A method for detecting aflatoxin B1 based on covalent organic framework nanochannels

Abstract

A method for detecting aflatoxin B1 based on covalent organic framework nanochannels. The present application relates to a covalent organic framework nanochannel membrane based on aptamer functionalization and its application in the detection of aflatoxin B1, belonging to the field of trace food contaminant detection and nanochannel analysis technology. The method comprises the following steps: modification of anodic aluminum oxide (AAO) nanochannels; in situ synthesis of a covalent organic framework (COF) membrane on the AAO membrane by a bottom-up approach; covalent bonding of an aptamer (Apt) with a specific recognition function to the COF membrane; using the Apt / COF / AAO membrane as a nanochannel and the Apt as a recognition unit to construct an Apt / COF / AAO sensor for sensitive and selective detection of trace AFB1 in food. The selective change of the surface charge of the nanochannel is reflected in a significant change in the ion current, thereby achieving selective and sensitive detection of trace aflatoxin B1 in food. The sensor has the advantages of high sensitivity, simple operation, low cost, and good economy; and has broad application prospects in the fields of medicine, food safety, and environmental science.

Description

Technical Field

[0001] The present application relates to a method for sensitively and selectively detecting trace amounts of aflatoxin B1 based on aptamer-functionalized covalent organic framework nanochannels, belonging to the technical field of trace mycotoxin detection and nanochannel analysis. Background Art

[0002] Mycotoxins are secondary metabolites produced by certain toxin-producing fungi under favorable conditions. They primarily include aflatoxins (AFs), deoxynivalenol (DON), ochratoxin A (OTA), zearalenone (ZEN), and fumonisins (FB). Aflatoxin B1 (AFB1), a cyclic difuran toxin produced by Aspergillus, is the most toxic of all aflatoxins and has been linked to hepatocellular carcinoma, growth suppression, immune system modulation, and malnutrition. Foods including peanuts, corn, wheat, and tree nuts can be contaminated with aflatoxin B1 during cultivation, harvesting, storage, and processing.

[0003] Currently, food contaminant detection technology is relatively mature, but in practice, food contaminant detection mainly relies on cumbersome instruments such as gas chromatography, high-performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Although these instruments all have certain detection accuracy and stability, they are limited by limitations such as bulky instruments, chemical pretreatment, and complex operations, making it impossible to achieve rapid or non-destructive on-site detection of food contaminants.

[0004] Nanochannel analysis, an emerging sensing technology that effectively integrates coulometric counting with channel ion current measurement, has attracted considerable attention due to its high sensitivity, fast transmission speed, and ease of miniaturization. Covalent organic frameworks (COFs) have shown great potential for food contaminant detection and analysis due to their porosity, structural scalability, functional tunability, and controllable pore size.

[0005] Therefore, a real-time, sensitive, and highly selective detection technology is needed for the detection of food contaminants to minimize the risk of food contaminants endangering food safety. Summary of the Invention

[0006] Aptamers (Apts) are widely used for the detection of trace amounts of AFB1 in food due to their high stability, high affinity and specificity, and ease of modification. COFs, with their ordered porous structure and tunable physicochemical properties, provide an ideal platform for immobilizing aptamers and enhancing their target recognition capabilities. Combining Apts with COFs is a promising approach to improve biosensor performance, particularly in the field of food safety, with the potential for efficient and specific capture of AFB1 in complex food matrices.

[0007] In order to solve the above problems, the present application provides a covalent organic framework nanochannel membrane based on aptamer functionalization, and based on this, provides a method for sensitive and selective detection of trace aflatoxin B1. First, a COF / AAO membrane is synthesized as a nanochannel by a bottom-up method; the aptamer covalently bound to the COF surface is used as a recognition unit to construct a new nanochannel sensing platform, which is applied to the detection of trace aflatoxin B1 in food matrices.

