COFs-based SERS (Surface Enhanced Raman Scattering) aptamer sensor as well as preparation method and application thereof

Abstract

The invention provides a COFs (Covalent Organic Frameworks)-based SERS (Surface Enhanced Raman Scattering) aptamer sensor as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing COF (at) Ag-cDNA (Complementary Deoxyribose Nucleic Acid) from a COFs probe loaded by silver nanoparticles, preparing Au (at) MBA (at) Ag-apt from a gold-silver core-shell probe embedded with 4-MBA, and mixing the COF (at) Ag-cDNA and the Au (at) MBA (at) Ag-apt to prepare the COFs-based SERS aptamer sensor. According to the SERS aptamer sensor based on the covalent organic framework material COFs, a COFs probe with silver nanoparticles growing on the surface and a gold-silver core-shell probe embedded with a Raman reporter molecule 4-MBA are accurately connected by utilizing a base complementary pairing principle of an aptamer and a complementary chain of the aptamer, so that the hot spot density is effectively increased, the signal intensity is remarkably enhanced, and the detection sensitivity is improved. By establishing a quantitative relation curve between Raman intensity of a characteristic signal peak and mycotoxin concentration, specific quantitative detection of patulin in fruits and vegetables is realized, and the method has the characteristics of simplicity and convenience in operation and quick response, has excellent sensitivity, stability and specificity, and has good accuracy and reliability in detection of fruit and vegetable samples.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapid food safety detection, and relates to a COFs-based SERS aptamer sensor, a preparation method and an application thereof, in particular to the application of detecting patulin (PAT). Background Art

[0002] Patulin (PAT), a fungal secondary metabolite, is widely present in fruits, vegetables, and their processed products. It exhibits severe biotoxicity, causing health hazards such as paralysis, convulsions, and gastrointestinal damage. With the rapid development of global trade, patulin contamination in fruits and vegetables has seriously impacted import and export security. Therefore, there is an urgent need to develop a rapid and highly sensitive patulin detection technology.

[0003] Currently, the detection of patulin relies primarily on traditional analytical methods, including liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC). While these methods offer high accuracy, they are generally limited by complex sample pretreatment, expensive instrumentation, and demanding technical skills. In contrast, immunoassays, while offering advantages such as high specificity and ease of use, still face challenges in practical applications, such as high cost and susceptibility to interference from impurities.

[0004] Surface-enhanced Raman scattering (SERS) technology, as an emerging detection method, shows great potential in food safety testing due to its rapid response, high sensitivity, and unique fingerprint characteristics. Precious metal nanoparticles, as a commonly used SERS substrate, can enhance the Raman signal. However, existing SERS substrates still have limitations in stability and sensitivity, which seriously restricts their application in the practical detection of mycotoxins in fruits and vegetables.

[0005] Therefore, developing a robust and sensitive patulin detection method remains a significant challenge. Breaking through this technical bottleneck will not only improve food safety testing capabilities but also significantly contribute to the healthy development of my country's food industry. Summary of the Invention

[0006] In view of the above technical problems, the present invention aims to provide a COFs-based SERS aptamer sensor and its preparation method and application.

[0007] The SERS aptamer sensor based on covalent organic frameworks (COFs) in the present invention utilizes the base complementary pairing principle of the aptamer and its complementary chain to precisely connect a COFs probe with silver nanoparticles grown on the surface and a gold-silver core-shell probe with an embedded Raman reporter molecule (4-MBA), effectively increasing the hotspot density and significantly enhancing the signal intensity. By establishing a quantitative relationship curve between the Raman intensity of the characteristic signal peak and the fungal toxin concentration, specific quantitative detection of patulin in fruits and vegetables is achieved. The present invention has the characteristics of simple operation and rapid response, and at the same time has excellent sensitivity, stability and specificity. It has good accuracy and reliability in detecting fruit and vegetable samples, providing an efficient solution for rapid food safety detection.

[0008] Note that the inclusion of these objectives does not preclude the existence of other objectives. One embodiment of the present invention does not necessarily achieve all of the above objectives. Objectives other than the above objectives may be extracted from the description of the specification, drawings, and claims.

[0009] This invention addresses the shortcomings of existing patulin detection technologies in terms of sensitivity, stability, and practicality by innovatively developing a COFs-based SERS aptamer sensor, its preparation method, and its application. The sensor's core structure consists of three key components: a COFs probe loaded with silver nanoparticles, a gold-silver core-shell probe modified with an embedded signal molecule, 4-MBA, and an aptamer-complementary chain system that specifically recognizes patulin. This unique structural design successfully enables the efficient detection of patulin in fruits and vegetables, such as apples, and their processed products.

[0010] The present invention addresses the key issue of insufficient substrate stability in existing SERS technology and innovatively uses covalent organic framework (COFs) materials as enhanced probes. COFs, as a new type of porous crystalline polymer, forms a highly ordered two-dimensional or three-dimensional network structure through covalent bonds, and has significant structural stability and controllable physical and chemical properties. Compared with traditional inorganic materials, COFs not only exhibit excellent stability, but their open porous structure and high specific surface area characteristics are more conducive to the uniform loading and stable fixation of metal nanoparticles. The present invention constructs a COF@Ag composite probe by compounding silver nanoparticles (Ag NPs) with COF materials, fully utilizing the structural advantages of COF to enhance the formation of SERS hotspots, while solving the problem of poor stability of traditional SERS substrates.

[0011] The present invention achieves the above technical objectives through the following technical means.

[0012] A method for preparing the COFs-based SERS aptamer sensor comprises the following steps:

[0013] Preparation of silver nanoparticle-loaded COFs probes:

[0014] Step (1) Synthesis of COF materials

[0015] 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxybenzaldehyde were dissolved in a mixed solvent of 1,4-dioxane, n-butanol and methanol and ultrasonically treated. Acetic acid was added for pre-reaction, and then transferred to an autoclave for reaction. The product was washed with tetrahydrofuran and vacuum dried to obtain the target COF material.

