A sers substrate prepared by using diatomite and a preparation method and application thereof

Abstract

The application discloses a method for preparing a SERS substrate by using diatomite, and comprises the following steps: (1) taking diatomite, adding metal salt, polyvinylpyrrolidone and ultrapure water, uniformly mixing, then adding o-dihydroxybenzene, and stirring at room temperature to form a diatomite-metal polyphenol network structure; (2) centrifuging the diatomite-metal polyphenol network structure, removing supernatant, washing, adding ultrapure water, and obtaining a diatomite-metal polyphenol network structure dispersion liquid; (3) adding 10x 3-morpholinopropanesulfonic acid buffer solution into the diatomite-metal polyphenol network structure dispersion liquid, uniformly mixing, adding chloroauric acid, vortexing, centrifuging, removing excess chloroauric acid, and diluting to obtain a SERS substrate. The method prepares MPN-Dia-Au with reducibility, the material has a high detection limit for common contaminative dyes, has a good detection and distinguishing ability for foodborne pathogenic microorganisms, and has a simple preparation process and low cost.

Description

Technical Field

[0001] This invention belongs to the field of surface-enhanced Raman scattering (SERS) technology, specifically relating to a SERS substrate prepared using diatomaceous earth, its preparation method, and its application. Background Technology

[0002] The increasingly broad field of nanomaterials research has led to the rapid development of nanotechnology, and research on gold nanoparticles has also gradually increased. Therefore, a thorough understanding of the synthesis and properties of gold nanoparticles will help people better develop and utilize gold nanomaterials. Gold nanoparticles occupy an extremely important position in research fields such as surface-enhanced Raman spectroscopy, surface-enhanced resonance reflection spectroscopy, and molecular biology spectroscopy, and are important materials for basic research. In order to better apply Raman spectroscopy and SERS technology in these fields, an important requirement is to fabricate a rationally designed, highly stable, reliable, and repeatable plasma matrix that can generate dense local electromagnetic (EM) fields, i.e., SERS "hot spots".

[0003] Methylene blue (MB), chemical formula C 16 H 18 N3ClS, a phenothiazine salt, is a dark green, bronze-lustered crystal or powder, soluble in water and ethanol, but insoluble in ethers. Methylene blue is relatively stable in air, but its aqueous solution is alkaline and toxic. It is widely used in aquaculture and transportation to treat fish diseases such as red mouth disease, septicemia, and ichthyosis. However, excessive use of MB can cause severe vomiting, shock, and limb paralysis in humans. Therefore, the EU and the US have banned the use of MB to treat aquatic diseases. Methylene blue is also widely used as a chemical indicator, dye, biological stain, and pharmaceutical.

[0004] Food safety is one of the most pressing public concerns. Foodborne pathogens, such as Listeria monocytogenes, Escherichia coli, Salmonella spp., and Campylobacter jejuni, are widespread and closely associated with disease outbreaks. Although improvements in food processing technology and strict industrial hygiene practices have limited the pathogenicity of these microorganisms, outbreaks of foodborne illnesses continue. Food spoilage caused by these microorganisms and their active substances results in significant economic losses to the food industry. Therefore, the ability to effectively detect disease-causing and spoilage-causing microorganisms in raw materials, food processing environments, and final products is crucial for preventing large-scale outbreaks of foodborne diseases and resource losses.

[0005] Existing technologies for detecting various dye pollutants mostly utilize reducing agents such as citric acid and sodium borohydride to reduce chloroauric acid under certain conditions, preparing core-shell structures or core-shell structures forming films between two phases for detecting common dye pollutants. For example, the article with patent number CN 115106096 A synthesized a magnetic iron tetroxide / manganese dioxide core-shell structure material, which showed good removal effect of methylene blue in water and was safe and stable; however, the detection limit issue involved was not mentioned.

[0006] The main drawbacks of existing technologies include the following:

[0007] 1) The detection limit for common polluting dyes is low, and the detection results are unstable;

[0008] 2) The preparation method is complex and the process is cumbersome, requiring high levels of skill from the operators;

[0009] 3) The substrate materials used for dye detection are rarely capable of detecting bacteria.