[0008] The first aspect of the present application proposes a method for preparing a covalent organic framework nanochannel membrane based on aptamer functionalization, which is characterized by comprising the following steps:

[0009] Step S11: modifying the anodic aluminum oxide film with an amino functional solution, washing it, and then baking it at a high temperature to obtain an amino functional AAO film;

[0010] Step S12: dissolving the triamine monomer and the dialdehyde monomer in an organic solvent, and then immersing the amino-functionalized AAO membrane prepared in step S11, and washing to obtain an amorphous COF / AAO membrane;

[0011] Step S13: preparing a reaction solvent required for the solvothermal reaction of COF to obtain solution B, and then immersing the amorphous COF / AAO membrane obtained in step S12 in solution B again for reaction, and then washing and drying to obtain a COF / AAO membrane;

[0012] The solution B is a mixture of acetonitrile and acetic acid;

[0013] Step S14: reacting the COF / AAO membrane obtained in step S13 with an aptamer buffer solution, and washing to obtain an Apt / COF / AAO membrane.

[0014] Furthermore, step S11 specifically includes: ultrasonically cleaning the anodic aluminum oxide film in ethanol and water respectively, soaking it in dilute hydrochloric acid, and drying it in a freeze dryer; modifying the cleaned anodic aluminum oxide film with an amino functional solution;

[0015] In some embodiments, step S11: ultrasonically cleaning the anodic aluminum oxide (AAO) membrane in ethanol and water, respectively, then soaking it in dilute hydrochloric acid, and finally drying it with a vacuum freeze dryer. The cleaned AAO membrane is modified with an amino-functionalized solution, and then baked at a high temperature after washing to obtain an amino-functionalized AAO membrane.

[0016] In some preferred embodiments, in step S11, the anodic aluminum oxide (AAO) membrane is ultrasonically cleaned in ethanol and water for 5 minutes each;

[0017] Preferably, in step S11, the concentration of the dilute hydrochloric acid is 5%; the soaking time is 60s;

[0018] In one embodiment, the amino-functionalized solution is a methanol solution of 3-aminopropyltrimethoxysilane in a volume ratio of 1:1;

[0019] In one embodiment, the concentration of 3-aminopropyltrimethoxysilane is 15%;

[0020] In one embodiment, in step S11, the baking temperature is 120° C. and the baking time is 2 hours.

[0021] Furthermore, in step S11, the amino functionalization solution is a methanol solution of 3-aminopropyltrimethoxysilane;

[0022] In some preferred embodiments, in step S11, the amino functionalization solution is a methanol solution of 3-aminopropyltrimethoxysilane with a concentration of 15% and a volume ratio of 1:1;

[0023] Furthermore, in step S11, the pore size of the anodized aluminum oxide membrane is 30±5 nm, the thickness is 60±5 nm, and the pore density is not less than 2.8×10 10 cm -2 ;

[0024] Furthermore, step S12 specifically comprises dissolving the triamine monomer and the dialdehyde monomer in an organic solvent under ultrasonic conditions, then immersing the amino-functionalized AAO membrane therein, heating for reaction, and then washing to obtain an amorphous COF / AAO membrane.

[0025] Furthermore, the triamine monomer is selected from at least one of 1,3,5-tris(4-aminophenyl)benzene, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine, tris(4-aminophenyl)amine, and 1,3,5-benzenetricarboxylic acid hydrazide;

[0026] Furthermore, the dialdehyde monomer is selected from at least one of 2,5-divinylterephthalaldehyde, terephthalaldehyde, and 2,5-dimethoxybenzene-1,4-dicarbaldehyde.

[0027] In some embodiments, in step S12, the triamine monomer and the dialdehyde monomer are 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinylterephthalaldehyde, respectively; 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinylterephthalaldehyde are dissolved in an organic solvent under ultrasonic conditions, and then the amino-functionalized AAO membrane is immersed in the solution and reacted in a water bath, and then washed to obtain an amorphous COF / AAO membrane; in step S12, the molar ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-divinylterephthalaldehyde is 2:3:

[0028] In one embodiment, the concentration of 1,3,5-tris(4-aminophenyl)benzene is 0.006-0.01 mol / L;

[0029] In one embodiment, the concentration of 2,5-divinylterephthalaldehyde is 0.008-0.02 mol / L;

[0030] In a preferred embodiment, the molar concentration of 1,3,5-tris(4-aminophenyl)benzene is 0.013 mmol;

[0031] In a preferred embodiment, the molar concentration of 2,5-divinylterephthalaldehyde is 0.019 mmol.

[0032] In a preferred embodiment, the 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinylterephthalaldehyde are dissolved in tetrahydrofuran / n-butanol (1:3, v / v) at a molar ratio of 2:3.