[0016] Step (2) Preparation of COF@Ag composite material

[0017] The COF material powder obtained in step (1) and the silver nitrate powder were fully dispersed by ultrasonic treatment in ultrapure water, and then magnetically stirred to form a uniform suspension. After heating to boiling, sodium citrate solution was quickly added and the boiling reaction was maintained to obtain a COF@Ag composite material;

[0018] Step (3) Preparation of aptamer-functionalized COF@Ag-cDNA

[0019] The cDNA was mixed with an equal volume of TCEP buffer and incubated for activation, and the activated cDNA was reacted with the COF@Ag composite material suspension obtained in step (2). The precipitate was centrifuged and resuspended in PBS buffer to obtain COF@Ag-cDNA, which was stored for later use;

[0020] Preparation of gold-silver core-shell probe embedded with 4-MBA:

[0021] Step 1) Synthesis of 4-MBA labeled Au cores

[0022] Ultrapure water and chloroauric acid trihydrate were mixed and boiled, trisodium citrate solution was added to react until the solution turned deep red, and then continued to heat and store to prepare Au core solution; the Au core solution was reacted with 4-MBA solution, the precipitate was centrifuged and resuspended in ultrapure water to obtain 4-MBA labeled Au core solution;

[0023] Step 2) Synthesis of silver-shelled Au@MBA@Ag core-shell nanomaterials

[0024] Trisodium citrate and ascorbic acid were added to the 4-MBA labeled Au core solution obtained in step 1) in sequence, and after mixing evenly, silver nitrate solution was slowly added dropwise to react until the solution turned orange. After centrifugation, the precipitate was resuspended in ultrapure water to prepare Au@MBA@Ag core-shell nanomaterials;

[0025] Step 3) Preparation of aptamer-functionalized Au@MBA@Ag-apt

[0026] After the aptamer apt was activated by incubation with an equal volume of TCEP buffer, the activated aptamer apt was reacted with the Au@MBA@Ag core-shell nanomaterial obtained in step 2), and the precipitate was resuspended in PBS buffer after centrifugation to obtain the aptamer-functionalized Au@MBA@Ag-apt, which was stored in the dark.

[0027] Preparation of COFs-based SERS aptasensors:

[0028] The COF@Ag-cDNA obtained in step (3) and the Au@MBA@Ag-apt obtained in step 3) were mixed and incubated at room temperature. The unbound Au@MBA@Ag-apt and COF@Ag-cDNA were removed by centrifugation, and the precipitate was resuspended in PBS solution to obtain a COFs-based SERS aptamer sensor.

[0029] In the above scheme, in the synthesis of the COF material in step (1), 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxybenzaldehyde are dissolved in a mixed solvent of 1,4-dioxane, n-butanol and methanol and ultrasonicated for 20 minutes. After adding acetic acid, the mixture is pre-reacted at 37°C for 2 hours, and then transferred to an autoclave and reacted at 75°C for 12 hours. The product is washed five times with tetrahydrofuran and then dried in vacuo at 65°C to obtain the target COF material;

[0030] In the preparation of the COF@Ag composite material in step (2), the COF material powder and the silver nitrate powder are ultrasonically dispersed in ultrapure water for 15 minutes, magnetically stirred at 37° C. for 4 hours to form a uniform suspension, heated to boiling, and then rapidly added with sodium citrate solution, and the boiling reaction is maintained for 30 minutes to obtain the COF@Ag composite material;

[0031] In the preparation of the aptamer-functionalized COF@Ag-cDNA in step (3), the cDNA is mixed with TCEP buffer and incubated for 1 hour for activation, the activated cDNA is reacted with the COF@Ag composite material suspension at 37° C. for 12 hours, and the precipitate is resuspended in PBS buffer after centrifugation to obtain COF@Ag-cDNA.

[0032] Furthermore, in step (1), the volume ratio of 1,3,5-tris(4-aminophenyl)benzene powder to 2,5-dimethoxybenzaldehyde powder is 20 mg:15 mg; the volume ratio of 1,4-dioxane solution, n-butanol and methanol solution is 4 mL:4 mL:1 mL; wherein the concentration of the acetic acid solution is 12 M;

[0033] In step (2), the volume ratio of COF powder to silver nitrate powder is 5 mg:30 mg; the volume ratio of ultrapure water to sodium citrate solution is 96 mL:4 mL, wherein the concentration of the trisodium citrate aqueous solution is 1 wt%;

[0034] In step (3), the volume ratio of cDNA, TCEP buffer and COF@Ag composite material is 300 μL:300 μL:4400 μL; the final concentration of the activated complementary chain cDNA in the COF@Ag solution is 600 nM;

[0035] The nucleotide sequence of the cDNA is:

[0036] SH-5'-GCG GGT TGG CGG GCC-3', modified with a thiol group at the 5' end.

[0037] The preparation method of the COFs-based SERS aptamer sensor includes the following steps: 1) in the synthesis of 4-MBA-labeled Au cores, ultrapure water and chloroauric acid trihydrate are mixed and boiled, trisodium citrate solution is added to react until the solution turns deep red, and the solution is heated for 30 minutes and then stored at 4°C to obtain an Au core solution; the Au core solution is reacted with the 4-MBA solution for 40 minutes, and the solution is centrifuged at 8200g for 10 minutes, and the precipitate is resuspended in ultrapure water to obtain a 4-MBA-labeled Au core solution;

[0038] In the synthesis of the silver-shelled Au@MBA@Ag core-shell nanomaterial in step 2), trisodium citrate and ascorbic acid were sequentially added to the 4-MBA-labeled Au core solution, and after uniform mixing, a silver nitrate AgNO3 aqueous solution was slowly added dropwise and reacted for 30 minutes until the solution turned orange. After centrifugation at 6800 g for 8 minutes, the precipitate was resuspended in ultrapure water to obtain the Au@MBA@Ag core-shell nanomaterial;

[0039] In the step 3) of preparing the aptamer-functionalized Au@MBA@Ag-apt, the aptamer apt was activated by incubating with TCEP buffer for 1 hour, and then the activated aptamer apt was reacted with the Au@MBA@Ag core-shell nanomaterial at 37° C. for 12 hours. After centrifugation, the precipitate was resuspended in PBS buffer to obtain the aptamer-functionalized Au@MBA@Ag-apt, which was then stored at 4° C. in the dark.

[0040] Furthermore, in step 1), the volume ratio of ultrapure water, chloroauric acid trihydrate solution and trisodium citrate solution is 99.5 mL:0.5 mL:1.6 mL; wherein the ratio of the Au core to the 4-MBA solution is 1 mL:20 μL;

[0041] In step 2), the volumes of the Au core solution labeled with 4-MBA, the sodium citrate aqueous solution, and the ascorbic acid aqueous solution are 1 mL: 20 μL: 50 μL; wherein the concentration of the trisodium citrate aqueous solution is 1 wt%; and the concentration of the ascorbic acid aqueous solution is 10 mM;

[0042] The final concentration of 4-MBA in the Au@MBA@Ag core-shell nanomaterial mixed solution was 1×10 -7~1×10 -5 M, preferably, the final concentration of 4-MBA in the mixed solution is 5×10 -6 M;

[0043] The concentration of the silver nitrate AgNO3 aqueous solution is 10 mM, and the volume is 20 μL to 70 μL. Preferably, the volume of the silver nitrate AgNO3 aqueous solution is 60 μL;

[0044] The final concentration of the activated aptamer apt in the Au@MBA@Ag solution in step 3) is 600 nM; the nucleotide sequence of the aptamer apt is:

[0045] 5'-GGC CCG CCA ACC CGC ATC ATC TAC ACT GAT ATT TTA CCT T-3'-SH, modified with a thiol group at the 3' end.

