Graphene oxide-silver nanoparticle modified porous silicon SERS substrate and its application in label-free detection of aflatoxin

Abstract

The application discloses a kind of graphene oxide-silver nanoparticle modified porous silicon SERS substrate and its application in label-free detection aflatoxin, the silver nanoparticle modified porous silicon is prepared by metal-assisted chemical etching, and after graphene oxide is functionalized by mercapto, it is loaded on silver nanoparticle modified porous silicon by Ag-S bond.Utilize the π-π effect of graphene oxide to enrich the benzene ring of AFB1, make it close to SERS "hot spot" to promote chemical enhancement.At the same time, introduce gold nanoparticles as enhancer, after mixing with sample, drop on substrate to form hydrophobic droplet to further enrich target molecule.The multiple coupling enhancement generated by gold nanoparticles, graphene oxide and silver nanoparticles in the application significantly enhances the Raman response signal of AFB1 molecule, and finally obtains the fingerprint spectrum of AFB1, realizes the fingerprint spectrum detection of AFB1.The method has the advantages of simple operation, rapid detection, low cost and the like.

Description

Technical Field

[0001] This invention belongs to the field of food safety, specifically relating to a graphene oxide-silver nanoparticle modified porous silicon SERS substrate and its application in label-free detection of aflatoxin. Background Technology

[0002] Aflatoxins (AFs) are toxic secondary metabolites produced by two fungi, Aspergillus flavus and Aspergillus parasiticus. Edible agricultural products such as peanuts, soybeans, corn, and their processed products are highly susceptible to contamination under warm and humid conditions, making them among the most harmful types of mycotoxins to human health. There are approximately 20 derivatives of aflatoxins, named B1, B2, M1, M2, G1, G2, Q1, P1, and GM, among which aflatoxin B1 (AFB1) is the most toxic. AFB1 is primarily a secondary metabolite produced by Aspergillus flavus and is a difuran cyclic toxin. It is most widely distributed in food and soil and possesses strong toxicity and carcinogenicity. AFB1 exhibits thermal and chemical stability, remaining difficult to degrade even at high temperatures of 268°C. The acute toxicity of AFB1 is 10 times that of potassium cyanide and 68 times that of arsenic. It can induce liver cell and liver function damage, leading to liver cancer and posing a significant risk to human health.

[0003] Currently, the main methods for detecting AFB1 in samples include chromatography and immunoassay. Chromatography primarily involves High Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS / MS). These methods offer high sensitivity and accurate results, but require complex sample pretreatment, expensive equipment, and time-consuming detection, making them unsuitable for rapid on-site detection and high-throughput sample screening. Furthermore, some toxin samples require derivatization before detection. Immunoassays mainly involve Enzyme-Linked Immunosorbent Assay (ELISA) and immunochromatography. These methods are simple to operate and fast, but they often only detect single toxins, making it difficult to achieve simultaneous high-throughput detection of multiple toxins. They also have low sensitivity, and cross-reactions and matrix interference can lead to false positives. Therefore, based on the mycotoxin limits proposed by various countries, and to ensure food safety and human health, there is an urgent need to develop rapid, economical, low-requirement, highly specific, and sensitive mycotoxin detection technologies. Summary of the Invention

[0004] Objective: To address the problems of complex operation, strong equipment dependence, and difficulty in field application of existing AFB1 detection technologies, this invention provides a graphene oxide-silver nanoparticle-modified porous silicon SERS substrate. This invention uses graphene oxide-silver nanoparticle-modified porous silicon as the SERS substrate. The π-π bonds of graphene oxide can adsorb AFB1 molecules containing benzene rings, resulting in excellent chemical enhancement. Simultaneously, AuNPs act as a reinforcing agent, mixing with AFB1 molecules and then dropping onto the SERS substrate to form hydrophobic droplets, further enriching the target molecules and directly obtaining the AFB1 fingerprint spectrum. This eliminates the need for complex labeling processes, enabling rapid and accurate detection of AFB1. This effectively solves the problems of complex operation, expensive equipment, high reagent consumption, and long detection time associated with existing detection methods.

[0005] This invention also provides a graphene oxide-silver nanoparticle modified porous silicon SERS substrate and its application in label-free detection of AFB1.

[0006] This invention utilizes metal-assisted chemical etching to prepare porous silicon modified with silver nanoparticles. The key features are the uniform and discrete distribution of silver nanoparticles generating numerous SERS "hot spots," the porous structure providing a large specific surface area, and the good biocompatibility of the silicon-based material. Using this as a carrier, graphene oxide and gold nanoparticles are combined to detect AFB1. Raman spectroscopy is used to acquire Raman signals from the silicon wafer surface, obtaining the fingerprint spectrum of AFB1 and achieving label-free detection of AFB1.

[0007] Technical solution: In order to achieve the above objectives, the present invention provides a graphene oxide-silver nanoparticle modified porous silicon SERS substrate, wherein the graphene oxide in the substrate is functionalized with thiol groups and stably loaded on the porous silicon surface modified with silver nanoparticles through Ag-S bonds, and the silver nanoparticles are grown in situ on the porous silicon surface to form silver nanoparticle modified porous silicon.

[0008] The graphene oxide-silver nanoparticle-modified porous silicon SERS substrate has a large number of SERS "hot spots" and generates a strong electromagnetic enhancement effect.