[0033] Further, in step S12, the organic solvent is selected from one or a combination of tetrahydrofuran, n-butanol and acetic acid;

[0034] In some preferred embodiments, in step S12, the reaction is heated in a water bath, and the reaction is carried out in a water bath at 60° C. for 7 hours;

[0035] In some embodiments, in step S12, washing is performed by washing with anhydrous ethanol three times.

[0036] Furthermore, in step S13, the solution B includes acetonitrile and acetic acid in a volume ratio of 50:7.

[0037] Furthermore, in step S13, the reaction solvent required for the solvothermal reaction includes acetonitrile and acetic acid in a volume ratio of 50:7. Preferably, the concentration of the catalyst acetic acid is 12M.

[0038] Furthermore, in step S13, the reaction is carried out in a water bath at 25° C. for 24 to 72 hours;

[0039] Furthermore, in step S13, washing and drying are performed using tetrahydrofuran and ethanol, which can effectively remove unreacted monomers, and the mixture is naturally dried overnight under a fume hood.

[0040] Furthermore, in step S14, the aptamer buffer solution is a Tris-HCl buffer solution containing the aptamer;

[0041] Furthermore, in step S14, the aptamer is CHO-5′-GTT GGG CAC GTG TTG TCT CTCTGTGTC TCG TGC CCT TCG CTAGGC CCACA-3′;

[0042] In some embodiments, in step S14, the aptamer buffer solution includes Tris-HCl buffer solution, NaCl solution and MgCl2 solution;

[0043] In some preferred embodiments, in step S14, the aptamer buffer solution includes a 1.5 μM aptamer solution; a Tris-HCl buffer solution with a concentration of 10 mM and a pH of 8; a 500 mM NaCl solution; and a 1 mM MgCl2 solution.

[0044] In some embodiments, in step S14, the COF / AAO membrane and solution C are reacted in a three-necked flask; the reaction conditions are: vacuuming for 2 hours, then adding the solution under vacuum, and reacting for 5 hours on a shaker at 25° C. and 80 rpm;

[0045] In some embodiments, in step S14, the washing is performed 3 to 6 times with a 10 mM Tris-HCl buffer solution at pH 7.0. The washing is performed with the Tris-HCl buffer solution to remove the incompletely reacted aptamers.

[0046] The second aspect of the present application provides a covalent organic framework nanochannel membrane based on aptamer functionalization prepared by the preparation method described above.

[0047] The third aspect of the present application provides a method for detecting aflatoxin B1 based on an aptamer-functionalized covalent organic framework nanochannel membrane, comprising:

[0048] Step S21: incubating the aflatoxin B1 solution to be tested with the Apt / COF / AAO membrane, and rinsing to obtain the Apt / COF / AAO-AFB1 membrane (i.e., the Apt / COF / AAO-AFB1 sensor);

[0049] Step S22: The Apt / COF / AAO-AFB1 membrane obtained in step S21 is placed in the middle of the flow cell, electrolyte is added to both sides of the Apt / COF / AAO-AFB1 membrane, voltage is applied, and the concentration value to be measured is obtained based on the electrical signal.

[0050] Furthermore, in step S21, the incubation time is ≥40 min;

[0051] Furthermore, in step S21, the rinsing step uses a Tris-HCl buffer solution for 3 to 6 times.

[0052] In a preferred embodiment, the washing is performed with a 10 mM Tris-HCl buffer solution at pH 7.0.

[0053] Furthermore, in step S22, the electrolyte in the electrolyte is selected from at least one of potassium chloride, sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate;

[0054] Furthermore, in step S22, the transmembrane potential on both sides of the Apt / COF / AAO-AFB1 membrane is -1V to 1V.

[0055] Furthermore, the scan rate was 10 mV / s.

[0056] In one embodiment, the electrolyte in the electrolyte solution is sodium chloride;

[0057] In one embodiment, the concentration of the electrolyte is 10 -5 M;

[0058] In one embodiment, the pH in the flow cell is 7.4.

[0059] In one embodiment, the material of the flow cell is tetrafluoroethylene or quartz glass.

[0060] In a preferred embodiment, the concentration of the aptamer solution is 1.5 μmol / L.

[0061] In a preferred embodiment, the incubation time with AFB1 is 40 min.