[0046] In the above scheme, the preparation of the COFs-based SERS aptamer sensor is specifically as follows:

[0047] The COF@Ag-cDNA obtained in step (3) and the Au@MBA@Ag-apt obtained in step 3) are mixed in a volume ratio of 1:1-1:6. Preferably, the volume ratio of the composite material COF@Ag-cDNA and Au@MBA@Ag-apt is 1:5; and incubated at room temperature for 1 hour. The unbound Au@MBA@Ag-apt and COF@Ag-cDNA are removed by centrifugation at 3600g for 5 minutes, and the precipitate is resuspended in PBS solution to obtain a COFs-based SERS aptamer sensor.

[0048] A COFs-based SERS aptamer sensor is prepared according to the preparation method of the COFs-based SERS aptamer sensor.

[0049] A COFs-based SERS aptamer sensor prepared according to the preparation method of the COFs-based SERS aptamer sensor is used in the detection of patulin.

[0050] In the above scheme, the detection of patulin using the COFs-based SERS aptamer sensor includes the following steps:

[0051] Step S(1) mixing the COFs-based SERS aptamer sensor with a patulin standard solution, capturing the reaction, and collecting Raman spectra;

[0052] Step S(2) establishing a standard curve based on the relationship between the concentration of the patulin standard solution and the characteristic Raman peak of the COFs-based SERS aptamer sensor;

[0053] Step S(3) detecting the content of patulin in the sample to be tested: mixing the sample solution to be tested with the COFs-based SERS aptamer sensor, collecting Raman spectra according to the method of step S(1), and calculating the content of patulin in the sample to be tested according to the standard curve of step S(2).

[0054] In the above scheme, the volume ratio of the SERS aptamer sensor to the patulin standard solution in step S(1) is 1:1; the concentration of the patulin standard solution is 0.1 ng / mL to 250 ng / mL; and the capture reaction time is 1 hour.

[0055] In the above scheme, step S(3) further calculates the content of patulin in the test sample based on the measured concentration of patulin in the test sample. In the present invention, the calculation is performed as follows:

[0056]

[0057] X: The content of patulin in the sample to be tested, in micrograms per kilogram (μg / kg),

[0058] ρ: concentration of patulin in the sample solution, in nanograms per mL (ng / mL),

[0059] V: Final volume of sample solution, in mL (mL).

[0060] m: The weight of the sample to be tested, in grams (g).

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] (1) The COFs-based SERS aptamer sensor of the present invention utilizes a controlled chemical synthesis method to uniformly grow silver nanoparticles on the surface of a COF material. Subsequently, a gold-silver core-shell structure modified with 4-MBA is assembled with an aptamer-complementary chain system, ultimately forming a COFs-based SERS aptamer with high stability and strong signal response. This sensor not only exhibits excellent detection sensitivity but also meets the requirements for rapid batch detection of patulin in fruits, vegetables, and their products, providing a reliable technical solution for food safety testing.

[0063] (2) The present invention constructs for the first time a binary synergistic enhancement system of COF@Ag and gold-silver core-shell, wherein the COF material, with its unique crystalline porous structure and rich surface functional groups, not only provides a stable anchoring site for silver nanoparticles, but also significantly enhances the local electromagnetic field intensity through π-π stacking. At the same time, an innovative embedding method is adopted to pre-embed the signal molecule in the gold-silver core-shell structure, and the local electromagnetic field enhancement effect generated by the nanogap is utilized to obtain a high-intensity and stable SERS signal. In particular, the "aptamer-complementary chain" dual recognition mechanism adopted by the present invention achieves precise regulation of molecular recognition and signal transduction of patulin through specific competitive binding, and the detection sensitivity is improved by about 150 times compared with the existing national standard method. This method effectively reduces external matrix interference by accurately analyzing the attribution of characteristic Raman peaks and establishing a standard curve, achieving high-sensitivity and high-specificity detection of patulin, solving key technical problems such as poor substrate stability and poor signal reproducibility in traditional SERS detection, and providing a reliable theoretical basis and technical support for food safety monitoring.

[0064] (3) During the sensor preparation process, the present invention modifies the aptamer complementary chain on the COFs probe loaded with silver nanoparticles, and simultaneously modifies the specific aptamer on the surface of the gold-silver core-shell probe containing the embedded signal molecule 4-MBA, forming a complete SERS aptamer sensor system. This system utilizes the aptamer's specific recognition of patulin and its competitive binding with the complementary chain to precisely control the enrichment degree of different numbers of probes in the electromagnetic field enhancement area of ​​the substrate surface, thereby achieving effective regulation of the nanoprobe assembly process. This precise regulation mechanism based on molecular recognition significantly improves the analytical performance and detection sensitivity of the SERS spectrum, providing a new technical approach for the efficient detection of patulin.

[0065] (4) The present invention achieves efficient and rapid detection of patulin. The method has a simple operation process. After appropriate pretreatment, the sample to be tested specifically binds to the pre-incubated SERS aptamer sensor. By collecting Raman spectra and analyzing the intensity of the characteristic peak of the signal molecule, a quantitative relationship with the concentration of patulin is established. Experimental results show that in the concentration range of 0.1ng / mL to 250ng / mL, the SERS signal intensity and the patulin concentration show a good linear negative correlation (R 2 =0.992), with a detection limit of 0.0544 ng / mL. In actual sample testing, the method demonstrated excellent anti-interference capabilities, with recoveries ranging from 93.95% to 106.43% and relative standard deviations ranging from 1.23% to 4.40%, fully demonstrating the accuracy and repeatability of its test results.

[0066] In summary, the SERS aptamer sensor system constructed in the present invention has significant advantages such as high sensitivity, good accuracy, and strong anti-interference ability, and provides a reliable technical solution for the rapid and intelligent detection of penicillin.

[0067] Note that the description of these effects does not preclude the existence of other effects. One embodiment of the present invention does not necessarily have all of the above effects. Effects other than the above can be clearly seen and extracted from the description of the specification, drawings, claims, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 Schematic diagram of the preparation process of the COFs-based SERS aptamer sensor and the method for quantitatively detecting patulin in the present invention.

[0069] Figure 2 The morphological characteristics of COF and COF@Ag prepared in Example 1, wherein: Figure 2 A is the morphological characteristics of COF, Figure 2 B is the morphological characteristics of COF@Ag.

