SERS (Surface Enhanced Raman Scattering) substrate for detecting pesticide residues as well as preparation method and application thereof

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) substrate for detecting pesticide residues as well as a preparation method and application thereof, belongs to the technical field of analysis and detection, and provides a preparation method of the SERS substrate for detecting the pesticide residues, which comprises the following steps: inoculating acetobacter xylinum into a culture medium, culturing to obtain a bacterial cellulose membrane, treating the bacterial cellulose membrane with a sodium hydroxide aqueous solution, and drying to obtain the SERS substrate for detecting the pesticide residues. Obtaining a bacterial cellulose substrate; mixing the metal salt aqueous solution with the reducing agent aqueous solution to obtain a precursor solution; and soaking the bacterial cellulose substrate in the precursor solution, standing in a dark place to obtain a bacterial cellulose substrate adsorbed with a precursor, carrying out low-temperature plasma treatment, brewing with ultrapure water, and drying to obtain the SERS substrate. According to the SERS substrate, bacterial cellulose serves as a supporting material, surface modification and nano-structure regulation and control are achieved through a low-temperature plasma technology, the high-density SERS substrate is prepared, and the requirement for rapid and sensitive detection of imidacloprid pesticide residues can be met.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a SERS substrate for detecting pesticide residues, a preparation method thereof, and an application thereof. Background Art

[0002] Imidacloprid is a widely used neonicotinoid insecticide, which is widely used in agricultural production due to its high efficiency and broad spectrum. However, the overuse of imidacloprid has led to increasingly serious residue problems in the environment. Imidacloprid has strong water solubility and persistence, and is easily diffused through soil and water bodies, causing toxicity to non-target organisms (such as bees and aquatic organisms). Research shows that imidacloprid has significant toxicity to the nervous system of bees, which may lead to colony collapse disorder (CCD), thereby affecting ecological balance and agricultural production. In addition, imidacloprid residues may enter the human body through the food chain, and long-term exposure may cause health problems such as neurotoxicity and immunosuppression. Therefore, developing a rapid and sensitive detection method for imidacloprid is of great significance for ensuring food safety and ecological environment.

[0003] Currently, the conventional methods for detecting imidacloprid mainly include high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and enzyme-linked immunosorbent assay (ELISA). Although these methods have high accuracy, they have disadvantages such as expensive equipment, complex operation, and long time consumption, and are difficult to meet the requirements of on-site rapid detection.

[0004] Surface-enhanced Raman scattering (SERS) technology is a spectral analysis technology based on the local electromagnetic field enhancement effect on the surface of nanostructures, which can significantly amplify the Raman signals of adsorbed molecules, thereby achieving single-molecule-level detection sensitivity. The core lies in the design and preparation of the SERS substrate, and the performance of the substrate directly determines the detection sensitivity, stability, and reproducibility. The preparation of noble metal nanostructures can be directly created on the substrate through lithography technology. However, lithography technology is hindered by the necessity of long-time treatment in a vacuum environment in practical applications. In addition, in-situ growth and electrochemical deposition are also used for the construction of the substrate surface, but they are not easy to control, and the manufactured nanostructures often face the problem of uniformity. The directly synthesized noble metal nanocolloids require reagents such as surfactant CTAB, which is toxic.

[0005] Chinese invention patent CN115612156A discloses an Ag NPs-BCM substrate for SERS detection of cancer markers, a preparation method thereof, and an application thereof. This method uses the silver mirror reaction to grow silver nanoparticles on a bacterial cellulose membrane, but ammonia water has strong irritation to eyes, nose, and skin. Compared with the above method, the method of reducing gold nanoparticles by low-temperature plasma technology is simple in operation, does not require toxic reducing agents, and has the characteristics of green and high efficiency. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a SERS substrate for detecting pesticide residues, its preparation method and application. The SERS substrate uses bacterial cellulose as a support material, and surface modification and nanostructure regulation are achieved through low-temperature plasma technology. The high-energy state composed of ions, electrons, and free radicals of low-temperature plasma is used to drive the in-situ growth of metal precursors on bacterial cellulose (BNC), load high-density metal nanoparticles, and prepare a high-density SERS substrate. The preparation method has the advantages of environmental friendliness, low cost, and simple process. The prepared SERS substrate can meet the requirements for rapid and sensitive detection of imidacloprid pesticide residues.