[0009] Furthermore, the present invention uses a graphene oxide-silver nanoparticle-modified porous silicon as a SERS enhancement substrate. The silver nanoparticle-modified porous silicon is formed by in-situ growth of silver nanoparticles on the porous silicon surface. The graphene oxide is loaded onto the silver nanoparticle-modified porous silicon surface through thiol functionalization. A mixture of gold nanoparticles and AFB1 is then dropped onto the surface to form hydrophobic droplets. This invention improves the sensitivity of label-free AFB1 SERS detection through a synergistic enhancement strategy of silver nanoparticles, gold nanoparticles, and graphene oxide, as well as the hydrophobic effect of the droplets.

[0010] The porous silicon modified with silver nanoparticles was prepared by metal-assisted chemical etching.

[0011] The thiol-functionalized graphene oxide captures AFB1 through large π bonds and generates chemical enhancement.

[0012] The hydrophobic droplets can effectively confine and enrich AFB1 molecules, thereby improving the sensitivity of SERS detection.

[0013] The method for preparing a graphene oxide-silver nanoparticle-modified porous silicon SERS substrate according to the present invention includes the following steps:

[0014] (1) Preparation of porous silicon modified with silver nanoparticles;

[0015] (2) Preparation of thiol-functionalized graphene oxide: thiol groups are modified on the surface of graphene oxide using silane reagents;

[0016] (3) Preparation of porous silicon modified with graphene oxide-silver nanoparticles: Take the thiol-functionalized graphene oxide prepared in step (2), add it to the porous silicon modified with silver nanoparticles, and carry out the reaction; after the reaction is completed, wash to obtain the porous silicon SERS substrate modified with graphene oxide-silver nanoparticles.

[0017] In step (1), after the silicon wafer is cut and cleaned, HF is added to react and remove the SiO2 on the surface of the silicon wafer to form Si-H bonds. Then, AgNO3 / HF solution is added to react and achieve in-situ growth of silver nanoparticles on the surface of the silicon wafer. After the reaction is completed, the wafer is washed and dried.

[0018] Preferably, in step (1), after cutting and cleaning the silicon wafer, 5% HF is added to remove SiO2 from the silicon wafer surface and form Si-H bonds. Then, AgNO3 / HF solution is added to continue the reaction, thereby achieving in-situ growth of silver nanoparticles on the silicon wafer surface. After completion, the wafer is rinsed several times with double-distilled water and dried with gentle N2 to remove any remaining moisture.

[0019] In step (2), the silane reagent is 3-mercaptopropyltrimethoxysilane, the reaction temperature is room temperature, the reaction time is 20-24 h, the reaction solution system is ethanol, and it is dissolved in water after washing.

[0020] As a preferred option, the silane reagent in step (2) is 3-mercaptopropyltrimethoxysilane, the reaction temperature is room temperature, the reaction time is 24 h, the reaction solution system is ethanol, and it is dissolved in water after washing.

[0021] In step (3), the concentration of the thiol-functionalized graphene oxide is 1-10000 ng / mL, the volume is 300-500 μL, the reaction temperature is room temperature, and the time is 4-5 h.

[0022] Preferably, the concentration of graphene oxide in step (3) is 1 ng / mL, the volume is 300 μL, the reaction temperature is room temperature, and the time is 4 h.

[0023] Furthermore, a porous silicon substrate modified with silver nanoparticles was placed in a centrifuge tube, and a fixed volume of graphene oxide was added for further modification.

[0024] The application of the graphene oxide-silver nanoparticle modified porous silicon SERS substrate fingerprint spectroscopy detection of AFB1 described in this invention.

[0025] The application involves dropping a mixed solution of gold nanoparticles and AFB1 onto a porous silicon SERS substrate modified with graphene oxide-silver nanoparticles. After drying, the Raman fingerprint information of AFB1 is obtained through SERS technology to directly detect AFB1, thereby achieving semi-quantitative analysis of AFB1's SERS fingerprint spectrum.

[0026] Furthermore, the detection method involves mixing the gold nanoparticles with the sample, dropping the mixture onto a porous silicon SERS substrate modified with graphene oxide-silver nanoparticles, drying the substrate, and then collecting the Raman signal on the silicon wafer surface using a microconfocal Raman spectrometer. Label-free detection is achieved based on the obtained AFB1 fingerprint spectrum.

[0027] The concentration factor of the gold nanoparticles is 0-10 times (the prepared gold nanoparticle solution is concentrated by centrifugation at different ratios), and the gold nanoparticles and the sample are mixed at a volume ratio of 1:1, with a volume of 5 μL, and dried at 60℃ for 20 min.

[0028] In this process, the graphene oxide-silver nanoparticles modify the porous silicon SERS substrate, adsorbing AFB1 containing benzene rings and generating a chemical enhancement effect. Meanwhile, during the thiol functionalization process, the hydrophilic oxygen-containing functional groups such as hydroxyl, carboxyl, and epoxy groups on the surface of graphene oxide are reacted, reducing the number of hydrophilic groups. At the same time, hydrophobic alkyl chains (-C3H6-) are introduced, giving the substrate a hydrophobic effect and confining the target molecules.

[0029] The porous silicon detection platform based on graphene oxide-silver nanoparticle modification of the present invention includes the graphene oxide-silver nanoparticle modified porous silicon SERS substrate and gold nanoparticles as a reinforcing agent.