[0070] Figure 3 This is the EDS element mapping diagram of COF@Ag prepared in Example 1, corresponding to total elements, carbon, nitrogen, oxygen, and silver elements respectively.

[0071] Figure 4 This is the UV-visible absorption spectrum characterization diagram of COF and COF@Ag in Example 1.

[0072] Figure 5 is the X-ray photoelectron spectrum of COF@Ag prepared in Example 1, wherein, Figure 5 A is the X-ray photoelectron spectrum, Figure 5 B is the Ag3d fine spectrum.

[0073] Figure 6 This is the Fourier infrared characterization image of COF@Ag prepared in Example 1.

[0074] Figure 7 The morphology and particle size distribution of Au@MBA@Ag prepared in Example 1 are shown in FIG. Figure 7 A is the morphological characteristics of Au@MBA@Ag, Figure 7 B is the particle size distribution diagram.

[0075] Figure 8 This is the UV-visible absorption spectrum characterization diagram of the Au core and Au@MBA@Ag in Example 1.

[0076] Figure 9The UV-visible spectra and Raman signal intensity changes of Au@MBA@Ag synthesized with different amounts of AgNO3 added in Example 1, where: Figure 9 A is the UV-visible spectrum, Figure 9 B is the change in Raman signal intensity.

[0077] Figure 10 The Raman signal intensity changes of Au@MBA@Ag prepared with different concentrations of the signal molecule 4-MBA in Example 1.

[0078] Figure 11 The Raman spectra of the probes synthesized from two composite materials, COF@Ag-cDNA and Au@MBA@Ag-apt, at different volume ratios are shown. Figure 11 A is the SERS spectra of the mixture of COF@Ag-cDNA and Au@MBA@Ag-apt with different volume ratios. Figure 11 B is the quantitative analysis result.

[0079] Figure 12 Raman characterization of COF@Ag, Au@MBA@Ag and SERS aptamer sensors prepared in Example 1.

[0080] Figure 13 This is the specificity verification result of the COFs-based SERS aptamer sensor detection in Example 2.

[0081] Figure 14 This is the stability verification result of the COFs-based SERS aptamer sensor detection in Example 3.

[0082] Figure 15 The COFs-based SERS aptamer sensor in Example 4 detects Raman spectra of patulin standard samples with different concentrations. The Raman spectra of patulin standard samples with different concentrations are at 1584 cm -1 The relationship curve between the peak value at the characteristic peak and its concentration, where Figure 15 A is the Raman spectrum, Figure 15 B is the relationship curve between the peak value and its concentration. DETAILED DESCRIPTION

[0083] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following is a detailed explanation with reference to specific embodiments, but the following embodiments do not limit the scope of protection of the present invention.

[0084] In the embodiments of the present invention, those that are not described in detail are all completed using conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail.

[0085] Example 1: Preparation of a SERS aptamer sensor for detecting patulin

[0086] like Figure 1 As shown, a method for preparing a COFs-based SERS aptamer sensor comprises the following steps:

[0087] (1) Synthesis of COF materials

[0088] First, 20 mg of 1,3,5-tris(4-aminophenyl)benzene and 15 mg of 2,5-dimethoxybenzaldehyde were dissolved in 9 mL of a mixed solvent consisting of 1,4-dioxane, n-butanol, and methanol (wherein the volume ratio of 1,4-dioxane, n-butanol, and methanol was 4 mL:4 mL:1 mL), and the mixture was fully dissolved by ultrasonic treatment for 20 min. Subsequently, 10 mL of 12 M acetic acid solution was added as a catalyst, and the mixture was allowed to react at 37°C for 2 h to complete the prepolymerization. The reaction system was then transferred to a high-pressure reactor and subjected to a solvent thermal reaction at 75°C for 12 h to construct the COF skeleton. After the reaction was completed, the obtained solid product was repeatedly washed five times with tetrahydrofuran to remove unreacted substances and by-products, and finally vacuum dried at 65°C overnight to obtain the target COF material.

[0089] (2) Synthesis of COF@Ag

[0090] 5 mg of COF powder and 30 mg of silver nitrate were fully dispersed in 96 mL of ultrapure water by ultrasonic treatment for 15 minutes, and then magnetically stirred for 4 hours at 37°C to form a uniform suspension. The suspension was then heated to boiling while stirring, and after the temperature stabilized, 4 mL of sodium citrate solution was quickly injected for reduction reaction. Finally, the boiling state was maintained and the reaction continued for 30 minutes to obtain the COF@Ag composite material.

[0091] (3) Preparation of aptamer-functionalized COF@Ag-cDNA

[0092] 300 μL of cDNA was thoroughly mixed with an equal volume of TCEP buffer and incubated under appropriate conditions for 1 hour to complete the activation of the cDNA. Subsequently, 600 μL of the activated cDNA solution was mixed with 4400 μL of COF@Ag suspension and incubated at a constant temperature of 37°C for 12 hours to promote the full binding of cDNA and COF@Ag material. After the reaction was completed, the unbound free cDNA was removed by centrifugation, the precipitate was collected and redispersed in 5 mL of PBS buffer, and the resulting product was finally stored at 4°C for use.

[0093] The nucleotide sequence of the complementary chain cDNA is: SH-5'-GCG GGT TGG CGG GCC-3', with a thiol group modified at the 5' end.

[0094] The present invention confirms the successful preparation and structural characteristics of COF@Ag composite materials through various characterization methods. Figure 2 As shown in A, the synthesized COF material presents a nearly spherical porous structure with typical covalent organic framework morphology characteristics. Figure 2 B shows that the silver nanoparticles in the COF@Ag composite material are evenly and densely distributed on the COF surface, forming a good composite structure.

[0095] EDS energy spectrum analysis Figure 3 As shown in Figure 2, the presence of silver was clearly detected, confirming the successful loading of silver nanoparticles. Figure 4 As shown in the Figure 3, COFs have a broad absorption band in the range of 300 nm to 700 nm, while COF@Ag exhibits an obvious surface plasmon resonance absorption peak at 422 nm, and the half-peak width is significantly reduced, which is attributed to the successful loading of silver nanoparticles and their interaction with the COF matrix.

[0096] X-ray photoelectron spectroscopy Figure 5 As shown in A, it shows characteristic peaks such as Ag3d, O1s, N1s and C1s, among which the Ag3d fine spectrum is as follows Figure 5 B can be fitted to Ag 0 (374.3 and 368.3 eV) and Ag + (375.1 and 368.9 eV), indicating that there are silver nanoparticles of different chemical states in the composite material.