[0007] To achieve the above object, the present invention provides a preparation method of a SERS substrate for detecting pesticide residues, comprising the following steps:

[0008] (1) Acetobacter xylinum is inoculated into a culture medium, cultured to obtain a bacterial cellulose membrane, and the bacterial cellulose membrane is soaked in an aqueous sodium hydroxide solution and boiled to obtain a bacterial cellulose substrate;

[0009] (2) An aqueous metal salt solution and an aqueous reducing agent solution are mixed to obtain a precursor solution;

[0010] (3) The bacterial cellulose substrate obtained in step (1) is soaked in the precursor solution obtained in step (2), allowed to stand in the dark, and the bacterial cellulose substrate adsorbed with the precursor is obtained. After low-temperature plasma treatment, rinsed with ultrapure water and dried to obtain the SERS substrate.

[0011] Preferably, the inoculation amount of Acetobacter xylinum in step (1) is calculated according to 8-12% of the volume of the culture medium; the temperature of the culture in step (1) is 28-30 °C, and the culture time is 7-14 d.

[0012] Preferably, the formula of the culture medium in step (1) is: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water.

[0013] Preferably, the mass concentration of the aqueous sodium hydroxide solution in step (1) is 0.5-1.5%; the boiling time in step (1) is 50-70 min.

[0014] Preferably, the concentration of the aqueous metal salt solution in step (2) is 1-10 mM, and the aqueous metal salt solution is one or more of an aqueous chloroauric acid solution, an aqueous silver nitrate solution, and an aqueous palladium chloride solution; the concentration of the aqueous reducing agent solution in step (2) is 0.5-20 mM, and the aqueous reducing agent solution is an aqueous sodium citrate solution and / or an aqueous ascorbic acid solution; the aqueous metal salt solution and the aqueous reducing agent solution are mixed in equal volume.

[0015] Preferably, the time for the light-shielding static placement in step (3) is 10 to 60 min.

[0016] Preferably, the working voltage of the low-temperature plasma treatment in step (3) is 20 to 40 V, the time of the low-temperature plasma treatment is 1 to 7 min, the working gas of the low-temperature plasma treatment is one or more of air, nitrogen, helium, and argon, the form of the low-temperature plasma treatment is one of dielectric barrier discharge, glow discharge, radio frequency discharge, corona discharge, and sliding arc discharge, and when the low-temperature plasma treatment is carried out, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbing the precursor is 0.1 to 2 cm.

[0017] The present invention also provides the SERS substrate prepared by the preparation method.

[0018] The present invention also provides the application of the SERS substrate in detecting imidacloprid.

[0019] The present invention also provides a method for detecting imidacloprid. The SERS substrate is immersed in 10 to 100 μL of the sample to be detected, and a Raman spectrometer is used to collect a spectrum to calculate the concentration of imidacloprid in the sample to be detected.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] 1. Green and environmentally friendly: The present invention adopts the low-temperature plasma technology and does not need to use toxic and harmful chemical reducing agents (such as sodium borohydride, etc.), avoiding the environmental pollution problems brought by the traditional chemical reduction method. At the same time, as a natural biological material, the bacterial cellulose substrate has degradability, further improving the environmental friendliness of the method.

[0022] 2. Simple process and low cost: The present invention uses low-temperature plasma to complete the reduction of metal ions and the construction of nanostructures in one step, without complex equipment and expensive raw materials. The process is simple and easy to realize large-scale production, significantly reducing the time cost and production cost.

[0023] 3. Excellent substrate performance: The SERS substrate prepared based on low-temperature plasma in the present invention has high sensitivity. After the bacterial cellulose loaded with gold nanoparticles is dried, the volume is reduced, and the gold nanoparticles are more densely packed to form high-density "hot spots", significantly enhancing the Raman signal. Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 Test result diagrams of the SERS substrates prepared in Examples 1 to 12 using R6G as the Raman signal molecule;

[0026] Figure 2 SEM diagram of the SERS substrate prepared in Example 1, with the scale bar being 3 μm;

[0027] Figure 3 Elemental analysis diagram of the SERS substrate prepared in Example 1;

[0028] Figure 4 Standard curve diagram drawn for the SERS substrate prepared in Example 1 with different concentrations of R6G at the characteristic peak of 1510 cm -1 ;

[0029] Figure 5 Effect diagram for verifying the uniformity of the SERS substrate prepared in Example 1 using R6G as the Raman signal molecule;

[0030] Figure 6 Raman spectrogram of imidacloprid tested on the SERS substrate prepared in Example 1. Detailed implementation manners

[0031] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of this invention, various modifications and variations can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0035] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] The formula of the culture medium is: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. The culture medium is sterilized at 121 °C for 30 min.