[0010] There are currently no reports on diatomaceous earth as a good SERS substrate for dye detection, and this invention aims to conduct in-depth research in this area. Summary of the Invention

[0011] The purpose of this invention is to provide a method for preparing SERS substrates using diatomaceous earth. This method prepares a reducing diatomaceous earth-metal polyphenol network (MPN-Dia) structure MPN-Dia-Au. This material has a high detection limit for common polluting dyes, a good ability to detect and distinguish foodborne pathogens, and the preparation process is simple and low in cost.

[0012] Another objective of this invention is to provide a SERS substrate prepared using the above method.

[0013] The final objective of this invention is to provide the application of the above-mentioned SERS substrate in the preparation of detection reagents for dye residues in water, dye residues in textiles, and foodborne pathogens.

[0014] The first objective of this invention can be achieved through the following technical solution: a method for preparing SERS substrates using diatomaceous earth, comprising the following steps:

[0015] (1) Take diatomaceous earth (Dia), add metal salt, polyvinylpyrrolidone (PVP) and ultrapure water, mix well, then add catechol, stir at room temperature until the generated metal polyphenol network structure (MPN) is fully attached to the surface and internal pores of diatomaceous earth (Dia), forming diatomaceous earth-metal polyphenol network structure (MPN-Dia).

[0016] (2) Centrifuge the diatomaceous earth-metal polyphenol network structure (MPN-Dia), remove the supernatant, wash, add ultrapure water, so that the diatomaceous earth-metal polyphenol network structure (MPN-Dia) is evenly distributed in the ultrapure water to obtain the diatomaceous earth-metal polyphenol network structure (MPN-Dia) dispersion.

[0017] (3) Add 10×3-morpholine propanesulfonic acid (MOPS) buffer to the diatomaceous earth-metal polyphenol network structure (MPN-Dia) dispersion, mix well, add chloroauric acid, vortex, centrifuge, remove excess chloroauric acid, and dilute to obtain SERS substrate (MPN-Dia-Au).

[0018] This invention synthesizes and designs a reducing MPN-Dia structure. When the metal polyphenol network structure (MPN) is attached to the surface and pores of diatomaceous earth (Dia), the originally non-reducible diatomaceous earth acquires a certain reducing ability due to the attachment of MPN. Adding a certain concentration of chloroauric acid causes in-situ reduction to gold nanoparticles. Because the natural pores contain very small gaps, sufficient "hot spots" are formed between particles. Therefore, this material MPN-Dia-Au exhibits excellent detection performance for methylene blue (MB) and IR-780, with detection limits of 10⁻⁶ and 10⁻⁶, respectively. -9 and 10 -11 It also has a good ability to detect and distinguish foodborne pathogens. In addition, raw materials such as diatomaceous earth are easy to obtain, inexpensive, and pollution-free.

[0019] In this method for preparing SERS substrates using diatomaceous earth:

[0020] Preferably, in step (1), the dosage relationship of diatomaceous earth, metal salt, polyvinylpyrrolidone, ultrapure water and catechol is 50-120 mg; 100 μL: 500 μL: 4.3-4.7 mL: 100 μL; wherein the concentration of the metal salt is 8-15 mg / mL, the concentration of the polyvinylpyrrolidone is 20-40 mg / mL, and the concentration of the catechol is 35-45 mg / mL.

[0021] In step (1), the metal salt reacts with catechol to generate a metal polyphenol network structure (MPN). All materials are added in the order of diatomaceous earth, metal salt, polyvinylpyrrolidone, ultrapure water and catechol to generate a metal polyphenol network structure (MPN) on the surface of diatomaceous earth.

[0022] Preferably, the metal salt in step (1) is FeCl3·6H2O.

[0023] This invention is applicable to a variety of natural polyphenols, not limited to tannic acid, but also including other catechols such as catechins, gallic acid, etc.

[0024] Preferably, the catechol in step (1) is tannic acid, catechol or gallic acid.

[0025] Preferably, during centrifugation in step (2), the rotation speed is 200-500 rpm and the number of washing cycles is 1-3.

[0026] Preferably, in step (3), the ratio of the amount of diatomaceous earth-metal polyphenol network structure (MPN-Dia) dispersion, 10×3-morpholine propanesulfonic acid (MOPS) buffer to chloroauric acid is 100:1:0.6-0.8, wherein the concentration of chloroauric acid is 100mM.