[0097] Figure 6 Fourier transform infrared spectroscopy showed that COF and COF@Ag have the highest peak at 1588 cm -1 The presence of distinct C=N stretching vibration peaks at both ends confirms the formation of imine bonds, while the appearance of characteristic new peaks in COF@Ag further verifies the successful loading of AgNPs. These characterization results reinforce each other and fully demonstrate the successful preparation and structural properties of the COF@Ag composite.

[0098] S2. Preparation of gold-silver core-shell probes embedded with 4-MBA

[0099] (1) Synthesis of 4-MBA-labeled Au cores

[0100] First, a hydrothermal method was used to prepare the Au core solution. 99.5 mL of ultrapure water was mixed with 0.5 mL of chloroauric acid trihydrate and heated to boiling with continuous stirring. 1.6 mL of trisodium citrate solution was then quickly added to initiate a reduction reaction. The solution's color changed significantly from pale yellow to grayish-black, purple, and finally deep red, indicating the successful formation of gold nanoparticles and the resulting Au core solution. After the reaction was complete, the solution was heated for an additional 30 minutes to stabilize the product. The Au core solution was then stored at 4°C until ready for use.

[0101] Then, the 4-MBA labeling step was carried out. 20 μL of 4-MBA solution was slowly added to 1 mL of the above-mentioned Au core solution and stirred continuously for 40 min to achieve full modification. After the reaction was completed, the product was collected by centrifugation at 8200 g for 10 min, and the precipitate was finally redispersed in 1 mL of ultrapure water to obtain the 4-MBA labeled Au core solution.

[0102] (2) Synthesis of Ag-shelled Au@MBA@Ag

[0103] 20 μL of trisodium citrate and 50 μL of ascorbic acid were added sequentially as stabilizers and reducing agents to the 4-MBA-labeled Au core solution, and the mixture was thoroughly mixed to form a uniform system. Subsequently, under continuous stirring, 60 μL of silver nitrate solution was slowly introduced into the reaction system at a precisely controlled dropwise addition rate. After 30 minutes of reaction, the color of the solution changed significantly and eventually exhibited a characteristic orange color, indicating the successful deposition of the silver shell on the Au core surface. After the reaction was completed, the product was centrifuged at 6800 g for 8 minutes to remove unreacted materials, and the collected precipitate was redispersed in 1 mL of ultrapure water to obtain Au@MBA@Ag nanomaterials with a uniform core-shell structure.

[0104] (3) Preparation of aptamer-functionalized Au@MBA@Ag-apt

[0105] 300 μL of apt was mixed with an equal volume of TCEP buffer and incubated under appropriate conditions for 1 hour to fully open the disulfide bond structure of the aptamer apt, achieving aptamer activation. Subsequently, 600 μL of the activated aptamer solution was mixed with 4400 μL of Au@MBA@Ag nanomaterial suspension and incubated at 37°C for 12 hours to covalently bind the aptamer to the nanomaterial surface via Au-S bonds. After the reaction was complete, unbound free aptamer was removed by centrifugation, and the collected nanoprobe precipitate was redispersed in 5 mL of PBS buffer. Finally, the functionalized nanoprobe Au@MBA@Ag-apt was stored at 4°C in the dark until further use.

[0106] The nucleotide sequence of the aptamer apt is: 5'-GGC CCG CCA ACC CGC ATC ATC TAC ACTGAT ATT TTA CCT T-3'-SH, and a thiol group is modified at the 3' end.

[0107] The present invention confirms the successful preparation and performance characteristics of Au@MBA@Ag core-shell nanomaterials through systematic material characterization and condition optimization. Figure 7 As shown in Figure A, transmission electron microscopy characterization shows that the synthesized Au@MBA@Ag nanoparticles have a regular spherical core-shell structure, showing good size uniformity and dispersion. It can be clearly observed that the darker gold core is evenly coated by the brighter silver shell. This result directly confirms the successful deposition of silver on the surface of the gold seed. Particle size statistical analysis results Figure 7 B shows that the average diameter of the original gold core is 28.2nm, while the average diameter of the final Au@MBA@Ag core-shell particles is 43.7nm. The average thickness of the silver shell calculated from this is 7.75nm. This data further verifies the formation of the core-shell structure.

[0108] The optical properties of Au core and Au@MBA@Ag were systematically characterized by UV-visible absorption spectroscopy, e.g. Figure 8 As shown, the absorption peak observed at 522 nm is attributed to the localized surface plasmon resonance of the Au core. After depositing an appropriate amount of silver on the gold core surface, characteristic changes in the UV absorption spectrum were observed, with the gold core plasmon resonance peak significantly blue-shifted to approximately 486 nm, and a new absorption band appeared at 404 nm. These findings indicate that the silver shell has successfully formed and grown on the gold core structure.

[0109] In order to enhance the Raman signal of the gold-silver core-shell probe embedded with 4-MBA, the amount of AgNO3 added was optimized. Figure 9 As shown in Figure 2, when the volume of AgNO3 increases from 20 μL to 70 μL, the absorption peak of the Au core shifts blue, accompanied by a gradual increase in the absorbance of the silver peak. The plasmonic properties of the silver shell become more pronounced, eventually masking the gold absorption peak. The SERS detection signal spectrum is shown in Figure 2. Figure 9 As shown in Figure B, the Raman signal intensity of Au@MBA@Ag shows a characteristic volcano-shaped dependence based on the change in the amount of AgNO3 added, reaching a maximum when the optimal AgNO3 volume is 60μL. Beyond this volume, the signal intensity decreases.

[0110] The concentration of 4-MBA, as a Raman marker, has a direct impact on the signal intensity of the aptamer sensor. Therefore, the concentration of 4-MBA was further optimized based on the optimal addition amount of AgNO3 solution. Figure 10As shown, as the 4-MBA concentration increased from 1×10 -7 M increased to 5×10 -6 M, the Raman signal gradually increases. In addition, it further increases to 1×10 -5 M, the signal increased only slightly. It is worth noting that the experiment showed that the 4-MBA concentration was 1×10 -5 M, it is easy to cause Au@MBA@Ag to aggregate. Therefore, 5×10 -6 M was determined to be the most effective 4-MBA concentration.

[0111] S3. Preparation of COFs-based SERS aptasensors

[0112] The COF@Ag-cDNA and Au@MBA@Ag-apt prepared above were mixed at a volume ratio of 1:5 and incubated undisturbed at room temperature for 1 hour. Unbound Au@MBA@Ag-apt and COF@Ag-cDNA were then removed by centrifugation at 3600g for 5 minutes, and the pellet was resuspended in PBS solution to obtain the COFs-based SERS aptasensor.

