERS substrate material. However, in real biological fluids such as urine and blood, as well as in lutein-adulterated samples, high ionic strength, complex matrix composition, and large amounts of interfering components such as proteins can easily cause uncontrolled aggregation of metal nanoparticles or non-specific adsorption and masking of surface active sites, leading to signal attenuation, uneven spatial distribution, and reduced repeatability.
[0004] In recent years, hydrogel materials have demonstrated unique advantages in the pretreatment of complex samples due to their three-dimensional hydrophilic network, high porosity, and molecular sieving capabilities. Natural polysaccharide gels such as alginate can efficiently exclude large molecular interferences such as proteins through a dual mechanism of electrostatic repulsion and steric hindrance, while selectively enriching small molecule analytes through electrostatic adsorption, hydrogen bonding, and hydrophobic interactions. Based on this, uniformly loading metal nanoparticles into a hydrogel matrix can form a composite system with both enrichment and sensing functions. Existing strategies have achieved enhanced sensitivity using anisotropic two-dimensional films and bulk hydrogel surface-enhanced Raman scattering (SERS) substrates by utilizing evaporation-induced shrinkage; however, the effects of asymmetric structure and hydrogel type on signal uniformity and reproducibility have not been fully investigated. These parameters can lead to signal inhomogeneity through mechanisms such as non-uniform evaporation, analyte redistribution, and substrate curling. Therefore, there is an urgent need to develop a SERS detection method based on geometrically regular spherical hydrogel composite beads. This method would leverage the isotropic and uniform shrinkage properties of these beads to fundamentally suppress the coffee ring effect, while simultaneously endowing the system with excellent resistance to salt, proteins, and multi-component interference. This would enable highly sensitive and reliable detection of target analytes in complex real-world samples. Such a hydrogel SERS platform, with its higher sensitivity and reproducibility, is of great significance for advancing trace analysis in complex real-world samples. Summary of the Invention
[0005] To address the shortcomings of the existing technologies, this invention provides a SERS detection method based on spherical hydrogel-metal nanoparticle composite beads. This method constructs geometrically regular and structurally uniform spherical hydrogel-metal nanoparticle composite beads as the SERS substrate material, which combines structural stability, interface controllability, and excellent resistance to salt, proteins, and multi-component interference. It is suitable for high-reliability surface-enhanced Raman detection under complex real-world sample conditions.
[0006] The specific technical solution is as follows:
[0007] One objective of this invention is to provide a SERS detection method based on spherical hydrogel-metal nanoparticle composite beads, comprising the following steps:
[0008] S1. Obtain silver nanoparticles (AgNPs);
[0009] S2. Silver nanoparticles are mixed with an aqueous alginate solution to obtain a hydrogel mixture; the hydrogel mixture is then dropped into an aqueous CaCl2 solution to carry out a cross-linking reaction, resulting in hydrogel-metal nanoparticle composite beads.
[0010] S3. Add the hydrogel metal nanoparticle composite beads to the sample solution to be tested for sample adsorption, and then take out the hydrogel metal nanoparticle composite beads and let them stand for 10~50 min to evaporate and concentrate them.
[0011] S4. SERS detection was performed on the hydrogel-metal nanoparticle composite beads after evaporation and concentration.
[0012] The mechanism of the above detection method is as follows: The core of this invention lies in constructing a geometrically regular and structurally uniform spherical hydrogel-metal nanoparticle composite bead as a SERS substrate material. This bead uses alginate as a framework, and silver nanoparticles are uniformly embedded within a three-dimensional network through a homogeneous spherical formation process, forming spherical sensing units with uniform macroscopic dimensions and symmetrical microscopic structures. Compared to the irregular gel carriers in existing technologies, this spherical composite bead exhibits ideal isotropic uniform shrinkage during evaporation and dehydration, simultaneously compressing the analyte molecules and silver nanoparticles distributed throughout the three-dimensional network to a tiny volume, generating high-density, spatially uniform SERS hotspots in situ within the bead. This uniform spherical structure not only fundamentally avoids the uncontrollable aggregation and coffee ring effect in traditional droplet evaporation but also endows the substrate material with excellent signal reproducibility and uniformity. Therefore, the hydrogel-metal nanoparticle composite bead SERS material system provided by this invention has structural stability, interface controllability, and excellent resistance to salt, protein, and multi-component interference, making it suitable for high-reliability surface-enhanced Raman detection under complex real sample conditions.
