Sponge-like chitosan composite material and preparation method thereof, SERS substrate material and application thereof

Abstract

This invention discloses a sponge-like chitosan composite material and its preparation method, a SERS substrate material and its application, belonging to the field of optoelectronic devices. The sponge-like chitosan SERS substrate material is composed of chitosan nanofibers (CS-NFs), noble metal nanoparticles (NB-NPs), polydopamine (PDA), and physically cross-linked molecular DNA, obtained through a layer-by-layer self-assembly method. Due to its 3D porous structure, it has a high specific surface area, ensuring the sensitivity and high repeatability of the SERS signal, superior to the bio-based characteristics of its raw materials. It can be applied in the field of traditional Chinese medicine identification. Samples are taken by wiping the surface of the medicinal material or placing it in the medicinal material soaking solution, and then performing Raman spectroscopy testing. Before use, an alkaline or acidic buffer solution can be sprayed onto the sponge-like chitosan SERS substrate material to further enhance the capture ability of cationic or anionic dyes in the stained medicinal material.

Description

Technical Field

[0001] This invention relates to the field of optoelectronic devices, and more particularly to a sponge-like chitosan composite material and its preparation method, SERS substrate material and its application. Background Technology

[0002] Traditional Chinese medicine (TCM) is a treasure of Chinese civilization and plays a vital role in public health. Zhejiang Province ranks third in the country in terms of total authentic medicinal herb resources. However, counterfeit and substandard TCM materials have long been a persistent problem hindering the development of the TCM industry, mainly due to adulteration through dyeing. TCM dyeing often uses artificial synthetic pigments, which have side effects such as high toxicity, carcinogenicity, teratogenicity, and high residue levels. These artificial pigments contain chromophores such as azo groups, vinyl groups, and benzene rings, as well as acid / base auxochromes, with varying hydrophilic and hydrophobic properties. They also contain some inorganic substances such as malachite green (lead chromate). Currently, the state has approved and released 83 methods for detecting adulteration through dyeing in TCM. Relatively mature methods include: ① Physicochemical identification methods, mainly referring to color changes and precipitation reactions. For example, commercial rapid detection kits mainly utilize the difference in adsorption capacity between edible and non-edible pigments on test cotton for qualitative identification; ② High-performance liquid chromatography (HPLC), which has advantages such as high sensitivity and good separation effect. It is currently the most widely used and researched method, and also a commonly used method in national standards. However, current national standards for HPLC mainly target 1 to 5 dyes in more than 10 single-herb medicinal materials, and HPLC results require comparison with standard samples, making them unsuitable for complex and unknown samples. In addition, recent years have seen reports on the use of infrared spectroscopy, mass spectrometry and their coupled techniques, fluorescence spectroscopy, and immunoassay for the detection of synthetic pigments. However, given the complex, variable, and time-sensitive nature of the monitored components, commonly used techniques are no longer sufficient to meet testing needs due to their complexity, time-consuming analysis, and high cost.

[0003] Surface-enhanced Raman spectroscopy (SERS) is a qualitative and quantitative analytical method based on the principle of Raman scattering. Over the past 50 years, with the development of laser technology, inductively coupled plasma detectors, and nanofabrication processes, it has matured and begun to emerge in environmental, chemical, and biological analysis fields. Given the characteristics of SERS, its application in the staining detection of traditional Chinese medicine (TCM) materials has the following potential advantages: ① Simple pretreatment: Solids, liquids, colloidal ointments, etc., can be directly measured, requiring small samples, especially suitable for valuable TCM samples; ② High information content: It possesses fingerprint spectral characteristics, enabling rapid detection of unknown samples; ③ Low detection limit: It can achieve ultra-trace component analysis and even single-molecule detection, avoiding potential major drug safety hazards. Currently, research on dye detection using SERS technology both domestically and internationally shows a year-on-year increasing trend. However, the batch, controllable preparation of SERS substrates with high sensitivity and good signal reproducibility remains a challenge.