[0030] Furthermore, this invention provides a label-free SERS detection method for aflatoxin B1 based on a graphene oxide-silver nanoparticle-modified porous silicon SERS substrate. The SERS substrate is composed of silver nanoparticles with electromagnetic enhancement effects and chemically enhanced graphene oxide. The graphene oxide is chemically modified to introduce thiol groups, which can be loaded onto the silver nanoparticle-modified porous silicon surface. In the label-free SERS detection platform, the target analyte solution is mixed with a gold nanoparticle solution and dropped onto the graphene oxide-silver nanoparticle-modified porous silicon. At a specific Raman excitation wavelength, the Raman signal on the substrate surface is detected, thereby achieving the acquisition of an AFB1 fingerprint and semi-quantitative detection.

[0031] The method for label-free detection of aflatoxin B1 based on a porous silicon SERS substrate modified with graphene oxide-silver nanoparticles, as described in this invention, includes the following steps:

[0032] Step 1: Prepare porous silicon modified with silver nanoparticles.

[0033] Step 2: Modify the surface of graphene oxide with thiol groups using silane reagents and disperse in water.

[0034] Step 3: React thiolized graphene oxide with porous silicon modified with silver nanoparticles to prepare graphene oxide-silver nanoparticle modified porous silicon.

[0035] Step 4: Prepare gold nanoparticles, mix them with toxins, and drop them onto a porous silicon surface modified with graphene oxide-silver nanoparticles for drying.

[0036] Step 5: Detection of Raman signal on substrate surface. Place the dried silicon wafer on a glass slide and use a microconfocal Raman spectrometer to collect the Raman signal on the surface of the silicon wafer.

[0037] In step one, the silver nanoparticle-modified porous silicon was prepared using a metal-assisted chemical etching method. The concentration of AgNO3 was 10 mM, the concentration of HF was 10%, the addition amount was 300 μL, the reaction temperature was 50℃, and the reaction time was 60 s.

[0038] In step two, the silane reagent is 3-mercaptopropyltrimethoxysilane, the reaction temperature is room temperature, and the reaction time is 24 h. The reaction solution system is ethanol, which is dissolved in water after washing.

[0039] Preferably, in step three, the concentration of the thiolized graphene oxide is 1 ng / mL, the reaction temperature is room temperature, and the reaction time is 4 h.

[0040] Preferably, in step four, the concentration factor of the gold nanoparticles is 0 times, the volume ratio of the gold nanoparticles to the toxin is 1:1, the volume of the mixed liquid added is 5 μL, and the mixture is dried at 60°C for 20 min.

[0041] In step five, during Raman detection, the excitation wavelength was 785 nm, the current intensity was 550 mA, and the integration time was 5 s. The AFB1 fingerprint spectrum on the silicon wafer surface was obtained by acquiring the Raman signal.

[0042] This invention first prepares porous silicon modified with silver nanoparticles, then loads graphene oxide, which can adsorb aflatoxin B1 near the SERS "hot spot" on the surface, and finally mixes gold nanoparticles with aflatoxin B1 and drops them onto the porous silicon surface modified with graphene oxide-silver nanoparticles and dries them. Raman signals are obtained using microconfocal Raman spectroscopy to achieve label-free detection of AFB1.

[0043] Specifically, this invention loads thiol-functionalized graphene oxide onto a porous silicon surface modified with silver nanoparticles using Ag-S bonds. The π-π bonds of the graphene oxide adsorb AFB1 molecules containing benzene rings, resulting in excellent chemical enhancement. Simultaneously, gold nanoparticles, acting as a reinforcing agent, are mixed with AFB1 molecules and dropped onto a SERS substrate to form hydrophobic droplets that confine the target molecules. The synergistic effect of gold nanoparticles, graphene oxide, and silver nanoparticles significantly enhances the Raman response signal of AFB1 molecules, ultimately obtaining an AFB1 fingerprint spectrum and achieving label-free detection of AFB1. The porous silicon modified with silver nanoparticles is prepared using a metal-assisted chemical etching method with 10 mM AgNO3 / 10% HF at 50°C.

[0044] This invention constructs a porous silicon SERS detection platform based on graphene oxide-silver nanoparticle modification, which can be used to obtain the fingerprint spectrum of AFB1. The principle is as follows: First, porous silicon modified with silver nanoparticles is prepared using metal-assisted chemical etching. Then, utilizing the principle that graphene oxide can adsorb AFB1 containing benzene rings through π-π bonds, the target analyte is enriched in the SERS "hotspot" region to enhance the chemical enhancement effect, thus constructing a porous silicon SERS substrate modified with graphene oxide-silver nanoparticles. Simultaneously, synthesized AuNPs are used as an enhancer, mixed with AFB1 molecules, and dropped onto the porous silicon substrate to form hydrophobic droplets, further enriching the target molecules. The gold nanoparticles, graphene oxide, and silver nanoparticles generate multiple coupling enhancements, which can greatly enhance the Raman response signal of AFB1 molecules, ultimately obtaining the fingerprint spectrum of AFB1. This develops a rapid, non-destructive, and accurate label-free SERS detection method for AFB1, providing a new technical platform for rapid on-site detection of fungal toxins.

[0045] The focus of this invention is to obtain the fingerprint spectrum of AFB1 using SERS technology, thereby achieving label-free detection of AFB1. This invention significantly enhances the Raman signal of AFB1 by optimizing the concentration of graphene oxide and the concentration factor of gold nanoparticles.

[0046] The porous silicon modified with graphene oxide and silver nanoparticles used in this invention can detect target toxins through a synergistic enhancement effect with gold nanoparticles. The detection principle involves adsorbing AFB1 onto "hot spots" using graphene oxide, and then obtaining the fingerprint spectrum of the target toxin through the superposition of multiple enhancement effects of silver nanoparticles, gold nanoparticles, and graphene oxide. This method is rapid and inexpensive, and compared with the SERS labeling method commonly used for AFB1 detection in the past, it significantly improves the ease of operation and real-time detection capability, making it suitable for rapid screening and analysis of fungal toxins.