[0013] Furthermore, in step S2: the alginate is preferably sodium alginate.
[0014] Furthermore, in step S2: the concentration of alginate in the hydrogel mixture is preferably 0.5wt%~2wt%, more preferably 1wt%~2wt%, and most preferably 1wt%. The concentration of the alginate aqueous solution is preferably 0.5wt%~4wt%.
[0015] Furthermore, in step S2, the concentration of the CaCl2 aqueous solution is preferably 1wt%~30wt%, more preferably 15wt%~20wt%, and most preferably 15wt%.
[0016] Furthermore, in step S2: the crosslinking reaction time is preferably 1~15 min, more preferably 5~15 min, and most preferably 5 min.
[0017] Specifically, in step S2: the hydrogel mixture is added dropwise to the CaCl2 aqueous solution using a syringe to carry out the cross-linking reaction. The inner diameter of the syringe needle is preferably 0.3~0.6 mm, and the dripping speed is preferably 100~300 μL / min.
[0018] Furthermore, in step S3, the sample adsorption time is preferably 10~40 min.
[0019] Specifically, in step S3: the sample is shaken using a shaker during adsorption.
[0020] Furthermore, in step S1: silver nanoparticles can be synthesized by reducing silver nitrate with sodium citrate. The specific method includes: adding sodium citrate to an aqueous solution of silver nitrate, reacting at 120-150℃ for 20-50 min, to obtain a silver nanoparticle suspension.
[0021] The preferred molar ratio of silver nitrate to sodium citrate is 1:(0.3~1.5). Specifically, sodium citrate is added in the form of an aqueous solution of sodium citrate, and the concentration of the aqueous solution of sodium citrate is preferably 0.5wt%~2wt%.
[0022] In step S1, the concentration of the silver nitrate aqueous solution is preferably 200~400 mg / L. Meanwhile, in step S2, the silver nanoparticle suspension obtained in step S1 is concentrated to 5%~15% of its original volume to obtain a concentrated suspension; the concentrated suspension is then mixed with an alginate aqueous solution at a volume ratio of (0.05~1):1 to obtain a hydrogel mixture.
[0023] The second objective of this invention is to provide the application of the above-mentioned SERS detection method based on spherical hydrogel metal nanoparticle composite beads in the detection of Nile blue, malachite green, or lutein.
[0024] The detection method of this invention is applicable to the detection of pollutants such as Nile Blue or Malachite Green under high-salt (sodium chloride content below 150 mM) and high-protein (protein content below 100 μg / mL) conditions, and is further applicable to the detection of target components in seawater and biological fluids. Simultaneously, this detection method is suitable for the identification and differentiation of adulterated lutein samples, distinguishing lutein from adulterants such as gardenia yellow, tartrazine, or curcumin. Examples of adulterated samples include mixed samples formed by mixing lutein ester powder with gardenia yellow, tartrazine, or curcumin in different proportions.
[0025] Compared with the prior art, the present invention has the following beneficial effects:
[0026] (1) This invention constructs a spherical hydrogel metal nanoparticle composite bead with uniform structure, and utilizes its isotropic uniform shrinkage characteristics generated during the evaporation and dehydration process to fundamentally solve the problems of coffee ring effect and uneven signal spatial distribution caused by different shrinkage behaviors and uncontrollable aggregation during the evaporation process of traditional irregular gel carriers or droplets, which significantly improves the signal uniformity and batch reproducibility of SERS detection.
[0027] (2) The composite beads prepared by the homogeneous spheroidization process of the present invention have the characteristics of uniform macroscopic size and symmetrical microstructure, which ensures the structural stability and interface controllability of the substrate material, ensures the stability of detection performance, and lays the foundation for its standardized application and large-scale preparation in actual sample detection.