[0004] Chitosan, as the most abundant biomass polymer in the ocean, has natural advantages such as being renewable, insoluble in common solvents, having good thermal stability, being biocompatible, and being biodegradable. At the same time, chitosan itself has a large number of functional groups, which facilitates chemical modification and is expected to achieve specific interactions with different target molecules, with a wide range of potential applications. Summary of the Invention

[0005] To address the problems of poor timeliness, complex pretreatment, and inconvenience for large-scale testing in existing methods for identifying genuine and counterfeit Chinese medicinal materials, this invention provides a sponge-like chitosan composite material and its preparation method, as well as a SERS substrate material and its application. Utilizing the abundant hydroxyl and amino groups of chitosan and the adhesion and trapping effect of polydopamine, a sponge-like DNA / NB-NPs@PDA@CS-NFs composite material is obtained through self-assembly. Taking advantage of the capillary action and high specific surface area of ​​the sponge-like material, identification can be performed by wiping the surface of the Chinese medicinal material or by adding a drop of water from the soaking solution of the Chinese medicinal material to the sponge surface, followed by Raman spectroscopy testing.

[0006] To achieve the above-mentioned technical objectives, this invention provides a sponge-like chitosan composite material and its preparation method. The specific technical solution is as follows:

[0007] A sponge-like chitosan composite material is composed of chitosan nanofibers (CS-NFs), noble metal nanoparticles (NB-NPs), polydopamine (PDA), and physically cross-linked DNA molecules. The CS-NFs have a length of 200 nm to 3 μm, the NB-NPs have a particle size of 10-100 nm, PDA coats the surface of CS-NFs to form a PDA@CS-NFs structure, and NB-NPs coat the outside of PDA@CS-NFs to form an NB-NPs@PDA@CS-NFs structure. The NB-NPs are cross-linked with DNA molecules and self-assemble to form a sponge-like DNA / NB-NPs@PDA@CS-NFs composite material.

[0008] Furthermore, in the spongy DNA / NB-NPs@PDA@CS-NFs composite material, the mass percentage of NB-NPs is 5-10%, and the mass percentage of DNA is 1-3%.

[0009] A method for preparing the sponge-like chitosan composite material includes the following steps:

[0010] Step 1: Disperse CS-NFs in a solution containing dopamine hydrochloride and Tris-HCl buffer, homogenize by sonication, let stand for 24 hours, and dialyze to obtain PDA@CS-NFs dispersion.

[0011] Step 2: Add silver nitrate or chloroauric acid dropwise to the PDA@CS-NFs dispersion separately, or add them dropwise to the PDA@CS-NFs dispersion sequentially, and then add sodium borohydride for reduction. Dialyze to obtain the NB-NPs@PDA@CS-NFs dispersion.

[0012] Step 3: Concentrate the NB-NPs@PDA@CS-NFs dispersion to a mass concentration of 15-25%, then add DNA, stir and let stand to perform self-assembly to obtain DNA / NB-NPs@PDA@CS-NFs dispersion, and then freeze-dry to obtain sponge-like DNA / NB-NPs@PDA@CS-NFs composite material.

[0013] Another objective of this invention is to provide a method for preparing and applying a sponge-like chitosan SERS substrate material. The specific technical solution is as follows:

[0014] A sponge-like chitosan SERS substrate material, made from sponge-like chitosan composite material.

[0015] Application of sponge-like chitosan SERS substrate material: The sponge-like chitosan SERS substrate material is used as a flexible SERS substrate material for the identification of Chinese medicinal materials.

[0016] Furthermore, the surface of the Chinese medicinal materials can be wiped with a flexible SERS substrate material or the soaking solution of the Chinese medicinal materials can be dropped onto the surface of the flexible SERS substrate material, and then Raman spectroscopy can be performed for identification.

[0017] Furthermore, before use, an alkaline or acidic buffer solution is sprayed onto the spongy chitosan SERS substrate material to enhance the capture capacity of cationic or anionic dyes in the dyed medicinal materials.

