Surface-enhanced raman substrate material, method for preparing the same, and use thereof

Abstract

This invention discloses a surface-enhanced Raman spectroscopy (SERS) substrate material, its preparation method, and its applications. The method includes the following steps: S1, extracting and pretreating silk material; S2, immersing the silk material in a chloroauric acid solution; S3, heating and reducing a mixture of silk material and chloroauric acid solution; S4, removing the silk material, washing, and drying to obtain the SERS substrate material. The SERS substrate of this invention significantly improves the detection sensitivity and selectivity for mucin through optimized nanoparticle distribution and porous structure. This invention uses silk fiber waste as a bio-substrate and utilizes sericin and fibroin for in-situ reduction, effectively reducing the use of toxic chemicals and improving the biocompatibility and environmental friendliness of the substrate. The preparation method of this invention is simple, low-cost, suitable for mass production, and convenient for on-site testing in resource-limited environments.

Description

Technical Field

[0001] This invention relates to the field of biosensors and detection technology, and in particular to a surface-enhanced Raman substrate material, its preparation method and application. Background Technology

[0002] Mucin is an important biomolecule, and its concentration variations in body fluids are closely related to various diseases, such as cancer and inflammation. Raman spectroscopy, especially surface-enhanced Raman scattering (SERS), has become an ideal tool for detecting mucin due to its high sensitivity and molecular specificity. However, traditional SERS substrate preparation often requires the use of chemical reducing agents, which may have adverse effects on the environment and biocompatibility.

[0003] With the development of green synthesis technologies, biosynthesized metal nanoparticles have attracted much attention due to their better biocompatibility. Sericin and silk fibroin from silkworm silk have been extensively studied due to their excellent biological properties. Sericin can serve as a reducing agent and stabilizer for gold nanoparticles (AuNPs), while silk fibroin, due to its porous structure and stability, is suitable for the green synthesis of silver nanoparticles (AgNPs). Furthermore, utilizing silkworm fiber waste (SFw) for green synthesis improves resource utilization and reduces the use of harmful chemicals. Therefore, using silk-based materials to prepare SERS substrates can not only improve their biocompatibility but also further optimize the detection sensitivity for mucin.

[0004] Raman spectroscopy, especially surface-enhanced Raman scattering (SERS), has become an ideal tool for detecting mucin due to its high sensitivity and molecular specificity. However, the preparation of traditional SERS substrates usually requires expensive precious metals and complex processes, and is not conducive to handling whole blood samples under mild conditions. Natural silk materials possess good biocompatibility and abundant three-dimensional porous structures, making them a potential substrate for novel SERS substrates. Summary of the Invention

[0005] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing a surface-enhanced Raman spectroscopy (SERS) substrate material, its preparation method, and its applications. This invention utilizes a silk substrate to achieve in-situ reduction and self-assembly of gold and silver nanoparticles under mild conditions. By controlling the concentrations of the precursor and reducing agent, as well as the pH value of the reaction, the distribution of gold and silver nanoparticles on the silk substrate material is optimized, resulting in a SERS substrate with excellent stability and sensitivity. Furthermore, the SERS substrate of this invention can sensitively and specifically detect mucin in whole blood samples, making it suitable for on-site testing in resource-constrained environments.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In its first aspect, the present invention provides a method for preparing a surface-enhanced Raman substrate material, comprising the following steps:

[0007] S1. Extract and pre-treat silk materials;

[0008] S2. The silk material is impregnated in chloroauric acid solution;

[0009] S3. The mixture of silk material and chloroauric acid solution is heated and reduced;

[0010] S4. Take out the silk material, clean and dry it to obtain the surface-enhanced Raman substrate material.

[0011] Preferably, step S1 specifically includes:

[0012] Silk fibroin or sericin is extracted from natural silk or silk fiber waste, cut into suitable sizes, and then washed and dried to obtain pretreated silk material.

[0013] Preferably, step S2 specifically involves: adding the silk material to a chloroauric acid solution and immersing it at room temperature.

[0014] Preferably, step S2 specifically involves adding silk material to a concentration of 1.5 × 10⁻⁶. -4 ~7.5×10 -4 In the chloroauric acid solution of M, the mass ratio of silk material to chloroauric acid solution is 1:50, and the silk material is immersed at room temperature for 30 minutes.

[0015] Preferably, step S3 specifically involves: heating the mixture of silk material obtained in step S2 and chloroauric acid solution in a water bath at 85°C with shaking for 30 minutes.

[0016] Preferably, step S4 specifically involves: removing the silk material from the chloroauric acid solution, rinsing it with deionized water, and drying it at room temperature to obtain a surface-enhanced Raman substrate material.

[0017] In a second aspect, the present invention provides a surface-enhanced Raman substrate material, which is prepared by the method described above.

[0018] A third aspect of the present invention provides the application of the surface-enhanced Raman substrate material as described above in the detection of mucin in whole blood samples.

[0019] Preferably, the application method is as follows:

[0020] An aptamer capable of specifically binding to mucin is modified onto the surface-enhanced Raman substrate material of claim 7 to obtain a SERS substrate;

[0021] The SERS substrate was incubated with the sample to be tested, and then the characteristic peaks of mucin were detected by Raman spectroscopy. The concentration of mucin was analyzed based on the intensity of the characteristic peaks.

[0022] The beneficial effects of this invention are:

[0023] This invention provides a surface-enhanced Raman spectroscopy substrate material, its preparation method, and its application, which have at least the following advantages:

[0024] 1. High sensitivity and selectivity: The SERS substrate of this invention significantly improves the detection sensitivity and selectivity for mucin through optimized nanoparticle distribution and porous structure.