[0028] (3) The hydrogel network of the present invention effectively screens out interference from macromolecules such as proteins. Its uniform swelling-shrinkage mechanism drives the directional enrichment of analytes, completely eliminating the coffee ring effect and realizing the controllable and uniform concentration of analytes in the hot spot area of fixed nanoparticles, thereby obtaining a highly repeatable and stable SERS signal (RSD < 4.39%).
[0029] (4) The hydrogel-metal nanoparticle composite beads of the present invention can achieve femtomolar level detection sensitivity for Nile Blue while resisting interference from high salt (150 mM sodium chloride) and high protein (100 μg / mL bovine serum albumin).
[0030] (5) The present invention can reliably identify and distinguish adulterated lutein samples in complex multi-component environments, including distinguishing and identifying mixed samples formed by adulteration of lutein ester powder with gardenia yellow, tartrazine or curcumin in different proportions, maintaining good signal stability and discrimination reliability, thereby expanding the practical application value of the SERS platform in the field of accurate detection of adulterated lutein. Attached Figure Description
[0031] Figure 1SEM image of the AgNPs prepared in Example 1;
[0032] Figure 2 Physical images of different types of hydrogel-metal nanoparticle composite beads for test 1;
[0033] Figure 3 SEM image of the hydrogel-metal nanoparticle composite beads after evaporation and concentration in Example 1 of Test 1;
[0034] Figure 4 Comparison of silver nanoparticle agarose SERS substrate and silver nanoparticle sodium alginate SERS substrate after wetting and drying;
[0035] Figure 5 Detection of silver nanoparticle agarose SERS substrate and preparation of silver nanoparticle sodium alginate SERS substrate (wet state, dry center position, dry edge position) 10 -7 SERS spectrum of Nile blue and 1619 cm⁻¹ -1 Characteristic peak intensity histogram;
[0036] Figure 6 To compare the initial morphology of the hydrogel-metal nanoparticle composite beads in Examples 1-5 of Test 2, the morphological evolution during the evaporation process, and the microstructure after evaporation and concentration;
[0037] Figure 7 To test the hydrogel-metal nanoparticle composite beads of different concentrations of sodium alginate in 2, the following was determined: 10 -7 Raman spectrum of M Nile blue and 1645 cm⁻¹ -1 A histogram of the intensity of the characteristic peak;
[0038] Figure 8 To test the cross-linking reaction time, CaCl2 concentration, and different ratios of AgNPs concentrated suspension / sodium alginate aqueous solution in hydrogel-metal nanoparticle composite beads, 10... -7 Raman spectrum of M Nile blue and 1645 cm⁻¹ -1 A histogram of the intensity of the characteristic peak.
[0039] Figure 9 To test the method of Example 1 in Test 3, the Nile blue solution (1×10) was tested. -6 M~1×10 -10 Raman spectrum, hotspot map and 1645 cm⁻¹ of M) -1 A histogram of the intensity of the characteristic peak;
[0040] Figure 10 To test the method of Example 1 in test 4, 10 were detected under different concentrations of sodium chloride. -7Raman spectrum of Nile blue and 1645 cm⁻¹ -1 A histogram of the intensity of the characteristic peak;
[0041] Figure 11 To test the method of Example 1 in 5, 10 were detected at different concentrations of bovine serum albumin. -7 Raman spectrum of Nile blue and 1645 cm⁻¹ -1 A histogram of the intensity of the characteristic peak;
[0042] Figure 12 To test the method of Example 1 in 6 cases, Raman spectra and 1619 cm⁻¹ spectra of malachite green at different concentrations were detected under 150 mM sodium chloride conditions. -1 The linear relationship between the intensity of the characteristic peak and the concentration of malachite green;
[0043] Figure 13 To test the method of Example 1 in 7, Raman spectra of different concentrations of malachite green were detected in seawater diluted 3 times and fish serum diluted 100 times.