[0062] In a preferred embodiment, the concentration of sodium chloride is 10 -5 mol / L, and the pH value in the flow cell was 7.4.

[0063] Furthermore, the detection limit of aflatoxin B1 did not exceed 0.11 pg / mL;

[0064] Furthermore, the detection range of aflatoxin B1 is 1 pg / mL to 500 pg / mL.

[0065] In some embodiments, the aflatoxin B1 detection method based on aptamer-functionalized covalent organic framework nanochannel membrane provided herein further comprises step S20, preparation of aflatoxin B1 solution to be tested;

[0066] Furthermore, when used for actual sample testing, the preparation method of the aflatoxin B1 solution to be tested is as follows: S20, after the food sample to be tested is crushed by a wall-breaking machine, the sample powder is weighed and added to a solvent, and then a certain concentration of AFB1 solution is added, ultrasonicated, centrifuged and extracted, the supernatant is collected, the pH is adjusted, and then filtered and diluted for later use;

[0067] Furthermore, in step S20, the actual sample includes but is not limited to one or a combination of wheat, corn, peanuts and rice.

[0068] Furthermore, in step S20, the ultrasonic treatment time is 30 minutes; centrifugation is performed for 10 minutes to collect the supernatant;

[0069] Furthermore, in step S20, the pH value is adjusted using a Tris-HCl buffer solution;

[0070] In one embodiment, in step S20, 1 g of each food sample powder is weighed;

[0071] In step S20, the concentration of the AFB1 solution is 1.6-16 μg kg -1 ;

[0072] In some embodiments, the AFB1 concentration is 1.6 μg kg -1 ,8.0μg kg -1 and 16 μg kg -1 ;

[0073] In some embodiments, in step S20, the solvent is ACN / water;

[0074] In a preferred embodiment, the volume of the solution is 4 mL, and the volume ratio of the two is 8:2.

[0075] In some embodiments, in step S20, the filtered supernatant is filtered using a filter membrane; the filtrate is diluted to 40 times using a volumetric flask for subsequent analysis.

[0076] In one embodiment, in step S20, the filter membrane used for filtering the supernatant is 0.22 μm.

[0077] In a preferred embodiment, the centrifugal speed is 10000 rpm;

[0078] In a preferred embodiment, the Tris-HCl buffer solution has a concentration of 10 mM and a pH of 7.4; the pH of the supernatant is adjusted to 7.4.

[0079] The fourth aspect of the present application provides a detection device based on the aflatoxin B1 detection method described above, wherein the aflatoxin B1 detection device includes a circulation cell, a first electrode, a second electrode, an Apt / COF / AAO membrane, an electrolyte, and an ammeter; the Apt / COF / AAO membrane is installed in the circulation cell, and the Apt / COF / AAO membrane divides the circulation cell into a first cell body and a second cell body; the first electrode is fixed in the first cell body, and the second electrode is fixed in the second cell body; the first electrode is connected to a power supply, the second electrode is connected to a power supply, and the ammeter is arranged in a loop formed by the first electrode and the second electrode.

[0080] In one embodiment, the first electrode is an Ag / AgCl electrode.

[0081] In one embodiment, the second electrode is an Ag / AgCl electrode.

[0082] The aptamer-functionalized covalent organic framework nanochannel membrane provided in this application can be expanded to other fields such as medicine, food safety and environmental science. Compared with other sensors, this sensor has the advantages of high sensitivity, simple operation, low cost and good economy.

[0083] Beneficial effects

[0084] The aptamer-functionalized covalent organic framework nanochannel membrane provided in this application can detect trace amounts of aflatoxin B1 with high sensitivity and selectivity. A COF membrane is synthesized on the surface of an AAO membrane through a bottom-up method. This method can conveniently and controllably obtain a COF / AAO membrane with good ionic current performance as a nanochannel. By preparing highly specific aptamers as recognition units, aflatoxin B1 in food matrices is specifically recognized, thereby causing selective changes in the surface charge of the nanochannel, resulting in significant changes in the ionic current, and achieving selective and sensitive detection of trace amounts of AFB1 in food. A novel nanochannel sensing platform has been constructed for the detection of trace amounts of aflatoxin B1 in food.