[0113] The present invention successfully constructed a highly sensitive SERS aptamer sensor through systematic experimental optimization and characterization analysis. Figure 11 As shown in Figure A, by testing the SERS spectra of COF@Ag-cDNA and Au@MBA@Ag-apt mixtures with different volume ratios, the effect of volume ratio on sensor performance was systematically investigated. Figure 11 The quantitative analysis results of B showed that when COF@Ag-cDNA and Au@MBA@Ag-apt were mixed at a volume ratio of 1:5, the characteristic peak at 1584 cm -1 The SERS signal intensity observed at 400 nm reached its maximum value, and this optimized ratio was determined to be the best reaction condition for sensor construction.

[0114] Figure 12 Raman spectroscopy further confirmed the feasibility of the sensor: COF@Ag alone exhibited no discernible characteristic Raman peaks, but when COF@Ag-cDNA formed a complex with Au@MBA@Ag-apt through base complementary pairing, the Raman signal was significantly enhanced. This significant signal enhancement fully demonstrated the successful construction of the SERS aptamer sensor and provided a reliable technical foundation for subsequent high-sensitivity detection.

[0115] Example 2: Specific detection of COFs-based SERS aptamer sensors

[0116] This example uses the detection of different types of toxin standards and their mixtures with patulin to verify the specificity of the COFs-based SERS aptamer sensor prepared in Example 1.

[0117] In response to the coexistence of multiple mycotoxins in fruit matrices, the present invention specifically selected Alternaria alternata (AOH) and ochratoxin A (OTA) as representative interfering substances to evaluate the specificity of the developed patulin (PAT) detection method. These two toxins are often present together with PAT in contaminated fruit products. The experiment prepared 10 ng / mL PAT, 10 ng / mL AOH, 10 ng / mL OTA, and mixed solutions of PAT with AOH or OTA (the final concentration of each toxin was 10 ng / mL). 100 μL of these sample solutions were mixed with 100 μL of the SERS aptamer sensor and the Raman spectrum was collected.

[0118] like Figure 13 As shown, at 1584cm -1 Analysis of the Raman signal intensity at the characteristic peaks showed that the COFs-based SERS aptasensor specifically recognized only PAT molecules. AOH and OTA, when present alone, did not bind to the aptamer, resulting in no significant difference in signal intensity compared to the blank control. However, in the presence of PAT, both PAT and other toxins, specifically bound to the aptamer and released signal molecules, significantly reducing the signal intensity. This result fully demonstrates that the COFs-based SERS aptasensor described in this invention has highly specific recognition capabilities for patulin and can effectively eliminate interference from other coexisting mycotoxins.

[0119] Example 3: Stability testing of COFs-based SERS aptamer sensors

[0120] In this embodiment, the COFs-based SERS aptamer sensor prepared in Example 1 was stored in a 4° C. environment, and the Raman signal intensity was detected at intervals to verify the detection stability of the sensor.

[0121] In order to evaluate the stability of the aptasensor in detecting patulin, the aptasensor formed by the ligation and incubation of Au@MBA@Ag-apt and COF@Ag-cDNA was stored at 4 °C for 20 days, and its fluorescence at 1584 cm was measured regularly at intervals of 5 days. -1 The Raman signal intensity at the characteristic peak. Figure 14 As shown in the figure, during the entire 20-day test period, the SERS signal intensity of the characteristic peak did not show significant fluctuations. This experimental result fully proves that the SERS aptamer sensor developed in the present invention has excellent long-term stability under low-temperature storage conditions and can meet the needs of actual detection applications.

[0122] Example 4: Application of COFs-based SERS aptamer sensor for quantitative detection of patulin in apple fruit

[0123] The present invention verifies the practical application performance of COFs-based SERS aptasensor in detecting patulin in real apple samples through systematic experiments. The specific steps are as follows:

[0124] (1) Testing of standard products

[0125] First, patulin standard solutions of varying concentrations were prepared. 100 μL of these solutions were reacted with an equal volume of SERS aptamer sensor solution for 1 hour. Detection was performed using a fully automated Raman microscope, initially using a 5x objective for coarse focus adjustment, then switching to a 10x objective for optimal imaging.

[0126] The acquisition parameters were set as follows: 638 nm excitation wavelength, 600 nm grating, 100% attenuation power, and 2 s integration time. Each standard was measured in parallel at least five times.

[0127] (2) Drawing of standard curve

[0128] Reliable quantitative and qualitative analysis of raw SERS spectra can effectively improve the accuracy of the detection method. LabSpec6 software pre-processes the raw spectra using chemometric methods to eliminate background drift caused by fluorescence absorption. The smoothing function smoothes the spectra to improve spectral quality.

[0129] Figure 15 A shows the Raman spectra collected when using a COFs-based SERS aptamer sensor to measure standard samples of patulin with different concentrations. During the detection process, it was found that as the concentration of patulin increased, the intensity of the Raman characteristic peak of the signal molecule steadily decreased.

[0130] In order to evaluate the detection effect of the SERS aptasensor for quantitative analysis, the signal molecule 4-MBA was plotted at 1584 cm -1 The functional relationship between the Raman peak intensity at and the concentration of patulin is shown in the figure. Figure 15 As shown in B, the OriginPro8.5 software was used to analyze the 1584 cm -1 The Raman peak intensity was linearly fitted, and the fitting results showed that the SERS aptamer sensor had a good linear relationship for the detection of patulin at different concentrations. The linear range of patulin quantification of the present invention was 0.1 ng / mL to 250 ng / mL, and the detection limit of patulin was calculated to be 0.0544 ng / mL.

[0131] (3) Pretreatment of apple fruit samples

[0132] Apples were chopped and crushed, and 5 grams of puree was spiked with different concentrations of patulin standard solution (10 μg / kg, 20 μg / kg, and 40 μg / kg) to simulate toxin-contaminated apple tissue. 10 mL of water and 20 mg of pectinase were added to the puree and enzymatic hydrolysis was performed in a 40°C waterbath for 1 hour. Then, 15 mL of the extraction solution (9:1 acetonitrile:water by volume) was added to different spiked tubes. The mixture was shaken for 15 minutes and then centrifuged at 5200 g for another 15 minutes. The precipitate was discarded, and the volume was made up to 25 mL with water to obtain the test sample solution.

[0133] (4) Testing of apple fruit samples

[0134] 100 μL of the sample solution to be tested was mixed with 100 μL SERS aptamer sensor and captured before collecting Raman spectra. The acquisition parameter settings and data processing and analysis methods were the same as those used to establish the standard curve. Based on the intensity of the acquired spectrum, the established standard curve was called to calculate the concentration of patulin in the sample solution.