[0038] (1) Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) is inoculated into the culture medium and cultured at 30 °C for 10 d. It is rinsed and soaked with distilled water, and squeezed to discharge the bacterial liquid to obtain a bacterial cellulose membrane. The bacterial cellulose membrane is soaked in a 1% sodium hydroxide aqueous solution and boiled for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. It is rinsed with deionized water until the eluate is neutral, soaked with ultrapure water, and the bacterial cellulose membrane is milky white and translucent. The water is blotted dry with filter paper to obtain a bacterial cellulose substrate;

[0039] (2) An aqueous solution of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and an aqueous solution of sodium citrate with a concentration of 5 mM are mixed at a volume ratio of 1:1 to obtain a precursor solution;

[0040] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass petri dish, and let it stand still in the dark for 60 min to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining a bacterial cellulose substrate adsorbed with the precursor. Place the glass petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.). Turn on the high-voltage power supply to activate the high-voltage electrode and generate low-temperature plasma. Perform low-temperature plasma treatment. The conditions for low-temperature plasma treatment are as follows: the working voltage is 35 V, the treatment time is 3 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is such that the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. A layer of dielectric barrier material (quartz glass) covers the upper electrode. Rinse with ultrapure water for 24 h to remove residual reaction by-products, and dry at 50 °C until dry to obtain the SERS substrate Plasma-AuNPs@BNC.

[0041] Example 2

[0042] The formula of the culture medium is as follows: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. Sterilize the culture medium at 121 °C for 30 min.

[0043] (1) Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) is inoculated into the culture medium and cultured at 30 °C for 10 d. Rinse and soak it with distilled water, and squeeze to drain the bacterial liquid to obtain a bacterial cellulose membrane. Immerse the bacterial cellulose membrane in a 1% sodium hydroxide aqueous solution and boil for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. Rinse with deionized water until the eluate is neutral, soak with ultrapure water, and the bacterial cellulose membrane is milky white and translucent. Blot the water with filter paper to obtain a bacterial cellulose substrate;

[0044] (2) Aqueous solutions of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and sodium citrate with a concentration of 10 mM are mixed in a volume ratio of 1:1 to obtain a precursor solution;

[0045] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass Petri dish, and let it stand still in the dark for 60 min to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining a bacterial cellulose substrate adsorbed with the precursor. Place the glass Petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.), turn on the high-voltage power supply to activate the high-voltage electrode to generate low-temperature plasma, and perform low-temperature plasma treatment. The conditions for the low-temperature plasma treatment are as follows: the working voltage is 35 V, the treatment time is 3 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is preferably when the plasma discharge area contacts the surface of the bacterial cellulose substrate adsorbed with the precursor. A layer of dielectric barrier material (quartz glass) is covered on the upper electrode, and it is rinsed with ultrapure water for 24 h to remove residual reaction by-products, and then dried at 50 °C until dry to obtain the SERS substrate.

[0046] Example 3

[0047] The formula of the culture medium is as follows: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. The culture medium is sterilized at 121 °C for 30 min.

[0048] (1) Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) is inoculated into the culture medium and cultured at 30 °C for 10 d. It is rinsed and soaked with distilled water, and squeezed to drain the bacterial liquid, obtaining a bacterial cellulose membrane. The bacterial cellulose membrane is immersed in a 1% sodium hydroxide aqueous solution and boiled for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. It is rinsed with deionized water until the eluate is neutral, soaked in ultrapure water, and the bacterial cellulose membrane is milky white and translucent. The water is blotted dry with filter paper to obtain the bacterial cellulose substrate;

[0049] (2) An aqueous solution of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and an aqueous solution of sodium citrate with a concentration of 20 mM are mixed according to a volume ratio of 1:1 to obtain the precursor solution;

[0050] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass Petri dish, and let it stand still for 60 min in the dark to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining a bacterial cellulose substrate adsorbed with the precursor. Place the glass Petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.). Turn on the high-voltage power supply to activate the high-voltage electrode to generate low-temperature plasma. Perform low-temperature plasma treatment. The conditions for low-temperature plasma treatment are as follows: the working voltage is 35 V, the treatment time is 3 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is such that the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. A layer of dielectric barrier material (quartz glass) is covered on the upper electrode. Rinse with ultrapure water for 24 h to remove residual reaction by-products, and dry at 50 °C until dry to obtain the SERS substrate.