[0047] This invention is the first to propose using porous silicon modified with graphene oxide and silver nanoparticles as a SERS substrate for label-free SERS detection of AFB1. Due to its excellent biocompatibility, easily controllable structure and size, good chemical stability, and excellent optical and electrical properties, porous silicon is considered an ideal high-quality and high-performance SERS substrate when combined with noble metal nanomaterials. This invention significantly enhances the ability of target analytes to accumulate at SERS "hot spots" by combining graphene oxide with silver nanoparticle-modified porous silicon, thereby enhancing the Raman signal of the target analytes and achieving label-free detection of aflatoxin B1. This invention utilizes the hydrophobicity of droplets to confine and further concentrate target molecules, allowing for the excitation of more analyte molecules within the same laser range, resulting in further Raman signal enhancement. The detection method of this invention is simple to operate, has a fast response speed, and is low in cost, making it widely applicable across various industries.

[0048] The detection of this invention does not require a complex labeling strategy, is simple to operate, and is fast. Compared with SERS labeling strategies such as SERS aptamer sensors or SERS antibody sensors, this invention is based on the fingerprint spectrum of the target molecule, does not require Raman reporter molecule labeling, and does not require competitive reaction steps. Detection can be completed with just one sample addition and signal spectrum acquisition, and the detection time is only 5 seconds.

[0049] The SERS substrate constructed in this invention has excellent surface plasmon enhancement effect and significant electromagnetic enhancement effect, which can efficiently amplify the intrinsic Raman signal of the analyte molecule, providing a solid foundation for achieving efficient, accurate and rapid label-free SERS detection.

[0050] This invention features strong signal specificity and high anti-interference capability. Its core lies in directly using the SERS fingerprint spectrum of the target analyte itself as the basis for qualitative and quantitative detection. Compared with the traditional strategy of indirectly detecting the target analyte by using the signal changes of Raman reporter molecules, it has higher accuracy and reliability.

[0051] The platform constructed by this invention has strong versatility and can be extended to the detection of other toxins, pesticides, veterinary drugs, food additives and other substances. It only requires incubating the target substance with a SERS substrate to obtain the fingerprint spectrum of the target substance for qualitative detection. At the same time, it can also perform quantitative detection based on the Raman signal intensity, and has a high market application capability.

[0052] This invention detects different concentrations of AFB1 in experiments. AFB1 exhibits distinct Raman characteristic peaks at concentrations of 5, 4, 3, 2, and 1 μg / mL, as shown in the Raman spectra. Furthermore, unlike SERS labeling detection which requires repeated washing and incubation steps, this invention only requires mixing the reaction and directly detecting the Raman signal response. The entire experimental process (including material preparation, sample drying, and final detection) can be completed within 30 minutes. Compared to the SERS labeling strategy, the steps are significantly simplified, enabling elution-free and rapid detection.

[0053] The silver nanoparticle-modified porous silicon used in this invention can be prepared in the laboratory using a metal-assisted chemical etching method. The reaction requires only HF and AgNO3 solutions, minimizing equipment and environmental requirements. The reaction process takes only 60 seconds, resulting in rapid preparation. Furthermore, the gold nanoparticle solution consumption is extremely low (only 2.5 μL per silicon wafer). Based on a chloroauric acid price of 1500 yuan / g, the reagent cost per application is approximately 0.075 yuan, making it a very cost-effective method suitable for various industries.

[0054] In this invention, silver nanoparticles are directly prepared using metal-assisted chemical etching (MEC). Graphene oxide (GO), functionalized with thiol groups, is loaded onto the silver nanoparticle-modified porous silicon via Ag-S bonds. The π-π interactions of GO enrich AFB1 containing benzene rings, bringing it closer to SERS "hot spots" to enhance chemical enhancement. Simultaneously, gold nanoparticles are introduced as an enhancer; after mixing with the sample, they are dropped onto the substrate to form hydrophobic droplets, further enriching the target molecules. This invention, through the multi-coupling enhancement generated by gold nanoparticles, graphene oxide, and silver nanoparticles, can significantly enhance the Raman response signal of AFB1 molecules, ultimately obtaining the fingerprint spectrum of AFB1 and achieving semi-quantitative detection of AFB1 fingerprint spectroscopy. This method has the advantages of simple operation, rapid detection, and low cost.

[0055] This invention prepares a specific composite SERS substrate and uses AFB1 fingerprint Raman spectroscopy for detection. The composite porous silicon SERS substrate, combined with AuNPs and target analyte premixing scheme, achieves excellent label-free detection results.

[0056] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0057] (1) This invention is the first to use chemical modification to prepare a multi-level composite SERS substrate of porous silicon modified with graphene oxide-silver nanoparticles, and amplifies the intrinsic Raman signal of AFB1 through the synergistic enhancement effect of the two noble metals, so as to realize the acquisition of label-free clear fingerprint spectrum and finally realize semi-quantitative detection.

[0058] (2) This invention utilizes metal-assisted chemical etching to prepare porous silicon modified with silver nanoparticles. The preparation method is simple and efficient, with low equipment requirements. The silver nanoparticles are uniformly distributed on the substrate surface, effectively forming a large number of highly active SERS "hot spots", resulting in good substrate reproducibility, stability, and enhancement effect.