[0044] Figure 14 Linear discriminant analysis plots and confusion matrices were used to test the method of Example 1 in 8 for detecting adulteration of lutein ester powder and pigments;
[0045] Figure 15 Linear discriminant analysis plots and confusion matrix plots were used to test the method of Example 1 in 8 to detect specific types of pigment adulteration;
[0046] Figure 16 To test the Raman spectra of lutein ester powder and samples adulterated with gardenia yellow, tartrazine or turmeric in different proportions using the method of Example 1 in 8;
[0047] Figure 17 Linear discriminant analysis plots were used to test the method of Example 1 in 8 to detect lutein ester powder and samples adulterated with gardenia yellow, tartrazine or turmeric at different proportions;
[0048] Figure 18 The confusion matrix diagram for detecting lutein ester powder and samples adulterated with gardenia yellow, tartrazine or turmeric in different proportions was obtained by testing the method of Example 1 in 8. Detailed Implementation
[0049] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0050] Example 1
[0051] A SERS detection method based on spherical hydrogel-metal nanoparticle composite beads includes the following steps:
[0052] S1. Dissolve 46 mg of silver nitrate in deionized water and bring the volume to 200 mL. Heat to 135 °C and add 4 mL of 1 wt% sodium citrate aqueous solution while stirring vigorously. Maintain heating at 135 °C for 30 min until the reaction solution turns gray-green to obtain a silver nanoparticle (AgNPs) suspension.
[0053] The prepared AgNPs were scanned by electron microscopy, and the resulting SEM images are shown below. Figure 1 As shown.
[0054] S2. Concentrate the suspension obtained in step S1 to 20 mL, and mix the concentrated suspension with a 2 wt% sodium alginate aqueous solution at a volume ratio of 1:1 to obtain a hydrogel mixture (sodium alginate concentration in the hydrogel mixture is 1 wt%). Using a syringe (needle inner diameter 0.45 mm) connected to an injection pump, add the hydrogel mixture dropwise to a 15 wt% CaCl2 aqueous solution at a constant rate of 200 μL / min, and carry out a cross-linking reaction for 5 min under stirring. Then wash with deionized water to remove residual ions to obtain hydrogel metal nanoparticle composite beads.
[0055] S3. Add the hydrogel-metal nanoparticle composite beads obtained in step S3 to 5 mL of the sample solution to be tested, place it in a shaker for 20 min for sample adsorption, and then take out the hydrogel-metal nanoparticle composite beads and place them on ordinary A4 paper for 30 min for natural evaporation and concentration.
[0056] S4. Perform SERS (Surface Enhanced Raman) detection on the hydrogel-metal nanoparticle composite beads obtained by natural evaporation and concentration in step S3.
[0057] Examples 2-19
[0058] Referring to Example 1, the difference from Example 1 is that the parameters in step S2 are shown in Table 1. Other technical features are the same as in Example 1.
[0059] Table 1 Parameter Table for Examples 2-19
[0060]
[0061] Comparative Examples 1-3
[0062] Referring to Example 1, the difference from Example 1 is that in step S2, sodium alginate is replaced in equal amounts with the materials shown in Table 2. Other technical features are the same as in Example 1.
[0063] Table 2. Hydrogel materials of Comparative Examples 1-3
[0064]
[0065] Test 1
[0066] The hydrogel-metal nanoparticle composite beads obtained in step S2 of Examples 1 and Comparative Examples 1-3 (before drying) and after drying were compared. The drying method was the same as the evaporation and concentration method in step S3 of Example 1. Images of the products before and after drying are shown below. Figure 2 As shown. Figure 2 In the middle section, from left to right, are Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3. The upper part shows the hydrogel-metal nanoparticle composite beads obtained in step S2, and the lower part shows the dried hydrogel-metal nanoparticle composite beads. The size comparison of the hydrogel-metal nanoparticle composite beads before and after drying is shown in Table 3.
[0067] The dried hydrogel-metal nanoparticle composite beads from Example 1 were subjected to electron microscopy scanning, and the resulting SEM images are shown below. Figure 3 As shown.