[0051] Example 4

[0052] The formula of the culture medium is as follows: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. The culture medium is sterilized at 121 °C for 30 min.

[0053] (1) Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) is inoculated into the culture medium and cultured at 30 °C for 10 d. Rinse and soak it with distilled water, and squeeze to drain the bacterial liquid to obtain a bacterial cellulose membrane. Immerse the bacterial cellulose membrane in a 1% sodium hydroxide aqueous solution and boil for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. Rinse with deionized water until the eluate is neutral, soak with ultrapure water. The bacterial cellulose membrane is milky white and semi-transparent. Blot the water with filter paper to obtain the bacterial cellulose substrate;

[0054] (2) An aqueous solution of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and an aqueous solution of sodium citrate with a concentration of 5 mM are mixed at a volume ratio of 1:1 to obtain the precursor solution;

[0055] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass petri dish, and let it stand still in the dark for 60 min to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining a bacterial cellulose substrate adsorbed with the precursor. Place the glass petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.), turn on the high-voltage power supply to activate the high-voltage electrode to generate low-temperature plasma. For the low-temperature plasma treatment, the conditions are as follows: the working voltage is 35 V, the treatment time is 1 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is such that the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. A dielectric barrier material (quartz glass) is covered on the upper electrode, and it is rinsed with ultrapure water for 24 h to remove residual reaction by-products, and then dried at 50 °C until dry to obtain the SERS substrate.

[0056] Example 5

[0057] The formula of the culture medium is: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. The culture medium is sterilized at 121 °C for 30 min.

[0058] (1) Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) is inoculated into the culture medium and cultured at 30 °C for 10 d. It is rinsed and soaked with distilled water and squeezed to discharge the bacterial liquid, obtaining a bacterial cellulose membrane. The bacterial cellulose membrane is immersed in a 1% sodium hydroxide aqueous solution and boiled for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. It is rinsed with deionized water until the eluate is neutral, soaked with ultrapure water, and the bacterial cellulose membrane is milky white and semi-transparent. The water is blotted dry with filter paper to obtain the bacterial cellulose substrate;

[0059] (2) An aqueous solution of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and an aqueous solution of sodium citrate with a concentration of 10 mM are mixed according to a volume ratio of 1:1 to obtain the precursor solution;

[0060] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass Petri dish, and let it stand still in the dark for 60 min to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining a bacterial cellulose substrate adsorbed with the precursor. Place the glass Petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.), turn on the high-voltage power supply to activate the high-voltage electrode to generate low-temperature plasma, and perform low-temperature plasma treatment. The conditions for low-temperature plasma treatment are as follows: the working voltage is 35 V, the treatment time is 1 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is such that the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. A layer of dielectric barrier material (quartz glass) is covered on the upper electrode, and it is rinsed with ultrapure water for 24 h to remove residual reaction by-products, and then dried at 50 °C until dry to obtain the SERS substrate.

[0061] Example 6

[0062] The formula of the culture medium is as follows: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. The culture medium is sterilized at 121 °C for 30 min.

[0063] (1) Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) is inoculated into the culture medium and cultured at 30 °C for 10 d. It is rinsed and soaked with distilled water and squeezed to discharge the bacterial liquid, obtaining a bacterial cellulose membrane. The bacterial cellulose membrane is immersed in a 1% sodium hydroxide aqueous solution and boiled for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. It is rinsed with deionized water until the eluate is neutral, soaked in ultrapure water, and the bacterial cellulose membrane is milky white and translucent. The water is blotted dry with filter paper to obtain the bacterial cellulose substrate;

[0064] (2) An aqueous solution of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and an aqueous solution of sodium citrate with a concentration of 20 mM are mixed at a volume ratio of 1:1 to obtain the precursor solution;