[0059] (3) In this invention, graphene oxide is introduced on the surface of a porous silicon substrate modified with silver nanoparticles. By utilizing its ultra-large specific surface area, rich oxygen-containing functional groups and excellent conjugated structure, it not only improves the biocompatibility of the substrate, but also synergizes with silver nanoparticles to produce a dual SERS enhancement effect of efficient chemical enhancement and electromagnetic enhancement. At the same time, graphene oxide can enrich trace amounts of AFB1 molecules through π-π stacking and hydrophobic interaction, so as to realize the adsorption and enrichment of target substances in the SERS "hot spot" region and improve detection sensitivity.

[0060] (4) Unlike label-based SERS detection strategies that rely on biorecognition elements such as antibodies and aptamers, this invention does not require the introduction of any exogenous markers and can directly obtain the fingerprint spectrum of AFB1. This fundamentally avoids problems such as complex immune competitive reactions, time-consuming incubation, non-specific adsorption interference, and signal deviations introduced by the labeling process, and significantly improves detection efficiency and the accuracy and reliability of results.

[0061] (5) Compared with traditional chromatography and immunoassay techniques, the label-free SERS detection strategy adopted in this invention does not require complex sample pretreatment, large precision instruments and long chromatographic separation process. It also avoids cumbersome steps such as antibody labeling, immune incubation and competitive reaction, and avoids defects such as easy inactivation of biological reagents and harsh detection conditions. It has the advantages of simpler detection process, faster detection speed, easy operation, low cost, non-destructive and strong specificity.

[0062] (6) The present invention enables rapid, micro-volume, and non-destructive detection of AFB1. Only a small amount of sample needs to be directly dropped onto the SERS substrate to complete the detection. The sample consumption for a single detection is only about 2.5 μL, which consumes a small amount of sample and does not damage the structure of the analyte. The entire detection process can be completed within 5 seconds, which can meet the needs of rapid on-site detection and high-throughput sample screening.

[0063] (7) By directly binding AFB1 to the SERS active substrate, the present invention successfully obtained AFB1 characteristic fingerprint spectra of 10, 5, 4, 3, 2 and 1 μg / mL, realizing label-free semi-quantitative analysis of AFB1 by SERS, and providing an efficient and feasible new solution for the rapid detection of AFB1 in grains and food. Attached Figure Description

[0064] Figure 1 This is a schematic diagram illustrating the principle of label-free detection of aflatoxin B1 based on a porous silicon SERS substrate modified with graphene oxide-silver nanoparticles according to the present invention.

[0065] Figure 2 Scanning electron microscope (SEM) images of silver nanoparticle-modified porous silicon PSi@AgNPs, graphene oxide-silver nanoparticle-modified porous silicon PSi@AgNPs@GO, and a mixed solution of gold nanoparticles and AFB1 dropped onto PSi@AgNPs@GO: (a) Surface of PSi@AgNPs; (b) Cross section of PSi@AgNPs; (c) PSi@AgNPs@GO; (d) PSi@AgNPs@GO + (AuNPs + AFB1).

[0066] Figure 3 A mixture of AFB1 and AuNPs was added to PSi@AgNPs@GO for detection. (a) Actual operation diagram; (b) Raman spectrum of AFB1;

[0067] Figure 4 Calculate the enhancement factor for the base;

[0068] Figure 5 The optimized results for graphene oxide concentration are: (a) 1 ng / mL; (b) 10 ng / mL; (c) 100 ng / mL; (d) 1000 ng / mL; (e) 10000 ng / mL.

[0069] Figure 6 The optimization results for the concentration factor of AuNPs are: (a) 0x; (b) 2x; (c) 5x; (d) 10x.

[0070] Figure 7The results of detection of different concentrations of AFB1 are shown in (a) 5 μg / mL; (b) 4 μg / mL; (c) 3 μg / mL; (d) 2 μg / mL; and (e) 1 μg / mL. Detailed Implementation

[0071] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0072] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0073] The monocrystalline silicon wafers were purchased from Hefei Kejing Technology Co., Ltd., all of which were P-type boron-doped with a crystal lattice orientation of [missing information]. <100> Its resistivity is 0.01-0.05 Ω.

[0074] Silver nitrate was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0075] The monolayer graphene oxide (GO) solution (sheet diameter < 500 nm, 2 mg / mL) was purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.

[0076] (3-Mercaptopropyl)trimethoxysilane and hydrofluoric acid were purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0077] Aflatoxin B1 (AFB1) was purchased from Qingdao Purybang Biotechnology Co., Ltd.; specification: ≥95%.

[0078] Example 1

[0079] Synthetic graphene oxide-silver nanoparticle modified porous silicon

[0080] 1. Preparation of porous silicon modified with silver nanoparticles

[0081] PSi@AgNPs were prepared using metal-assisted chemical etching: Two clean single-crystal silicon wafers (0.5 cm × 0.5 cm) were placed back-to-back (wafer side out) in a 1.5 mL centrifuge tube. 300 μL of 5% HF was added, and the wafers were soaked for approximately 30 min to remove SiO2 from the silicon surface and form Si-H bonds. A 10 mM AgNO3 / 10% HF mixed solution was prepared. The solution in the centrifuge tube was removed, and 300 μL of the prepared mixed solution was rapidly added at 50°C. The reaction was allowed to proceed at room temperature for 60 s. Silver ions were reduced by Si-H bonds, achieving in-situ growth of silver nanoparticles on the silicon wafer surface. After the reaction, the solution in the centrifuge tube was removed, and the wafers were rinsed 3-4 times with double-distilled water. The surface was then dried with gentle N2. Scanning electron microscopy results are shown below. Figure 2As shown in (a) and 2(b), the surface images show that silver nanoparticles are uniformly and discretely distributed on the porous silicon surface, and the cross-sectional images show a clear bilayer structure, with the upper layer being a densely packed AgNPs layer and the lower layer being a porous silicon channel structure.