[0068] Table 3. Volume retention rates of different hydrogel-metal nanoparticle composite beads before and after drying
[0069]
[0070] Figures 2 to 3 The study demonstrated the successful preparation of hydrogel-metal nanoparticle composite beads. Figure 2 Table 3 shows that the sodium alginate hydrogel-metal nanoparticle composite beads had the lowest volume retention rate, at 1.77%. Beads prepared from agarose and gellan gum collapsed during the drying process and could not be evaporated and concentrated into microspheres.
[0071] Silver nanoparticle agarose SERS substrate and silver nanoparticle sodium alginate SERS substrate were prepared. The silver nanoparticle agarose SERS substrate was prepared as follows: 2 mL of 2wt% agarose, 400 μL of concentrated silver nanoparticle suspension (preparation method as in Example 1), and 1.6 mL of water were mixed, heated and stirred until fully mixed, then poured into a glass dish (10 cm in diameter) and allowed to cool and solidify. The silver nanoparticle sodium alginate SERS substrate was prepared as follows: 2 mL of 2wt% sodium alginate, 400 μL of concentrated silver nanoparticle suspension (preparation method as in Example 1), and 1.6 mL of water were mixed, stirred until fully mixed, then poured into a glass dish (10 cm in diameter), and 2wt% calcium chloride was added along the edge. Crosslinking was carried out for 30 min. The above SERS substrates and their dried states were compared. The drying method was the same as the evaporation and concentration method in step S3 of Example 1. The concentration of the above SERS substrate was measured to be 10. -7 The Nile blue M was detected using the same method as in Example 1, with an excitation wavelength of 532 nm and a laser intensity of 1%. Comparison images of the above SERS substrate and its dried state are shown below. Figure 4 . Figure 4 In the image: A is a silver nanoparticle agarose SERS substrate, and B is a silver nanoparticle sodium alginate SERS substrate. The above SERS substrates (wet state, dry center position, dry edge position) were analyzed 10 times. -7 SERS spectrum of Nile blue and 1619 cm⁻¹ -1 The characteristic peak intensity histogram is shown below. Figure 5 . Figure 5 In the diagram: C represents the silver nanoparticle agarose SERS substrate, and D represents the silver nanoparticle sodium alginate SERS substrate. The silver nanoparticle agarose SERS substrate and the silver nanoparticle sodium alginate SERS substrate were analyzed after drying. -7 The SERS signal of Nile blue was significantly enhanced compared to the wet state, but due to molecular redistribution caused by evaporation, the SERS signal intensity at the dried edge was significantly higher than that at the dried center. Simultaneously, substrate curling caused by drying shrinkage further disrupted the surface smoothness and structural uniformity. These results indicate that it is difficult to obtain stable and reproducible detection signals on silver nanoparticle agarose SERS substrates and silver nanoparticle sodium alginate SERS substrates.
[0072] Test 2
[0073] The hydrogel-metal nanoparticle composite beads obtained in step S2 of Examples 1-5 were dried using the same evaporation and concentration method as in step S3 of Example 1. A comparison of the initial morphologies of the hydrogel-metal nanoparticle composite beads from Examples 1-5 is shown below. Figure 6 (A), See the morphological evolution during the evaporation process. Figure 6(B) The microstructure after evaporation and concentration is shown in Figure 1. Figure 6 (C). Figure 6 In the middle: 0.5%, 0.75%, 1%, 1.5%, and 2% correspond to Example 2, Example 3, Example 1, Example 4, and Example 5, respectively.
[0074] The concentration was detected using the methods described in Examples 1-5, and the concentration was 10. -7 The Nile blue sample of M was excited at a wavelength of 532 nm with a laser intensity of 1%. The resulting Raman spectrum and 1645 cm⁻¹ were obtained. -1 The histogram of the characteristic peak intensity is shown below. Figure 7 . Figure 7 In (A): 0.5%, 0.75%, 1%, 1.5%, and 2% correspond to Example 2, Example 3, Example 1, Example 4, and Example 5, respectively. Figure 7 (B) The horizontal axis represents the concentration of sodium alginate in the hydrogel mixture.