[0065] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass petri dish, and let it stand still in the dark for 60 min to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining the bacterial cellulose substrate adsorbed with the precursor. Place the glass petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.). Turn on the high-voltage power supply to activate the high-voltage electrode and generate low-temperature plasma. Perform low-temperature plasma treatment. The conditions for low-temperature plasma treatment are: the working voltage is 35 V, the treatment time is 1 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is such that the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. Cover the upper electrode with a layer of dielectric barrier material (quartz glass), rinse with ultrapure water for 24 h to remove residual reaction by-products, and dry at 50 °C until dry to obtain the SERS substrate.

[0066] Example 7

[0067] The formula of the culture medium is: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. The culture medium is sterilized at 121 °C for 30 min.

[0068] (1) Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) is inoculated into the culture medium and cultured at 30 °C for 10 d. It is rinsed and soaked with distilled water and squeezed to discharge the bacterial liquid, obtaining a bacterial cellulose membrane. Immerse the bacterial cellulose membrane in a 1% sodium hydroxide aqueous solution and boil for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. Rinse with deionized water until the eluate is neutral, soak with ultrapure water. The bacterial cellulose membrane is milky white and semi-transparent. Blot the water with filter paper to obtain the bacterial cellulose substrate;

[0069] (2) Aqueous solutions of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and sodium citrate with a concentration of 5 mM are mixed in a volume ratio of 1:1 to obtain the precursor solution;

[0070] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass petri dish, and let it stand still in the dark for 60 min to allow the precursor solution to adsorb into the bacterial cellulose substrate, obtaining a bacterial cellulose substrate adsorbed with the precursor. Place the glass petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.). Turn on the high-voltage power supply to activate the high-voltage electrode to generate low-temperature plasma. Perform low-temperature plasma treatment. The conditions for low-temperature plasma treatment are as follows: the working voltage is 35 V, the treatment time is 5 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is such that the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. Cover the upper electrode with a layer of dielectric barrier material (quartz glass), rinse with ultrapure water for 24 h to remove residual reaction by-products, and dry at 50 °C until dry to obtain the SERS substrate.

[0071] Example 8

[0072] The formula of the culture medium is: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. The culture medium is sterilized at 121 °C for 30 min.

[0073] (1) Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) is inoculated into the culture medium and cultured at 30 °C for 10 d. Rinse and soak it with distilled water, and squeeze to discharge the bacterial liquid to obtain a bacterial cellulose membrane. Immerse the bacterial cellulose membrane in a 1% sodium hydroxide aqueous solution and boil for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. Rinse with deionized water until the eluate is neutral, soak with ultrapure water until the bacterial cellulose membrane is milky white and translucent, and blot the water dry with filter paper to obtain the bacterial cellulose substrate;

[0074] (2) Aqueous solutions of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and sodium citrate with a concentration of 10 mM are mixed at a volume ratio of 1:1 to obtain the precursor solution;

[0075] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass Petri dish, and let it stand still in the dark for 60 min to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining a bacterial cellulose substrate adsorbed with the precursor. Place the glass Petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.), turn on the high-voltage power supply to activate the high-voltage electrode to generate low-temperature plasma, and perform low-temperature plasma treatment. The conditions for low-temperature plasma treatment are as follows: the working voltage is 35 V, the treatment time is 5 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is preferably when the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. A dielectric barrier material (quartz glass) is covered on the upper electrode, and it is rinsed with ultrapure water for 24 h to remove residual reaction by-products, and then dried at 50 °C until dry to obtain the SERS substrate.

[0076] Example 9

[0077] The formula of the culture medium is as follows: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. The culture medium is sterilized at 121 °C for 30 min.