[0082] 2. Thiol functionalization of graphene oxide

[0083] 0.5 mL of 2 mg / mL GO dispersion, 9.5 mL of ethanol, and 0.1 mL of MPTMS were added sequentially to a glass bottle, and the mixture was stirred continuously at room temperature for 24 h. After the reaction was complete, the sample was washed three times with anhydrous ethanol by centrifugation to remove unreacted excess MPTMS. Finally, the obtained thiol-functionalized GO precipitate was redispersed in 1 mL of double-distilled water to obtain thiol-functionalized graphene oxide with a concentration of 1 mg / mL.

[0084] 3. Preparation of porous silicon SERS substrates modified with graphene oxide-silver nanoparticles

[0085] The thiol-functionalized graphene oxide solution was diluted to 1 ng / mL, and 300 μL was added to a 2 mL centrifuge tube. The silver nanoparticle-modified porous silicon from step 1 was then immersed in the solution using tweezers. The reaction was carried out with shaking at room temperature for 4 h. After the reaction, the liquid in the centrifuge tube was removed, and the silicon wafer was washed 3-4 times with double-distilled water. The remaining moisture on the silicon wafer surface was then dried with a gentle nitrogen stream to obtain graphene oxide-silver nanoparticle-modified porous silicon. Scanning electron microscopy results are shown below. Figure 2 As shown in (c), mercapto-modified graphene oxide is coated on a porous silicon surface modified with silver nanoparticles using Ag-S covalent bonds, and its unique thin and wrinkled sheet structure is clearly visible.

[0086] Example 2

[0087] AFB1 was directly detected using the substrate prepared in Example 1.

[0088] 1. Preparation of gold nanoparticles (AuNPs)

[0089] Measure 50 mL of double-distilled water into a clean conical flask, bring to a boil, and then add 250 µL of a pre-prepared 2% chloroauric acid solution while stirring rapidly. After boiling for 2 minutes, add 375 µL of a freshly prepared 1% trisodium citrate solution. Observe the solution color gradually change from colorless to dark purple and finally to a clear wine red. Then reduce the stirring speed and continue heating for 10 minutes. After 10 minutes, remove the heat source, reduce the stirring speed, and allow the solution to cool to room temperature. Store in a glass bottle at 4°C in the dark.

[0090] 2. Electron microscopy characterization of porous silicon modified with silver nanoparticles, porous silicon modified with graphene oxide-silver nanoparticles, and porous silicon combined with gold nanoparticles for detecting AFB1.

[0091] The porous silicon modified with silver nanoparticles was characterized by scanning electron microscopy, such as... Figure 2 As shown in (a), most of the area in the figure contains spherical particles with relatively uniform particle size. These are AgNPs grown in situ on the porous silicon surface, and their cross-sections are as shown in the figure. Figure 2 (b) It can be seen that a porous structure of porous silicon has been formed. Furthermore, the electron microscopy characterization of the porous silicon modified with graphene oxide-silver nanoparticles is as follows: Figure 2 As shown in (c), graphene oxide was successfully loaded onto the porous silicon surface modified with silver nanoparticles. Finally, 5 μL of the unconcentrated gold nanoparticle solution prepared in step (1) and 10 μg / mL AFB1 were mixed at a volume ratio of 1:1 and dropped onto the graphene oxide-silver nanoparticle modified porous silicon. After drying, the surface was characterized by electron microscopy, as shown in (c). Figure 2 As shown in (d), the composite substrate exhibits a clear three-layer structure: AgNPs are distributed in the bottom layer, GO sheets serve as the middle layer, and AuNPs are mainly distributed on the GO surface. The layers are tightly bonded, and no obvious particle shedding or aggregation is observed, indicating the successful preparation of the composite substrate.

[0092] 3. Detection of AFB1 by dropping a mixed solution of AuNPs and AFB1 onto porous silicon modified with graphene oxide-silver nanoparticles.

[0093] The unconcentrated AuNPs prepared in step (1) were mixed with 10 μg / mL AFB1 diluted in 50% methanol at a volume ratio of 1:1. 5 μL of the mixed solution was then dropped onto the porous silica modified with graphene oxide-silver nanoparticles prepared in step 3 of Example 1. Figure 3 (a) The mixed droplets exhibit a hydrophobic state. This is because when MPTMS modifies graphene oxide, it reacts with hydrophilic oxygen-containing functional groups such as hydroxyl, carboxyl, and epoxy groups on the GO surface, while simultaneously introducing hydrophobic alkyl chains (-C3H6-). This reduces the number of hydrophilic groups on the graphene oxide surface and introduces hydrophobic components. Therefore, after loading GO onto the porous silicon modified with silver nanoparticles using Ag-S bonds, the mixed solution can maintain a hydrophobic state on the silicon wafer surface. The mixture was dried in a 60℃ oven for 20 min. The silicon wafer was placed under a Raman microscope, focused under a 20X objective lens, and the laser position was adjusted to acquire Raman signals. The results are as follows: Figure 3 (b). AFB1 at 626 cm -1 684 cm -1 747 cm -1 821 cm -1 933 cm -1 991 cm -1 1088 cm -1 1244 cm-1 1268 cm -1 1298 cm -1 1351 cm -1 1437 cm -1 1489 cm -1 1550 cm -1 1591 cm -1 1618 cm -1 1753 cm -1 The presence of distinct characteristic peaks at all locations indicates that this method can directly obtain the fingerprint spectrum of AFB1. The detection principle diagram is shown below. Figure 1 .