[0075] like Figure 6 As shown, a comparative analysis of the initial morphology, morphological evolution during evaporation, and microstructure of hydrogel metal nanoparticle composite beads with different sodium alginate concentrations indicates that moderately increasing the sodium alginate concentration can effectively maintain the structural integrity of the microbeads and reduce the anisotropic shrinkage phenomenon caused by evaporation. Figure 7 Further evidence shows that when the sodium alginate concentration in the hydrogel mixture is 1 wt%, the hydrogel-metal nanoparticle composite beads achieve the optimal balance between structural stability and SERS signal enhancement.
[0076] Test 3
[0077] The concentration of 10 was detected using the methods described in Examples 1 and 6-19. -7 The Nile blue sample of M was excited at a wavelength of 532 nm with a laser intensity of 1%. The resulting Raman spectrum and 1645 cm⁻¹... −1 The histogram of the characteristic peak intensity is shown below. Figure 8 . Figure 8 In (A): 1 min, 3 min, 5 min, 10 min, and 15 min correspond to Examples 6, 7, 1, 8, and 9, respectively. Figure 8 In (B): 1%, 5%, 10%, 15%, 20%, and 30% correspond to Examples 10, 11, 12, 1, 13, and 14, respectively. Figure 8 In (C): 0.01, 0.05, 0.1, 0.3, 0.5, and 1.0 correspond to Examples 15, 16, 17, 18, 19, and 1, respectively.
[0078] Depend on Figure 8 It can be seen that the optimal preparation conditions for hydrogel metal nanoparticle composite beads are: crosslinking time of 5 min, CaCl2 solution concentration of 15 wt%, and AgNPs suspension to sodium alginate aqueous solution volume ratio of 0.5:1, thus successfully constructing a hydrogel metal nanoparticle composite bead SERS substrate with optimal performance and stable structure.
[0079] The method in Example 1 detected a concentration of 1×10⁻⁶. -6 M to 1×10 -10 The Nile blue solution of M was excited at a wavelength of 532 nm with a laser intensity of 1%. The Raman spectrum is shown below. Figure 9 (A), concentration 1×10 -7 The mapping heatmap of M's Nile blue solution is shown below. Figure 9 (B), concentration 1 × 10 -7 M's Nile Blue Solution at 1645 cm −1 The histogram of the characteristic peak intensity is shown below. Figure 9 (C).
[0080] Figure 9 The results showed that the detection limit for Nile blue by the hydrogel-metal nanoparticle composite beads was as low as 10. -10 M, in 10 -6 M ~10 -10 Within the concentration range of M, Nile blue at 1645 cm⁻¹ -1 The characteristic peak signal intensity and concentration at that location exhibit a good linear relationship (R0). 2 =0.99); furthermore, the spatially resolved SERS plot showed a relative standard deviation of 4.36%, demonstrating good homogeneity. Simultaneously, parallel detection of eight different batches of SERS substrates showed consistent SERS spectral characteristic peaks, with an RSD of 4.39% for the characteristic peak intensity. These results fully demonstrate that the hydrogel-metal nanoparticle composite bead SERS substrate exhibits excellent performance in terms of structural uniformity, signal uniformity, and batch reproducibility, laying a solid foundation for reliable SERS detection in complex multi-component environments.
[0081] Test 4
[0082] Using the method of Example 1, 10 [units of something] were subjected to sodium chloride concentrations of 0–300 mM. -7 SERS analysis was performed on the M Nile Blue sample using an excitation wavelength of 532 nm and a laser intensity of 1%. The results are shown below. Figure 10 Raman spectra can be found Figure 10 (A), 1645cm -1 The histogram of the characteristic peak intensity is shown below. Figure 10(B). The results showed that the hydrogel-metal nanoparticle composite bead SERS substrate could resist sodium chloride up to 150 mM without significant signal intensity attenuation, demonstrating superior salt resistance.