[0025] 2. Green biosynthesis: This invention uses silk fiber waste as a biological substrate and utilizes sericin and fibroin for in-situ reduction, which effectively reduces the use of toxic chemicals and improves the biocompatibility and environmental friendliness of the substrate.

[0026] 3. Mild preparation conditions: The preparation process of this invention is carried out at near-neutral pH and room temperature, which is suitable for processing temperature-sensitive biological samples and ensures that the biomolecules in the samples will not denature or degrade.

[0027] 4. Economic and practical: The preparation method of the present invention is simple, low in cost, suitable for mass production, and convenient for on-site testing in resource-limited environments. Attached Figure Description

[0028] Figure 1 The image shows a TEM image of the surface-enhanced Raman substrate material prepared in Example 1. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0030] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0031] 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. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0032] Example 1

[0033] A surface-enhanced Raman spectroscopy substrate material, the preparation method of which includes the following steps:

[0034] S1. Extract and pre-treat silk materials;

[0035] Silk fibroin is extracted from silk fiber waste (SFw), cut into suitable sizes, and then washed and dried to obtain pretreated silk material.

[0036] S2. The silk material is impregnated in chloroauric acid solution;

[0037] Silk materials were added to chloroauric acid (HAuCl4) solutions of different concentrations (1.5 × 10⁻⁶). -4 M, 2.5 × 10 -4 M, 3.0×10 -4 M, 4.5 × 10 -4 M, 6.0×10 -4 M and 7.5×10 -4 M), controlling the mass ratio of silk material to chloroauric acid solution to 1:50, and immersing at room temperature for 30 minutes to adsorb AuCl4. - Ions cause the silk to gradually turn light yellow.

[0038] S3, In-situ heating and reduction:

[0039] The mixture of silk material obtained in step S2 and chloroauric acid solution was heated with shaking in a water bath at 85°C for 30 minutes. After the heating reaction was completed, the color of the silk sample changed from light yellow to red or brown, indicating that gold nanoparticles had been generated in situ on the silk fibers.

[0040] S4. Remove the silk material from the chloroauric acid solution, rinse with deionized water, and air dry at room temperature to obtain surface-enhanced Raman substrate materials with different gold particle sizes.

[0041] During the reaction, gold and silver nanoparticles self-assemble on the three-dimensional porous structure of silk material, forming a uniform SERS-active substrate. Au NPs uniformly attached to paper fibers can generate dense hot spots on the fibers. By equipping appropriate aptamers, this SERS-active substrate can quantitatively detect the cancer biomarker Mucin-1 in whole blood samples.

[0042] I. Structural Characterization

[0043] Reference Figure 1 This is a TEM image of the surface-enhanced Raman substrate material prepared in this embodiment.

[0044] II. Mucin detection:

[0045] An aptamer capable of specifically binding to mucin is modified onto a surface-enhanced Raman spectroscopy (SERS) substrate to obtain an SERS substrate. The specific method is as follows:

[0046] The SERS substrate was incubated with the whole blood sample to be tested, and then the characteristic peaks of mucin were detected by Raman spectroscopy. The concentration of mucin was analyzed based on the intensity of the characteristic peaks.

[0047] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for preparing a surface-enhanced Raman spectroscopy substrate material, characterized in that, Includes the following steps: S1. Extract and pre-treat silk materials; S2. The silk material is impregnated in chloroauric acid solution; S3. The mixture of silk material and chloroauric acid solution is heated and reduced; S4. Take out the silk material, clean and dry it to obtain the surface-enhanced Raman substrate material.

2. The method for preparing the surface-enhanced Raman substrate material according to claim 1, characterized in that, Step S1 is as follows: Silk fibroin or sericin is extracted from natural silk or silk fiber waste, cut into suitable sizes, and then washed and dried to obtain pretreated silk material.

3. The method for preparing the surface-enhanced Raman substrate material according to claim 1, characterized in that, Step S2 specifically involves adding the silk material to a chloroauric acid solution and immersing it at room temperature.

4. The method for preparing the surface-enhanced Raman substrate material according to claim 3, characterized in that, Step S2 specifically involves adding silk material to a concentration of 1.5 × 10⁻⁶. -4 ~7.5×10 -4 In the chloroauric acid solution of M, the mass ratio of silk material to chloroauric acid solution is 1:50, and the silk material is immersed at room temperature for 30 minutes.

5. The method for preparing the surface-enhanced Raman substrate material according to claim 1, characterized in that, Step S3 specifically involves heating the mixture of silk material obtained in step S2 and chloroauric acid solution in a water bath at 85°C with shaking for 30 minutes.

6. The method for preparing the surface-enhanced Raman substrate material according to claim 1, characterized in that, Step S4 specifically involves: removing the silk material from the chloroauric acid solution, rinsing it with deionized water, and air-drying it at room temperature to obtain the surface-enhanced Raman substrate material.

7. A surface-reinforced Raman substrate material, characterized in that, It is prepared by the method described in any one of claims 1-6.

8. The application of the surface-enhanced Raman substrate material as described in claim 7 in the detection of mucin in whole blood samples.

9. The application according to claim 8, characterized in that, The application method is as follows: An aptamer capable of specifically binding to mucin is modified onto the surface-enhanced Raman substrate material of claim 7 to obtain a SERS substrate; The SERS substrate was incubated with the whole blood sample to be tested, and then the characteristic peaks of mucin were detected by Raman spectroscopy. The concentration of mucin was analyzed based on the intensity of the characteristic peaks.

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