[0094] 4. Calculation of the enhancement factor

[0095] 20 μL 10 -1 A solution of Nell Blue A (NBA) was added dropwise onto a regular monocrystalline silicon wafer. 10 -7 M's NBA aqueous solution was dropped onto the unmodified graphene SERS substrate from step 1 of Example 1. Additionally, the unconcentrated gold nanoparticles from step 1 of Example 2 were mixed with 10... -7 An equal volume of M's NBA aqueous solution was added dropwise to the SERS substrate of modified graphene oxide obtained in step 3 of Example 1, and then dried in an oven at 60°C for 20 min. The silicon wafer was placed under a Raman microscope, and the laser position was adjusted by focusing under a 20X objective lens to acquire Raman signals. The results are as follows: Figure 4 On a regular silicon wafer, the concentration is 10. -1 The M-type NBA molecule has a Raman characteristic peak signal intensity of approximately 417 au; when the concentration is 10... -7 When the NBA solution of M is directly dropped onto a porous silicon substrate modified with silver nanoparticles, it is located at 592 cm⁻¹. -1 The characteristic peak SERS signal intensity at the point can reach 11857 ​​au; when NBA molecules are premixed with AuNPs and then dropped onto a porous silicon substrate modified with graphene oxide-silver nanoparticles, the characteristic peak signal intensity of NBA reaches as high as 114414 au. This is attributed to the electromagnetic enhancement effect generated by AuNPs and AgNPs, as well as the synergistic contribution of the chemical enhancement effect provided by graphene oxide. Substituting the above intensity values ​​into the formula...

[0096]

[0097] Calculate the enhancement factor, where I SERS and I bulk Representing the Raman characteristic peak intensities of the signal molecule on the SERS substrate and ordinary silicon wafer, respectively, N bulk and NSERS This corresponds to the number of signal molecules adsorbed on the ordinary silicon wafer and the SERS substrate under laser irradiation, where S and V are the laser irradiation area and the detection solution volume, respectively. Based on the formula, the EF of the silver nanoparticle-modified porous silicon substrate is 2.84 × 10⁻⁶. 7 After the introduction of AuNPs and GO, the substrate EF increased to 2.75 × 10⁻⁶. 8 Based on the substrate enhancement effect and AFB1 elution characteristics, a porous silicon SERS substrate modified with graphene oxide-silver nanoparticles combined with gold nanoparticles can be used for label-free detection of AFB1. This embodiment compares the detection signals of porous silicon SERS substrates modified with silver nanoparticles, silver nanoparticles, graphene oxide, and gold nanoparticles, and calculates the EF (exposure response). The composite substrate achieves an order-of-magnitude higher enhancement factor, indicating a better enhancement effect.

[0098] Example 3

[0099] The optimization of the conditions for detecting AFB1 using the SERS substrate in Example 1 is as follows:

[0100] 1. Optimization of graphene oxide concentration

[0101] Place the silver nanoparticle-modified porous silicon prepared in Example 1 into centrifuge tubes. Dilute the thiolized graphene oxide from step 2 of Example 1 to 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL, respectively. Take two 300 μL portions of each concentration and add them to the centrifuge tubes containing the silver nanoparticle-modified porous silicon. Shake the mixture at room temperature for 4 h, wash with double-distilled water 3-4 times, and dry with N2. Five μL of an equal volume mixture of unconcentrated AuNPs and 10 μg / mL AFB1-50% methanol was added to each of the prepared porous silicon SERS substrates modified with graphene oxide-silver nanoparticles of different concentrations in Example 2, step 1. For the blank control, five μL of the same mixture was added to the corresponding SERS substrate. The substrates were dried in an oven at 60°C for 20 min. After drying, the silicon wafers were placed under a Raman microscope, focused under a 20X objective lens, with a current intensity of 550 mA and an integration time of 5 s, to acquire Raman signals. The results are as follows: Figure 5As shown, by comparing with the blank Raman peak spectrum, when the SERS substrate contained 1 ng / mL and 10 ng / mL GO, the Raman peak spectrum of AFB1 showed obvious characteristic peaks. However, excessive GO sheets can stack to form a multilayer structure, completely covering the AgNPs deposited on the SERS substrate surface. This prevents the AgNPs from contacting the subsequently added AuNPs, eliminating the "hot spots" of the AgNPs themselves and blocking the electromagnetic synergistic enhancement effect of Ag-Au, resulting in unsatisfactory peak elution of AFB1. Therefore, 1 ng / mL thiolized graphene oxide was selected to prepare porous silicon modified with graphene oxide-silver nanoparticles.