[0083] Test 5
[0084] Using the method of Example 1, bovine serum albumin (BSA) concentrations of 0–1000 μg / mL were used to treat 10 -7 SERS analysis was performed on the M Nile Blue sample using an excitation wavelength of 532 nm and a laser intensity of 1%. The results are shown below. Figure 11 Raman spectra can be found Figure 11 (A), 1645 cm -1 The histogram of the characteristic peak intensity is shown below. Figure 11 (B). The results showed that the SERS characteristic peak intensity of the hydrogel-metal nanoparticle composite bead SERS substrate remained stable even at bovine serum albumin concentrations as high as 100 μg / mL, demonstrating excellent anti-protein ability. This is attributed to the unique porous mesh "sieving" effect of the hydrogel, which allows small molecule targets (such as Nile Blue) to effectively penetrate and contact the internal AgNP hotspots, while hindering interference from large molecule proteins.
[0085] Test 6
[0086] To evaluate the application potential of hydrogel-metal nanoparticle composite beads for pollutant detection in real complex matrices, malachite green, a typical antibacterial agent, was selected as a model pollutant. A 150 mM sodium chloride solution was used to simulate seawater diluted three times. The method described in Example 1 was applied to detect different concentrations (0–10 mM) of seawater under 150 mM sodium chloride conditions. -6 M) Malachite green sample was subjected to SERS detection with an excitation wavelength of 532 nm and a laser intensity of 1%. Results are shown below. Figure 12 . Figure 12 Malachite green is shown at 1619 cm. -1 The characteristic peak intensity shows a good linear relationship with concentration (R0). 2 =0.99), confirming the high sensitivity of SERS detection capability of hydrogel-metal nanoparticle composite beads in high-salt environments.
[0087] Test 7
[0088] To further evaluate the anti-interference performance of this substrate in real, complex environmental samples, SERS analysis was performed on malachite green in real seawater diluted 3 times and fish serum diluted 100 times. The excitation wavelength was 532 nm and the laser intensity was 1%. The test results are shown below. Figure 13 Raman spectra of different concentrations of malachite green under three times the seawater environment are shown below. Figure 13(A) Raman spectra of different concentrations of malachite green under 100x fish serum conditions are shown in [reference needed]. Figure 13 (B). For example Figure 13 As shown, thanks to the effective shielding of salt ions and proteins by the hydrogel network, the hydrogel-metal nanoparticle composite beads can achieve malachite green levels as low as 10 in seawater and fish serum. -7 Sensitive detection of M.
[0089] Test 8
[0090] SERS detection was performed on adulterated lutein samples with different doping ratios (40%, 70%, and 100%) using the method described in Example 1. The excitation wavelength was 532 nm and the laser intensity was 1%. The adulterated samples consisted of lutein ester powder and dopant, or consisted solely of dopant; the dopant was gardenia yellow, tartrazine, or curcumin; 100% lutein ester powder was used as a reference. When the dopant was gardenia yellow, it was a mixture of gardenia yellow and maltodextrin in a mass ratio of 4:1.
[0091] To identify whether pigment adulteration exists in lutein samples, this study used linear discriminant analysis (LDA) to model and validate all 396 samples. The sample set included a blank group (36 samples), a pure lutein group (36 samples), and a pigment adulteration group (324 samples). The data were randomly divided into a training set (297 samples) and a prediction set (99 samples) at a ratio of 75%:25% for model training and prediction. The specific composition and discrimination results of the prediction set are as follows... Figure 14 As shown, 7 out of 9 blank samples were correctly identified; 8 out of 9 pure lutein samples were accurately identified; and among 81 adulterated samples, the model successfully identified 77, with only 4 false positives. Overall, the model achieved an accuracy rate of over 95% in binary classification for "adulteration." The results demonstrate that this method can not only effectively identify adulterated samples but also clearly distinguish pure lutein from the blank matrix, providing reliable data support for subsequent precise traceability analysis.
[0092] Based on the confirmation of adulteration in the samples, this study further precisely identified the specific types of adulterant pigments. The experiment targeted three common adulterant pigments: turmeric, tartrazine, and gardenia yellow, preparing 108 samples from each, for a total of 324 samples. The data were randomly divided into a training set (243 samples) and a prediction set (81 samples) at a ratio of 75% to 25% for constructing and validating a linear discriminant analysis (LDA) classification model. The results are as follows: Figure 15 As shown, the prediction set contains 27 samples for each pigment category. The model correctly classified all 27 samples from the turmeric, lemon yellow, and gardenia yellow groups, achieving 100% accuracy in each category with no misclassifications.