[0135] The content of patulin in the sample to be tested is further calculated based on the calculated patulin concentration, and in the present invention, it is calculated by the following formula:

[0136]

[0137] X: The amount of patulin in the sample to be tested, in micrograms per kilogram (μg / kg)

[0138] ρ: The concentration of patulin in the test sample, in nanograms per milliliter (ng / mL)

[0139] V: Final volume of test sample, in milliliters (mL)

[0140] m: The weight of the sample to be tested, in grams (g).

[0141] The detection values ​​and recovery rates determined by the method of the present invention are compared with those by liquid chromatography, as shown in Table 1. The results show that the recovery rates are between 95.93% and 104.85%, which are close to the results of HPLC, verifying the reliability of the method of the present invention.

[0142] Table 1 Comparison between the method of the present invention and liquid chromatography detection

[0143]

[0144] Example 5: Application of COFs-based SERS aptasensor for quantitative detection of patulin in commercial apple juice

[0145] In this example, a standard substance was added to commercial fruit juice that did not contain patulin to verify the practical applicability of the method for detecting patulin of the present invention.

[0146] First, 5 mL of commercially available juice sample was spiked with 15, 25, and 45 μg / kg of patulin standard solution. An appropriate amount of ultrapure water was added to thoroughly mix the mixture and the volume was adjusted to 25 mL to prepare a series of patulin extracts. Next, 100 μL of the extract was mixed with an equal volume of COFs-based SERS aptamer sensor solution. After sufficient capture, Raman spectra were collected. Spectral acquisition employed the same parameters and data processing as those used to establish the standard curve, including a 638 nm excitation wavelength, a 600 nm grating, 100% attenuation power, and a 2 s integration time. Finally, quantitative analysis was performed based on the measured spectral characteristic peak intensities, using a pre-established standard curve to accurately calculate the actual patulin concentration in the test sample.

[0147] The patulin content in spiked commercial juice samples was calculated using the same formula as in Example 4. The detection values ​​and recoveries determined by the present method are shown in Table 2. The recoveries ranged from 93.95% to 106.43%, similar to those obtained by HPLC. This method is simple to operate and provides accurate and reliable results, providing an effective technical tool for monitoring patulin contamination in juice products.

[0148] Table 2 Test results of spiked samples by the method of the present invention

[0149]

[0150] The present invention designs a COFs-based SERS aptamer sensor. A COFs probe with a uniform surface load of silver nanoparticles is prepared by a hydrothermal synthesis method. At the same time, a gold-silver core-shell probe modified with an embedded signal molecule 4-MBA is prepared by a seed growth method. By modifying the surfaces of these two probes with a patulin-specific aptamer and its complementary chain, respectively, a recognition system based on the principle of base complementary pairing is constructed. This system precisely regulates the formation process of the nanoprobe through a specific competitive recognition mechanism, effectively reducing nonspecific interference and significantly improving the analytical performance and detection sensitivity of the SERS spectrum. In addition, the present invention establishes a reliable quantitative detection method that can achieve high-sensitivity and rapid detection of patulin with only a trace amount of sample, with a detection limit of 0.0544 ng / mL, providing an efficient and reliable technical means for food safety monitoring.

[0151] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents. All technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for preparing a COFs-based SERS aptamer sensor, characterized in that: The steps include: Preparation of silver nanoparticle-loaded COFs probes: Step (1) Synthesis of COF materials 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxybenzaldehyde were dissolved in a mixed solvent of 1,4-dioxane, n-butanol and methanol and ultrasonically treated. Acetic acid was added for pre-reaction, and then transferred to an autoclave for reaction. The product was washed with tetrahydrofuran and vacuum dried to obtain the target COF material. Step (2) Preparation of COF@Ag composite material The COF material powder obtained in step (1) and the silver nitrate powder were fully dispersed by ultrasonic treatment in ultrapure water, and then magnetically stirred to form a uniform suspension. After heating to boiling, sodium citrate solution was quickly added and the boiling reaction was maintained to obtain a COF@Ag composite material; Step (3) Preparation of aptamer-functionalized COF@Ag-cDNA The cDNA was mixed with an equal volume of TCEP buffer and incubated for activation, and the activated cDNA was reacted with the COF@Ag composite material suspension obtained in step (2). The precipitate was centrifuged and resuspended in PBS buffer to obtain COF@Ag-cDNA, which was stored for later use; Preparation of gold-silver core-shell probe embedded with 4-MBA: Step 1) Synthesis of 4-MBA labeled Au cores Ultrapure water and chloroauric acid trihydrate were mixed and boiled, trisodium citrate solution was added to react until the solution turned deep red, and then continued to heat and store to prepare Au core solution; the Au core solution was reacted with 4-MBA solution, the precipitate was centrifuged and resuspended in ultrapure water to obtain 4-MBA labeled Au core solution; Step 2) Synthesis of silver-shelled Au@MBA@Ag core-shell nanomaterials Trisodium citrate and ascorbic acid were added to the 4-MBA labeled Au core solution obtained in step 1) in sequence, and after mixing evenly, silver nitrate solution was slowly added dropwise to react until the solution turned orange. After centrifugation, the precipitate was resuspended in ultrapure water to prepare Au@MBA@Ag core-shell nanomaterials; Step 3) Preparation of aptamer-functionalized Au@MBA@Ag-apt After the aptamer apt was activated by incubation with an equal volume of TCEP buffer, the activated aptamer apt was reacted with the Au@MBA@Ag core-shell nanomaterial obtained in step 2), and the precipitate was resuspended in PBS buffer after centrifugation to obtain the aptamer-functionalized Au@MBA@Ag-apt, which was stored in the dark. Preparation of COFs-based SERS aptasensors: The COF@Ag-cDNA obtained in step (3) and the Au@MBA@Ag-apt obtained in step 3) were mixed and incubated at room temperature. The unbound Au@MBA@Ag-apt and COF@Ag-cDNA were removed by centrifugation, and the precipitate was resuspended in PBS solution to obtain a COFs-based SERS aptamer sensor.

2. The method for preparing a COFs-based SERS aptamer sensor according to claim 1, wherein: In the synthesis of the COF material in step (1), 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxybenzaldehyde were dissolved in a mixed solvent of 1,4-dioxane, n-butanol, and methanol, and ultrasonicated for 20 minutes. After adding acetic acid, the mixture was pre-reacted at 37° C. for 2 hours, and then transferred to an autoclave and reacted at 75° C. for 12 hours. The product was washed five times with tetrahydrofuran and then dried in vacuo at 65° C. to obtain the target COF material. In the preparation of the COF@Ag composite material in step (2), the COF material powder and the silver nitrate powder are ultrasonically dispersed in ultrapure water for 15 minutes, magnetically stirred at 37° C. for 4 hours to form a uniform suspension, heated to boiling, and then rapidly added with sodium citrate solution, and the boiling reaction is maintained for 30 minutes to obtain the COF@Ag composite material; In the preparation of the aptamer-functionalized COF@Ag-cDNA in step (3), the cDNA is mixed with TCEP buffer and incubated for 1 hour for activation, the activated cDNA is reacted with the COF@Ag composite material suspension at 37° C. for 12 hours, and the precipitate is resuspended in PBS buffer after centrifugation to obtain COF@Ag-cDNA.