[0102] 2. Optimization of the concentration factor of gold nanoparticles

[0103] Gold nanoparticle solutions with concentrations of 0, 2, 5, and 10 times were obtained by centrifuging at 10,000 rpm for 10 min and discarding different volumes of supernatant. These solutions were then mixed with AFB1-50% methanol solution at a 1:1 volume ratio and added dropwise to porous silicon substrates modified with graphene oxide-silver nanoparticles. A blank control was prepared by adding 5 μL of a mixture of AuNPs and 50% methanol to the corresponding SERS substrate. The substrates were dried in a 60℃ oven for 20 min. After drying, the silicon wafers were placed under a Raman microscope, focused under a 20X objective lens, with a current intensity of 550 mA and an integration time of 5 s to acquire Raman signals. The results are as follows: Figure 6 As shown, the SERS signal of AFB1 decreased continuously with the increase of AuNPs concentration factor. Therefore, considering the experimental cost and the peak characteristics of AFB1 SERS signal, unconcentrated AuNPs were premixed with AFB1 and dropped onto porous silicon modified with graphene oxide-silver nanoparticles for AFB1 detection.

[0104] Example 4

[0105] Using the optimized conditions of Example 3, label-free detection of AFB1 was performed on a porous silicon SERS substrate modified with graphene oxide-silver nanoparticles.

[0106] A 1 mg / mL AFB1 solution was diluted with 50% methanol to concentrations of 5 μg / mL, 4 μg / mL, 3 μg / mL, 2 μg / mL, and 1 μg / mL. Equal volumes of each concentration were mixed with unconcentrated AuNPs. 5 μL of each solution was added dropwise to the prepared SERS substrate. A blank control was prepared by mixing AuNPs with 50% methanol at a 1:1 ratio. The solutions were dried in a 60°C oven for 20 min. The silicon wafer was placed under a Raman microscope, the laser position was adjusted, and the laser was focused under a 20X objective lens. The current intensity was 550 mA, and the integration time was 5 s. Raman signals from different concentrations of AFB1 were acquired. The results are as follows: Figure 7 As shown, AFB1 exhibited distinct Raman characteristic peaks at concentrations of 5, 4, 3, 2, and 1 μg / mL. This result confirms that the porous silicon SERS substrate modified with graphene oxide-silver nanoparticles prepared in this invention, with the synergistic effect of gold nanoparticle enhancers, can effectively excite the characteristic fingerprint spectrum of AFB1 molecules, enabling direct label-free detection of this fungal toxin.

Claims

1. A graphene oxide-silver nanoparticle-modified porous silicon SERS substrate, characterized in that, After being functionalized with thiol groups, the graphene oxide in the substrate is stably loaded onto the porous silicon surface modified with silver nanoparticles through Ag-S bonds. The silver nanoparticles are grown in situ on the porous silicon surface to form porous silicon modified with silver nanoparticles.

2. The graphene oxide-silver nanoparticle-modified porous silicon SERS substrate according to claim 1, characterized in that, The graphene oxide-silver nanoparticle-modified porous silicon SERS substrate has a large number of SERS "hot spots", generating a strong electromagnetic enhancement effect.

3. A method for preparing a porous silicon SERS substrate modified with graphene oxide-silver nanoparticles as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of porous silicon modified with silver nanoparticles; (2) Preparation of thiol-functionalized graphene oxide: thiol groups are modified on the surface of graphene oxide using silane reagents; (3) Preparation of porous silicon modified with graphene oxide-silver nanoparticles: Take the thiol-functionalized graphene oxide prepared in step (2), add it to the porous silicon modified with silver nanoparticles, and carry out the reaction; after the reaction is completed, wash to obtain the porous silicon SERS substrate modified with graphene oxide-silver nanoparticles.

4. The preparation method according to claim 3, characterized in that, In step (1), after the silicon wafer is cut and cleaned, HF is added to react and remove the SiO2 on the surface of the silicon wafer to form Si-H bonds. Then, AgNO3 / HF solution is added to react and achieve in-situ growth of silver nanoparticles on the surface of the silicon wafer. After the reaction is completed, the wafer is washed and dried.

5. The preparation method according to claim 3, characterized in that, In step (2), the silane reagent is 3-mercaptopropyltrimethoxysilane, the reaction temperature is room temperature, the reaction time is 20-24 h, the reaction solution system is ethanol, and it is dissolved in water after washing.

6. The preparation method according to claim 3, characterized in that, The preferred step (3) has a concentration of 1-10000 ng / mL, a volume of 300-500 μL, a reaction temperature of room temperature, and a reaction time of 4-5 h.

7. An application of the graphene oxide-silver nanoparticle modified porous silicon SERS substrate fingerprint spectroscopy detection of AFB1 based on the method described in claim 1.

8. The application according to claim 8, characterized in that, The application involves dropping a mixed solution of gold nanoparticles and AFB1 onto a porous silicon SERS substrate modified with graphene oxide-silver nanoparticles. After drying, the Raman fingerprint information of AFB1 is obtained through SERS technology to directly detect AFB1, thereby achieving semi-quantitative analysis of AFB1's SERS fingerprint spectrum.

9. The application according to claim 8, characterized in that, The graphene oxide-silver nanoparticles modify the porous silicon SERS substrate. The graphene oxide adsorbs AFB1 containing benzene rings and produces a chemical enhancement effect. At the same time, during the thiol functionalization process, the hydrophilic oxygen-containing functional groups such as hydroxyl, carboxyl, and epoxy groups on the surface of the graphene oxide are reacted, reducing the number of hydrophilic groups on the surface and introducing hydrophobic alkyl chains (-C3H6-). The substrate exhibits a hydrophobic effect and confines the target molecules.

10. A porous silicon detection platform based on graphene oxide-silver nanoparticle modification, characterized in that, It includes the graphene oxide-silver nanoparticle modified porous silicon SERS substrate and the reinforcing agent gold nanoparticles as described in claim 1.

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