[0027] More preferably, in step (3), the ratio of the amount of diatomaceous earth-metal polyphenol network structure (MPN-Dia) dispersion, 10×2,3-morpholine propanesulfonic acid (MOPS) buffer to chloroauric acid is 100:1:0.7, wherein the concentration of chloroauric acid is 100mM.

[0028] Preferably, in step (3), the vortexing time is 10-15 min, the centrifugation speed is 3000 rpm, and the centrifugation time is 15-20 min.

[0029] Preferably, in step (3), ultrapure water is used to dilute the diatomaceous earth-metal polyphenol network structure (MPN-Dia) dispersion to its original volume.

[0030] This invention utilizes the reducing properties of MPN attached to the surface of diatomaceous earth to reduce chloroauric acid of a certain concentration on the surface and pores of diatomaceous earth, thereby preparing an MPN-Dia-Au structure with reducing properties.

[0031] The second objective of the present invention can be achieved by the following technical solution: a SERS substrate prepared using diatomaceous earth, obtained by any of the above methods.

[0032] The last objective of the present invention can be achieved by the following technical solution: the application of the above-mentioned SERS substrate in the preparation of detection reagents for dye residues in water, dye residues in textiles, and foodborne pathogens.

[0033] Preferably, the material of the present invention can perform label-free detection of various dyes (IR-780, MB, R6G, etc.) through electrostatic adsorption.

[0034] Furthermore, the MPN-Dia-Au material synthesized in this invention serves as an excellent SERS substrate. Due to its extremely high detection limit, it can be applied to the detection of dye residues in water, the detection of dye residues in textiles, and the detection of foodborne bacteria residues in the cleanliness of various food packaging and tableware.

[0035] The present invention has the following advantages:

[0036] (1) This invention is the first to synthesize a novel MPN-Dia-Au material, which is widely available, simple to prepare, and low in cost;

[0037] (2) The present invention has a very low detection limit for a variety of dyes, such as methylene blue (MB) and IR-780, and can also detect and distinguish two common foodborne pathogens, such as Escherichia coli and Staphylococcus aureus. Attached Figure Description

[0038] Figure 1 The different amounts of chloroauric acid used in Example 1 correspond to a concentration of 10. -5 The test results of M's IR-780;

[0039] Figure 2 These are representative scanning electron microscope images of different morphologies of diatomaceous earth in Embodiment 2 of the present invention. Images A and B show diatomaceous earth with two-dimensional periodic pores. Image C shows the pore diameter of the diatomaceous earth after calculation using Image J, which is approximately 559.1 ± 131.3 nm. Image D shows the surface of the diatomaceous earth as very clean when observed under high magnification.

[0040] Figure 3 This is a scanning electron microscope image of AuNPs on the surface of MPN-Dia after reduction of chloroauric acid prepared in Example 2 of this invention;

[0041] Figure 4 This is the surface-enhanced Raman scattering (SERS) spectrum of MB prepared by MPN-Dia after reducing chloroauric acid in Example 2 of this invention. The spectrum shows that the detection limit of MPN-Dia-Au for MB can reach 10. -9 M;

[0042] Figure 5 This is the surface-enhanced Raman scattering (SERS) spectrum of MPN-Dia-Au after reduction of chloroauric acid in Example 2 of this invention, measured on IR-780. The spectrum shows that the detection limit of MPN-Dia-Au for MB can reach 10. -11 M;

[0043] Figure 6This is a surface-enhanced Raman scattering (SERS) spectrum of the MPN-Dia prepared in Example 2 of this invention after reducing chloroauric acid to foodborne pathogens SA (Staphylococcus aureus) and E. coli (Escherichia coli). Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only descriptive and not limiting, and should not be used to limit the scope of protection of the present invention. Unless otherwise specified, the test methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0045] Example 1

[0046] Accurately weigh 100 mg of diatomaceous earth. First, take 100 μL of FeCl3·6H2O (12 mg / mL) and 500 μL of polyvinylpyrrolidone (PVP) (30 mg / mL). Then add 4.7 mL of ultrapure water and turn on the magnetic stirrer (low speed, 150–300 rpm, 200 rpm in this example). Next, add 100 μL of tannic acid (40 mg / mL) and stir at room temperature for 12 h until the generated metal polyphenol network structure MPN is fully attached to the surface and internal pores of MPN, forming the diatomaceous earth-metal polyphenol network structure MPN-Dia.