3. The method for preparing a COFs-based SERS aptamer sensor according to claim 2, wherein: In step (1), the volume ratio of 1,3,5-tris(4-aminophenyl)benzene powder to 2,5-dimethoxybenzaldehyde powder is 20 mg:15 mg; the volume ratio of 1,4-dioxane solution, n-butanol and methanol solution is 4 mL:4 mL:1 mL; wherein the concentration of the acetic acid solution is 12 M; In step (2), the volume ratio of COF powder to silver nitrate powder is 5 mg:30 mg; the volume ratio of ultrapure water to sodium citrate solution is 96 mL:4 mL, wherein the concentration of the trisodium citrate aqueous solution is 1 wt%; In step (3), the volume ratio of cDNA, TCEP buffer and COF@Ag composite material is 300 μL:300 μL:4400 μL; the final concentration of the activated complementary chain cDNA in the COF@Ag solution is 600 nM; The nucleotide sequence of the cDNA is: SH-5'-GCG GGT TGG CGG GCC-3', modified with a thiol group at the 5' end.

4. The method for preparing a COFs-based SERS aptamer sensor according to claim 1, wherein: In the step 1) of synthesizing the 4-MBA-labeled Au core, ultrapure water and chloroauric acid trihydrate are mixed and boiled, trisodium citrate solution is added and reacted until the solution turns deep red, and the mixture is heated for 30 minutes and then stored at 4° C. to obtain an Au core solution; the Au core solution is reacted with the 4-MBA solution for 40 minutes, and the mixture is centrifuged at 8200 g for 10 minutes, and the precipitate is resuspended in ultrapure water to obtain a 4-MBA-labeled Au core solution; In the synthesis of the silver-shelled Au@MBA@Ag core-shell nanomaterial in step 2), trisodium citrate and ascorbic acid were sequentially added to the 4-MBA-labeled Au core solution, and after uniform mixing, a silver nitrate AgNO3 aqueous solution was slowly added dropwise and reacted for 30 minutes until the solution turned orange. After centrifugation at 6800 g for 8 minutes, the precipitate was resuspended in ultrapure water to obtain the Au@MBA@Ag core-shell nanomaterial; In the step 3) of preparing the aptamer-functionalized Au@MBA@Ag-apt, the aptamer apt was activated by incubating with TCEP buffer for 1 hour, and then the activated aptamer apt was reacted with the Au@MBA@Ag core-shell nanomaterial at 37° C. for 12 hours. After centrifugation, the precipitate was resuspended in PBS buffer to obtain the aptamer-functionalized Au@MBA@Ag-apt, which was then stored at 4° C. in the dark.

5. The method for preparing a COFs-based SERS aptamer sensor according to claim 4, wherein: In the step 1), the volume ratio of ultrapure water, chloroauric acid trihydrate solution and trisodium citrate solution is 99.5 mL:0.5 mL:1.6 mL; wherein the ratio of the Au core to the 4-MBA solution is 1 mL:20 μL; In step 2), the volumes of the Au core solution labeled with 4-MBA, the sodium citrate aqueous solution, and the ascorbic acid aqueous solution are 1 mL: 20 μL: 50 μL; wherein the concentration of the trisodium citrate aqueous solution is 1 wt%; and the concentration of the ascorbic acid aqueous solution is 10 mM; The final concentration of 4-MBA in the Au@MBA@Ag core-shell nanomaterial mixed solution was 1×10 -7 ~1×10 -5 M; The concentration of the silver nitrate AgNO3 aqueous solution is 10 mM, and the volume is 20 μL to 70 μL. Preferably, the volume of the silver nitrate AgNO3 aqueous solution is 60 μL; Preferably, the final concentration of 4-MBA in the mixed solution is 5×10 -6 M; The final concentration of the activated aptamer apt in the Au@MBA@Ag solution in step 3) is 600 nM; the nucleotide sequence of the aptamer apt is: 5'-GGC CCG CCA ACC CGC ATC ATC TAC ACT GAT ATT TTA CCT T-3'-SH, modified with a thiol group at the 3' end.

6. The method for preparing a COFs-based SERS aptamer sensor according to claim 1, wherein: The preparation of the COFs-based SERS aptamer sensor is specifically as follows: The COF@Ag-cDNA obtained in step (3) and the Au@MBA@Ag-apt obtained in step 3) were mixed in a volume ratio of 1:1-1:6 and incubated at room temperature for 1 h. The unbound Au@MBA@Ag-apt and COF@Ag-cDNA were removed by centrifugation at 3600 g for 5 min, and the precipitate was resuspended in PBS solution to obtain a COFs-based SERS aptamer sensor; Preferably, the volume ratio of COF@Ag-cDNA and Au@MBA@Ag-apt is 1:

5.

7. A COFs-based SERS aptamer sensor, characterized in that: The COFs-based SERS aptamer sensor is prepared according to the preparation method of any one of claims 1-6.

8. Use of a COFs-based SERS aptamer sensor prepared according to the preparation method of a COFs-based SERS aptamer sensor according to claim 7 or any one of claims 1 to 6 in detecting patulin.

9. Use of the COFs-based SERS aptamer sensor in detecting patulin according to claim 8, characterized in that: The detection of patulin using the COFs-based SERS aptamer sensor includes the following steps: Step S(1) mixing the COFs-based SERS aptamer sensor with a patulin standard solution, capturing the reaction, and collecting Raman spectra; Step S(2) establishing a standard curve based on the relationship between the concentration of the patulin standard solution and the characteristic Raman peak of the COFs-based SERS aptamer sensor; Step S(3) detecting the content of patulin in the sample to be tested: mixing the sample solution to be tested with the COFs-based SERS aptamer sensor, collecting Raman spectra according to the method of step S(1), and calculating the content of patulin in the sample to be tested according to the standard curve of step S(2).

10. Use of the COFs-based SERS aptamer sensor in detecting patulin according to claim 9, characterized in that: In step S(1), the volume ratio of the SERS aptamer sensor to the patulin standard solution is 1:1; the concentration of the patulin standard solution is 0.1 ng / mL to 250 ng / mL; and the capture reaction time is 1 hour.

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