[0078] (1) Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) is inoculated into the culture medium and cultured at 30 °C for 10 d. It is rinsed and soaked with distilled water and squeezed to drain the bacterial liquid, obtaining a bacterial cellulose membrane. The bacterial cellulose membrane is immersed in a 1% sodium hydroxide aqueous solution and boiled for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. It is rinsed with deionized water until the eluate is neutral, soaked in ultrapure water, and the bacterial cellulose membrane is milky white and semi-transparent. The water is blotted dry with filter paper to obtain the bacterial cellulose substrate;

[0079] (2) An aqueous solution of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and an aqueous solution of sodium citrate with a concentration of 20 mM are mixed according to a volume ratio of 1:1 to obtain the precursor solution;

[0080] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass Petri dish, and let it stand still in the dark for 60 min to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining the bacterial cellulose substrate adsorbed with the precursor. Place the glass Petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.). Turn on the high-voltage power supply to activate the high-voltage electrode to generate low-temperature plasma. Conduct low-temperature plasma treatment. The conditions for low-temperature plasma treatment are as follows: the working voltage is 35 V, the treatment time is 5 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is preferably when the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. A layer of dielectric barrier material (quartz glass) covers the upper electrode. Rinse with ultrapure water for 24 h to remove residual reaction by-products, and dry at 50 °C until dry to obtain the SERS substrate.

[0081] Example 10

[0082] The formula of the culture medium is as follows: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. Sterilize the culture medium at 121 °C for 30 min.

[0083] (1) Inoculate Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) into the culture medium, culture at 30 °C for 10 d, rinse and soak with distilled water, and squeeze to drain the bacterial liquid to obtain a bacterial cellulose membrane. Immerse the bacterial cellulose membrane in a 1% sodium hydroxide aqueous solution and boil for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. Rinse with deionized water until the eluate is neutral, soak with ultrapure water. The bacterial cellulose membrane is milky white and semi-transparent. Blot the water with filter paper to obtain the bacterial cellulose substrate;

[0084] (2) Mix an aqueous solution of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and an aqueous solution of sodium citrate with a concentration of 5 mM in a volume ratio of 1:1 to obtain the precursor solution;

[0085] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass petri dish, and let it stand still in the dark for 60 min to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining a bacterial cellulose substrate adsorbed with the precursor. Place the glass petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.), turn on the high-voltage power supply to activate the high-voltage electrode, generate low-temperature plasma, and perform low-temperature plasma treatment. The conditions for low-temperature plasma treatment are as follows: the working voltage is 35 V, the treatment time is 7 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is such that the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. A layer of dielectric barrier material (quartz glass) covers the upper electrode. Rinse with ultrapure water for 24 h to remove residual reaction by-products, and dry at 50 °C until dry to obtain the SERS substrate.

[0086] Example 11

[0087] The formula of the culture medium is as follows: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. The culture medium is sterilized at 121 °C for 30 min.

[0088] (1) Inoculate Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) into the culture medium, culture at 30 °C for 10 d, rinse and soak with distilled water, and squeeze to discharge the bacterial liquid to obtain a bacterial cellulose membrane. Immerse the bacterial cellulose membrane in a 1% sodium hydroxide aqueous solution and boil for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. Rinse with deionized water until the eluate is neutral, soak with ultrapure water, and the bacterial cellulose membrane is milky white and translucent. Blot the water with filter paper to obtain the bacterial cellulose substrate;

[0089] (2) Mix an aqueous solution of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and an aqueous solution of sodium citrate with a concentration of 10 mM in a volume ratio of 1:1 to obtain the precursor solution;

[0090] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass Petri dish, and let it stand still in the dark for 60 min to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining a bacterial cellulose substrate adsorbed with the precursor. Place the glass Petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.), turn on the high-voltage power supply to activate the high-voltage electrode to generate low-temperature plasma, and perform low-temperature plasma treatment. The conditions for low-temperature plasma treatment are as follows: the working voltage is 35 V, the treatment time is 7 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is such that the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. A layer of dielectric barrier material (quartz glass) is covered on the upper electrode, and it is rinsed with ultrapure water for 24 h to remove residual reaction by-products, and then dried at 50 °C until dry to obtain the SERS substrate.

[0091] Example 12

[0092] The formula of the culture medium is as follows: 20 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 2.1 g of disodium hydrogen phosphate, 1.2 g of citric acid, 8 g of trisodium citrate, 1.0 g of anhydrous magnesium sulfate, and 1 L of water. The culture medium is sterilized at 121 °C for 30 min.