[0047] The prepared MPN-Dia was centrifuged at 300 rpm for 5 minutes. After removing the supernatant, it was washed 3 times with ultrapure water. 2 mL of ultrapure water was added to the system to ensure that the MPN-Dia material was evenly distributed in the ultrapure water for later use.

[0048] Take 200 μL of the prepared MPN-Dia into an EP tube, add 2 μL of 10×3-morpholinopropanesulfonic acid (MOPS) buffer to maintain the stability of MPN in the system, mix thoroughly, and then add 1.0 μL, 1.2 μL, 1.4 μL, and 1.6 μL of 100 mM chloroauric acid, respectively. Vortex for about 10 min to complete the synthesis. Centrifuge at 3000 rpm for 15 min to remove excess chloroauric acid from the reaction system, and dilute back to the original volume of 200 μL to obtain a series of SERS substrates (MPN-Dia-Au).

[0049] Different amounts of chloroauric acid affect the concentration of 10 -5 The test results of M's IR-780 are as follows Figure 1 As shown, from Figure 1 As can be seen, adding 1.4 μL of 100 mM chloroauric acid (i.e., a concentration of 0.7 mM) had the best effect. Therefore, the experimental group with 1.4 μL of chloroauric acid was selected for subsequent experiments.

[0050] Example 2

[0051] The other steps are the same as in Example 1.

[0052] Take 200 μL of the prepared MPN-Dia into an EP tube, add 2 μL of 10×3-morpholinopropanesulfonic acid (MOPS) to maintain the stability of MPN in the system, mix thoroughly, then add 1.4 μL of 100 mM chloroauric acid, vortex for about 10 min. The solution changes from the grayish-white color of MPN-Au to purple-red, indicating the end of the synthesis. Centrifuge at 3000 rpm for 15 min to remove excess chloroauric acid from the reaction system, dilute back to the original volume of 200 μL, and obtain the SERS substrate (MPN-Dia-Au).

[0053] This embodiment utilizes the natural porous structure of diatomite. Representative scanning electron microscope images of different morphologies of diatomite are shown below. Figure 2 As shown, Figures A and B show diatomaceous earth with two-dimensional periodic pores. Figure C shows the pore diameter of the diatomaceous earth after calculation using ImageJ, which is approximately 559.1 ± 131.3 nm. Figure D shows the surface of the diatomaceous earth as very clean when observed under a high-power microscope.

[0054] Scanning electron microscopy images of the SERS substrate (MPN-Dia-Au) are shown below. Figure 3 As shown, AuNPs are present on the surface of MPN-Dia after the reduction of chloroauric acid.

[0055] Therefore, this embodiment, for the first time, combines diatomaceous earth (hereinafter referred to as Dia) with a metal polyphenol network structure (MPN) to synthesize and design a reducing MPN-Dia structure. When MPN adheres to the surface and pores of diatomaceous earth, the originally non-reducible diatomaceous earth acquires a certain reducing ability due to the adhesion of MPN. Adding a certain concentration of chloroauric acid causes in-situ reduction to gold nanoparticles. Because the natural pores contain very small gaps, sufficient "hot spots" (such as gold nanoparticles) are formed between the particles. Figure 3 ).

[0056] Take the optimized MPN-Dia-Au obtained above, which is MPN-Dia-Au obtained from 0.7 mM chloroauric acid. Accurately pipette 20 μL of the prepared MPN-Dia-Au and add 20 μL of ultrapure water to dilute it 1:1 (a fixed dilution ratio; 1:2 and 1:3 are not as effective as 1:1). After thorough mixing, take 2 μL and drop it onto an aluminum plate to air dry. After the MPN-Dia-Au has air-dried, drop 2 μL of MB and IR-780 of the corresponding concentrations onto it for detection. Use the same method to detect Staphylococcus aureus and Escherichia coli, and then perform SERS detection.

[0057] Surface-enhanced Raman scattering (SERS) spectra of MB, such as Figure 4 As shown in the spectrum, the detection limit of MPN-Dia-Au for MB can reach 10. -9 M (mol / L).

[0058] Surface-enhanced Raman scattering (SERS) spectrum of IR-780 as follows Figure 5 As shown in the spectrum, the detection limit of MPN-Dia-Au for MB can reach 10. -11 M.