[0093] The results show that the established LDA model can not only effectively complete the initial screening of "whether it is adulterated", but also accurately distinguish the specific types of adulterated pigments, realizing the accurate identification and classification of pigment adulterants in lutein products, and providing reliable technical support for subsequent quality monitoring.
[0094] Figure 16 The images show the Raman spectra of each adulterated mixed sample. Figure 17 Linear discriminant analysis plots for each adulterated mixed sample; Figure 18 This is a confusion matrix diagram of each adulterated mixed sample. Figure 17 In the middle: from top to bottom, the doping ratios are 40%, 70%, and 100%. Figure 18 In the diagram: (A) represents a doping ratio of 40%, (B) represents a doping ratio of 70%, and (C) represents a doping ratio of 100%. Figure 16 As shown, different doped systems exhibit significant differences in characteristic Raman peak positions and relative peak intensity distributions at different doping ratios (40%, 70%, and 100%). This result demonstrates that the proposed detection substrate can effectively amplify the molecular fingerprint information of each component, achieving a highly sensitive response to complex multi-component systems. The LDA model established based on Raman spectroscopy data further validates its discriminative ability. Figure 17 As shown, a clear clustering and separation trend is observed, indicating that the model can not only distinguish the types of adulterated pigments but also effectively differentiate between different adulterant concentrations within the same type. Furthermore, Figure 18 The confusion matrix shown validates the classification results, and the recognition accuracy remains high at all doping ratios, further confirming the robustness and reliability of the method.
[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A SERS detection method based on spherical hydrogel-metal nanoparticle composite beads, characterized in that, Includes the following steps: S1. Obtain silver nanoparticles; S2. Silver nanoparticles are mixed with an aqueous alginate solution to obtain a hydrogel mixture; the hydrogel mixture is then dropped into an aqueous CaCl2 solution to carry out a cross-linking reaction, resulting in hydrogel-metal nanoparticle composite beads. S3. Add the hydrogel metal nanoparticle composite beads to the sample solution to be tested for sample adsorption, and then take out the hydrogel metal nanoparticle composite beads and let them stand for 10~50 min to evaporate and concentrate them. S4. SERS detection was performed on the hydrogel-metal nanoparticle composite beads after evaporation and concentration.
2. The SERS detection method according to claim 1, characterized in that, In step S2: the alginate is sodium alginate.
3. The SERS detection method according to claim 1, characterized in that, In step S2: the concentration of alginate in the hydrogel mixture is 0.5wt%~2wt%.
4. The SERS detection method according to claim 1, characterized in that, In step S2: the concentration of the CaCl2 aqueous solution is 1wt%~30wt%.
5. The SERS detection method according to claim 1, characterized in that, In step S2: the cross-linking reaction time is 1~15 min.
6. The SERS detection method according to claim 1, characterized in that, In step S3: the sample adsorption time is 10~40 min.
7. The SERS detection method according to claim 1, characterized in that, In step S1: Silver nanoparticles are synthesized by reducing silver nitrate with sodium citrate.
8. The SERS detection method according to claim 7, characterized in that, In step S1: Sodium citrate is added to the silver nitrate aqueous solution, and the mixture is reacted at 120~150℃ for 20~50 min to obtain a silver nanoparticle suspension.
9. The SERS detection method according to claim 8, characterized in that, In step S1: the concentration of the silver nitrate solution is 200~400 mg / L; In step S2: the silver nanoparticle suspension obtained in step S1 is concentrated to 5%~15% of its original volume to obtain a concentrated suspension. The concentrated suspension was mixed with an aqueous alginate solution at a volume ratio of (0.05~1):1 to obtain a hydrogel mixture.
10. The application of the SERS detection method based on spherical hydrogel metal nanoparticle composite beads as described in any one of claims 1 to 9 in the detection of Nile blue, malachite green or lutein.