[0093] (1) Acetobacter xylinum (the inoculation amount is calculated according to 10% of the volume of the culture medium) is inoculated into the culture medium and cultured at 30 °C for 10 d. It is rinsed and soaked with distilled water and squeezed to drain the bacterial liquid, obtaining a bacterial cellulose membrane. The bacterial cellulose membrane is immersed in a 1% sodium hydroxide aqueous solution and boiled for 60 min to wash away the residual culture medium and Acetobacter xylinum cells. It is rinsed with deionized water until the eluate is neutral, soaked with ultrapure water, and the bacterial cellulose membrane is milky white and translucent. The water is blotted dry with filter paper to obtain the bacterial cellulose substrate;

[0094] (2) An aqueous solution of chloroauric acid (HAuCl4·3H2O) with a concentration of 10 mM and an aqueous solution of sodium citrate with a concentration of 20 mM are mixed according to a volume ratio of 1:1 to obtain the precursor solution;

[0095] (3) Immerse the bacterial cellulose substrate in the precursor solution, place it in a glass Petri dish, and let it stand in the dark for 60 min to allow the precursor solution to be adsorbed into the bacterial cellulose substrate, obtaining a bacterial cellulose substrate adsorbed with the precursor. Place the glass Petri dish containing the bacterial cellulose substrate adsorbed with the precursor in the plasma discharge area (electrode area) of a low-temperature plasma device (Shanghai Wanmuchun Bioengineering Co., Ltd.). Turn on the high-voltage power supply to activate the high-voltage electrode to generate low-temperature plasma. For the low-temperature plasma treatment, the conditions are as follows: the working voltage is 35 V, the treatment time is 7 min, the working gas is air, and the treatment form is atmospheric pressure dielectric barrier discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate adsorbed with the precursor is 0.5 cm. The best distance is such that the plasma discharge area is in contact with the surface of the bacterial cellulose substrate adsorbed with the precursor. A layer of dielectric barrier material (quartz glass) covers the upper electrode. Rinse with ultrapure water for 24 h to remove residual reaction by-products, and dry at 50 °C until dry to obtain the SERS substrate.

[0096] Experimental Example 1

[0097] Collect the SERS spectrum using a Renishaw inVia confocal micro-Raman spectrometer. Select a 785 nm laser with a power of 300 mW. For spectrum acquisition, use a 20× objective lens, a 10× eyepiece, an exposure time of 10 s, and a laser power of 1%. After calibrating the Raman spectrometer with a silicon wafer of 520 cm -1 , due to the specific chemical and spectral properties of R6G and being a commonly used reference substance in Raman spectra, R6G is used to verify the enhancement ability and sensitivity of the SERS substrate. Prepare a series of concentration gradients of R6G as Raman probe molecules, immerse the SERS substrates prepared in Examples 1 to 12 in R6G solutions with different concentration gradients, and detect the SERS performance of the substrates.

[0098] As Figure 1 shown, the figure shows the test results of the SERS substrates prepared in Examples 1 to 12 using R6G as the Raman signal molecule. In Example 1, when the low-temperature plasma treatment time is 3 min and the molar ratio of chloroauric acid to the reducing agent is 2:1, the prepared SERS substrate Plasma-AuNPs@BNC shows the strongest Raman intensity and is thus considered the optimal condition.

[0099] As Figure 2 shown, the gold nanoparticles in the bacterial cellulose substrate of the SERS substrate prepared in Example 1 are evenly and densely distributed. This is because high-energy electrons in the plasma reduce Au 3+ to generate gold nanoparticles. When high-energy electrons collide with H2O in the precursor solution, ionization occurs to produce H2O2 and OH - , and the generated H2O2 and OH- Reacts with Au 3+ to undergo a reduction reaction. As Figure 3 shown, it also verifies the large number of gold nanoparticles present on the SERS substrate bacterial cellulose substrate prepared in Example 1.

[0100] As Figure 4 shown, the standard curve is plotted using the spectral data of the SERS substrate Plasma-AuNPs@BNC prepared in Example 1 for testing R6G. R 2 = 0.984, and the enhancement factor is calculated to be 8.36×10 5 , indicating its good enhancement property.

[0101] As Figure 5 shown, the Raman spectra of 20 randomly selected test points are taken, and the RSD at the characteristic peak of 1510 cm -1 is calculated to be 10.1%, proving the good uniformity of the SERS substrate Plasma-AuNPs@BNC prepared in Example 1.