[0059] The surface-enhanced Raman scattering (SERS) spectra of SA (Staphylococcus aureus) and E. coli (Escherichia coli) are as follows: Figure 6 As shown, from Figure 6 As can be seen from the results, the material MPN-Dia-Au has a good ability to detect and distinguish foodborne pathogens.

[0060] The results showed that the material MPN-Dia-Au had excellent detection performance for methylene blue (MB) and IR-780, with detection limits of 10 Ω and 10 Ω, respectively. -9 and 10 -11 (like Figure 4 , 5 It also has a good ability to detect and distinguish foodborne pathogens (such as...). Figure 6 In addition, raw materials such as diatomaceous earth are easy to obtain, inexpensive, and pollution-free.

[0061] Therefore, the SERS substrate prepared in this embodiment can be used in the preparation of detection reagents for dye residues in water, dye residues in textiles, and foodborne pathogens.

[0062] Example 3

[0063] Unlike Example 1, gallic acid was used instead of catechol.

[0064] For those skilled in the art, modifications to the concepts and technical points addressed in this invention, and the corresponding changes, should fall within the scope of the claims made herein. This invention is not limited to the specific embodiments described above; these embodiments are merely for illustrating the use of the invention in detail, and equivalent production methods and technical details are also part of the invention. In fact, those skilled in the art, based on the foregoing description, can find different adjustments according to their own needs, and these adjustments should all fall within the scope of the appended claims.

Claims

1. A method for preparing SERS substrates using diatomaceous earth, characterized in that: Includes the following steps: (1) Take diatomaceous earth, add metal salt, polyvinylpyrrolidone and ultrapure water, mix well, then add catechol, stir at room temperature until the generated metal polyphenol network structure is fully attached to the surface and internal pores of diatomaceous earth, forming a diatomaceous earth-metal polyphenol network structure. (2) Centrifuge the diatomaceous earth-metal polyphenol network structure, remove the supernatant, wash, add ultrapure water, so that the diatomaceous earth-metal polyphenol network structure is evenly distributed in the ultrapure water, and obtain the diatomaceous earth-metal polyphenol network structure dispersion. (3) Add 10×3-morpholine propanesulfonic acid (MOPS) buffer to the diatomaceous earth-metal polyphenol network structure dispersion, mix well, add chloroauric acid, vortex, centrifuge, remove excess chloroauric acid, and dilute to obtain SERS substrate.

2. The method for preparing SERS substrates using diatomaceous earth according to claim 1, characterized in that: In step (1), the dosage relationship of diatomaceous earth, metal salt, polyvinylpyrrolidone, ultrapure water and catechol is 50-120 mg; 100 μL: 500 μL: 4.3-4.7 mL: 100 μL; wherein the concentration of the metal salt is 8-15 mg / mL, the concentration of the polyvinylpyrrolidone is 20-40 mg / mL, and the concentration of the catechol is 35-45 mg / mL.

3. The method for preparing SERS substrates using diatomaceous earth according to claim 1, characterized in that: The metal salt mentioned in step (1) is FeCl3·6H2O.

4. The method for preparing SERS substrates using diatomaceous earth according to claim 1, characterized in that: The catechol mentioned in step (1) is tannic acid, catechol or gallic acid.

5. The method for preparing SERS substrates using diatomaceous earth according to claim 1, characterized in that: In step (2), the centrifugation speed is 200-500 rpm, and the washing is performed 1-3 times.

6. The method for preparing SERS substrates using diatomaceous earth according to claim 1, characterized in that: In step (3), the volume ratio of the diatomaceous earth-metal polyphenol network structure dispersion, the 10×3-morpholine propanesulfonic acid buffer solution, and the chloroauric acid is 100:1:0.6-0.8, wherein the concentration of the chloroauric acid is 100mM.

7. The method for preparing SERS substrates using diatomaceous earth according to claim 1, characterized in that: In step (3), the vortexing time is 10-15 minutes, the centrifugation speed is 3000 rpm, and the centrifugation time is 15-20 minutes.

8. A SERS substrate prepared using diatomaceous earth, characterized in that: It is prepared by any one of the methods of claims 1-7.

9. The application of the SERS substrate according to claim 8 in the preparation of detection reagents for dye residues in water, dye residues in textiles, and foodborne pathogens.

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