[0018] The raw materials used in this invention are all bio-based, which have significant advantages in terms of environmental protection and biosafety. They are very suitable for identifying the authenticity of precious medicinal materials, so as to avoid contamination of the medicinal materials during the testing process.

[0019] The substrate material used in this invention is a porous 3D sponge-like material, mainly composed of chitosan nanofibers. The material's excellent porosity, specific surface area, and flexibility allow for direct wiping of the surface of Chinese medicinal materials for sampling, or the addition of extracts from Chinese medicinal materials to the substrate material for sampling, followed by Raman spectroscopy for detection. The pretreatment is simple and suitable for real-time monitoring of high-throughput, large-volume samples. It enables the controllable preparation of a 3D high-density "hot spot" framework, improving the detection limit while solving the problem of signal recurrence. Based on the material's high biocompatibility and non-toxicity of hot spots, it is particularly suitable for the identification of genuine and counterfeit precious Chinese medicinal materials.

[0020] The substrate material used in this invention is obtained through layer-by-layer self-assembly. The uniform distribution of high-density nanoparticles in the sponge ensures high sensitivity and good repeatability of the test results. At the same time, the polydopamine, DNA, and hydroxyl, amino, and phosphate groups in the chitosan nanofibers in the material are responsive to acidic and alkaline environments. By changing the pH value of the test environment, acidic or alkaline dyes can be selectively captured, further improving the sensitivity of the test. Attached Figure Description

[0021] Figure 1 TEM electron micrograph of DNA / NB-NPs@PDA@CS-NFs dispersion suspension;

[0022] Figure 2 SEM image of DNA / NB-NPs@PDA@CS-NFs sponge aerogel;

[0023] Figure 3 Digital photograph of DNA / NB-NPs@PDA@CS-NFs sponge aerogel;

[0024] Figure 4 A diagram of a cyclic compression experiment of DNA / NB-NPs@PDA@CS-NFs sponge aerogel;

[0025] Figure 5a SERS spectra of goldamine O solutions at different concentrations;

[0026] Figure 5b Linear fit plot of SERS intensity versus log[Auramine O (mg / ml)];

[0027] Figure 5c SERS spectra of 10⁻³ mg / ml auramine O solution obtained from 6 randomly selected points;

[0028] Figure 5d At 1272cm -1 The SERS intensity observed at [location]. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings.

[0030] 0.15 g Tris-HCl was added to the CS-NFs suspension, along with 0.3 g dopamine hydrochloride, and the mixture was stirred for 12 hours. After dialysis, a PDA@CS-NFs suspension was obtained for later use. 0.2 M AgNO3 solution was added to the PDA@CS-NFs suspension, and 0.01 M NaBH4 solution was slowly added dropwise while stirring. After dialysis, an Ag-NPs@PDA@CS-NFs dispersion was obtained. The Ag-NPs@PDA@CS-NFs dispersion was concentrated to 18% by mass, and then 0.01 g / ml DNA was added. After stirring and standing, self-assembly was performed to obtain a DNA / NB-NPs@PDA@CS-NFs dispersion. The suspension was poured into a mold and placed in a -40°C freezer overnight. The DNA / NB-NPs@PDA@CS-NFs sponge aerogel was obtained by freeze-drying in a vacuum freeze dryer.

[0031] like Figure 1-3 As shown, the pore size of the sponge aerogel sample was concentrated in the range of 1-5 nm, with some areas in the range of 4-6 nm, and the average pore size was 1.7 nm, as determined by nitrogen adsorption-desorption curves.

[0032] like Figure 4 As shown, the mechanical properties of the aerogel were tested by measuring the stress-strain curves of the sponge aerogel under different compression deformations. The DNA / NB-NPs@PDA@CS-NFs aerogel exhibits strong recovery properties, maintaining excellent compression resilience even under 33 kPa pressure during 80% deformation compression.

[0033] Different concentrations of R6G dye aqueous solutions were added dropwise to DNA / NB-NPs@PDA@CS-NFs sponge aerogels, followed by Raman spectroscopy analysis. The enhancement factor of the substrate material was found to be 5.4 × 10⁻⁶. 7 The detection limit reaches 10. -8 The linear relationship between the signal peak intensity and concentration of R6G dye aqueous solution of different concentrations reached 0.9621.