[0102] Experimental Example 2

[0103] Use the SERS substrate Plasma-AuNPs@BNC prepared in Example 1 to detect imidacloprid in tea leaves. Prepare a gradient concentration imidacloprid standard solution with tea water as the solvent. The gradient concentrations of the imidacloprid standard solution are set to 10 -2 mg / mL, 10 -3 mg / mL, 10 -4 mg / mL, 10 -5 mg / mL, 10 -6 mg / mL to detect the reliability of the SERS substrate prepared in Example 1.

[0104] Immerse the cut SERS substrate prepared in Example 1 in the imidacloprid standard solution (100 μL) and let it stand for 15 min. SERS measurements are performed using a Renishaw inVia confocal Raman microspectrometer equipped with a 785 nm laser. All SERS experiments use a 20× objective lens, the laser power is about 1%, the integration time is 10 s, and it is accumulated 1 time. To reduce measurement errors, the average intensity of the Raman signals at 20 random positions on the substrate is used.

[0105] The results are as Figure 6 shown. In the range of 10 -6 ~10 -2 mg / mL, the SERS substrate prepared in Example 1 has good signals for imidacloprid.

[0106] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the design of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a SERS substrate for detecting pesticide residues, characterized in that: The following steps are involved: (1) inoculating Acetobacter xylinus into a culture medium, culturing to obtain a bacterial cellulose film, and soaking the bacterial cellulose film in a sodium hydroxide aqueous solution, boiling to obtain a bacterial cellulose substrate; (2) mixing a metal salt aqueous solution with a reducing agent aqueous solution to obtain a precursor solution; (3) soaking the bacterial cellulose substrate obtained in step (1) in the precursor solution obtained in step (2), keeping it still in the dark to obtain a bacterial cellulose substrate adsorbing the precursor, treating it with low-temperature plasma, brewing it with ultrapure water, and drying it to obtain a SERS substrate.

2. The preparation method according to claim 1, characterized in that: The inoculation amount of Acetobacter xylinum in step (1) is calculated as 8-12% of the volume of the culture medium; the culture temperature in step (1) is 28-30° C., and the culture time is 7-14 days.

3. The preparation method according to claim 1, characterized in that: The formula of the culture medium in step (1) is: 20 g glucose, 10 g peptone, 10 g yeast extract powder, 2.1 g disodium hydrogen phosphate, 1.2 g citric acid, 8 g trisodium citrate, 1.0 g anhydrous magnesium sulfate, and 1 L water.

4. The preparation method according to claim 1, characterized in that: The mass concentration of the sodium hydroxide aqueous solution in step (1) is 0.5-1.5%; the boiling time in step (1) is 50-70 minutes.

5. The preparation method according to claim 1, characterized in that: The concentration of the metal salt aqueous solution in step (2) is 1 to 10 mM, and the metal salt aqueous solution is one or more of chloroauric acid aqueous solution, silver nitrate aqueous solution, and palladium chloride aqueous solution; the concentration of the reducing agent aqueous solution in step (2) is 0.5 to 20 mM, and the reducing agent aqueous solution is sodium citrate aqueous solution and / or ascorbic acid aqueous solution; the metal salt aqueous solution and the reducing agent aqueous solution in step (2) are mixed in equal volumes.

6. The preparation method according to claim 1, characterized in that: The time for standing still in the dark in step (3) is 10 to 60 minutes.

7. The preparation method according to claim 1, characterized in that: The working voltage of the low-temperature plasma treatment in step (3) is 20 to 40 V, the time of the low-temperature plasma treatment is 1 to 7 min, the working gas of the low-temperature plasma treatment is one or more of air, nitrogen, helium, and argon, and the form of the low-temperature plasma treatment is one of dielectric barrier discharge, glow discharge, radio frequency discharge, corona discharge, and sliding arc discharge. During the low-temperature plasma treatment, the distance between the high-voltage electrode and the surface of the bacterial cellulose substrate on which the precursor is adsorbed is 0.1 to 2 cm.

8. The SERS substrate prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the SERS substrate as claimed in claim 8 in detecting imidacloprid.

10. A method for detecting imidacloprid, characterized in that: Take the SERS substrate described in claim 8 and immerse it in 10 to 100 μL of the sample to be tested, use a Raman spectrometer to collect the spectrum, and calculate the concentration of imidacloprid in the sample to be tested.

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