[0034] The medicinal herb Phellodendron amurense was stained with different concentrations of auramine O dye.

[0035] DNA / NB-NPs@PDA@CS-NFs sponge aerogel was used to wipe the surface of stained Chinese medicinal materials, followed by Raman spectroscopy, with a detection limit as low as 10. -5 The linear relationship between the signal peak intensity and concentration of auramine O dye at different concentrations reached 0.949 mg / ml, and the relative standard deviation of repeated tests reached 9.28%.

[0036] The selectivity of the sponge aerogel substrate material for dyes with different charges was not detected. Sunset Yellow (SY, negative charge) and Methylene Blue (MB, positive charge) were selected as research subjects, and 1×10 -6 When SY / MB aqueous solutions of equal concentrations were mixed, the pH was adjusted to 4 and 11 by adding 0.1M HCl or NaOH, respectively. It was found that at pH=4 and pH=11, the signal peak intensity of SY was 100 times that of MB. At pH=11 and pH=4, the signal peak intensity of MB was 50 times that of MB.

Claims

1. A sponge-like chitosan composite material, wherein the sponge-like chitosan composite material is composed of chitosan nanofibers (CS-NFs), noble metal nanoparticles (NB-NPs), polydopamine (PDA), and physically cross-linked DNA molecules; characterized in that: The CS-NFs have a length of 200 nm to 3 μm, and the NB-NPs have a particle size of 10-100 nm. PDA is coated on the surface of CS-NFs to form a PDA@CS-NFs structure, and NB-NPs are coated on the outside of PDA@CS-NFs to form an NB-NPs@PDA@CS-NFs structure. They are cross-linked with DNA molecules and self-assembled to form a sponge-like DNA / NB-NPs@PDA@CS-NFs composite material.

2. The sponge-like chitosan composite material according to claim 1, characterized in that: The spongy DNA / NB-NPs@PDA@CS-NFs composite material contains 5-10% NB-NPs by mass and 1-3% DNA by mass.

3. A method for preparing a sponge-like chitosan composite material as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Disperse CS-NFs in a solution containing dopamine hydrochloride and Tris-HCl buffer, homogenize by sonication, let stand for 24 hours, and dialyze to obtain PDA@CS-NFs dispersion. Step 2: Add silver nitrate or chloroauric acid dropwise to the PDA@CS-NFs dispersion separately, or add them dropwise to the PDA@CS-NFs dispersion sequentially, and then add sodium borohydride for reduction. Dialyze to obtain the NB-NPs@PDA@CS-NFs dispersion. Step 3: Concentrate the NB-NPs@PDA@CS-NFs dispersion to a mass concentration of 15-25%, then add DNA, stir and let stand to perform self-assembly to obtain DNA / NB-NPs@PDA@CS-NFs dispersion, and then freeze-dry to obtain sponge-like DNA / NB-NPs@PDA@CS-NFs composite material.

4. A sponge-like chitosan SERS substrate material, characterized in that: It is made using the spongy chitosan composite material of claim 1 or 2.

5. The application of the sponge-like chitosan SERS substrate material as described in claim 4, characterized in that: The spongy chitosan SERS substrate material is used as a flexible SERS substrate material for the identification of Chinese medicinal materials.

6. The application of the sponge-like chitosan SERS substrate material as described in claim 5, characterized in that: The surface of Chinese medicinal materials is wiped with a flexible SERS substrate or the soaking solution of the Chinese medicinal materials is dropped onto the surface of the flexible SERS substrate, and then Raman spectroscopy is performed for identification.

7. The application of the sponge-like chitosan SERS substrate material as described in claim 5, characterized in that: Before use, an alkaline or acidic buffer solution is sprayed onto the spongy chitosan SERS substrate material to improve the capture ability of cationic or anionic dyes in the dyeing Chinese medicinal materials.

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