[0085] The method presented in this application offers the advantages of high throughput and excellent ionic current performance, facilitating the development of novel nanochannel sensing platforms. Compared to other detection methods, it boasts high sensitivity, ease of operation, low cost, and economical efficiency, enabling high-throughput and highly selective detection of trace amounts of aflatoxin B1.

[0086] The detection method and device based on the present application can achieve a wide detection range and low detection limit for AFB1; the detection limit is as low as 0.11 pg / mL and shows a good linear relationship within the range of 1 pg / mL to 500 pg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 This is a schematic diagram of the method for sensitive and selective detection of trace aflatoxin B1 based on aptamer-functionalized covalent organic framework nanochannels in the present application;

[0088] Figure 2 These are SEM images of the COF / AAO membrane process prepared in Example 1 (a: AAO; b: AAO-NH2; c reaction 7h; d reaction 24h; e reaction 48h; f reaction 72h);

[0089] Figure 3 is the XPS spectrum of the Apt / COF / AAO film prepared in Example 1 (a: Si 2p; b: N 1s; c: P 2p; d: Zeta potential diagram);

[0090] Figure 4 is a schematic diagram of the current-voltage curve for detecting different concentrations of AFB1;

[0091] Figure 5 Schematic diagram of the current-voltage curve for the specific detection of AFB1 in the presence of different fungal toxins. DETAILED DESCRIPTION

[0092] The following is a clear and complete description of the technical solution of the present invention in conjunction with the accompanying drawings and implementation examples. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and do not limit the claims of the present invention in any way. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified. The following examples are given based on the contents contained in the claims;

[0093] Sources:

[0094] In the examples of the present application, the AAO membrane used was purchased from Hefei Puyuan Nanotechnology Co., Ltd.; fungal toxins such as AFB1, AFB2, AFG2, OTA, ZEN and DON were all from Shanghai Anpu Laboratory Technology Co., Ltd.; AFB1 aptamer was purchased from Shanghai Sangon Bioengineering Co., Ltd.; the electrochemical test system consisted of a 2450 SourceMeter and a version 2.06 data acquisition system (Keithley, USA).

[0095] Example 1: Preparation of COF / AAO membrane

[0096] First, the amino-functionalized AAO membrane was prepared. The membrane was ultrasonically cleaned in ethanol and ultrapure water for 5 minutes each, then immersed in 5% HCl for 60 seconds. After rinsing with ultrapure water, the membrane was dried in a vacuum freeze dryer. The dried membrane was then reacted in a 15% v / v methanol solution of 3-aminopropyltriethoxysilane (APTES) at room temperature for 8 hours. After rinsing with methanol, the membrane was baked at 120°C for 2 hours to obtain the amino-functionalized AAO membrane (AAO-NH2).

[0097] In order to achieve in situ uniform growth of COF on the AAO-NH2 film, 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinylterephthalaldehyde (molar concentration ratio 2:3) were dissolved in a mixed solution of tetrahydrofuran and n-butanol (2 mL) under ultrasonic conditions; then the amino-functionalized AAO membrane was immersed in the above solution and reacted at 60°C for 7 hours to obtain amorphous COF / AAO.

[0098] The obtained amorphous COF / AAO membrane was washed with ethanol and then immersed in the reaction solvent (ACN / 12M acetic acid solution, 50:7, v / v) again and reacted at 25°C for 3 days; the highly crystalline COF / AAO membrane was obtained; the unreacted monomer on the membrane was thoroughly washed with tetrahydrofuran and anhydrous ethanol, and finally dried naturally under a fume hood overnight.

[0099] from Figure 2 a and Figure 2 b It can be seen that after reacting with APTES, the pores of AAO-NH2 membrane are smoother than those of AAO membrane. In order to study the effect of COF on the pore size of AAO-NH2 membrane under different reaction times, its morphology was characterized, as shown in Figure 2. Figure 2 Figures (cf) are shown. From Figures (c) to (f), the COF reaction times on the AAO-NH2 membrane are 7 hours, 24 hours, 48 ​​hours, and 72 hours, respectively. It is clearly evident that with increasing reaction time, fewer and fewer original solid channels are exposed, and the surface of the prepared COF becomes increasingly smooth. After 72 hours of reaction, all channels are uniformly covered with COF. Therefore, the highly crystalline COF / AAO membrane constructed after 72 hours was selected for subsequent nanochannel analysis.

[0100] Example 2: Preparation of Apt / COF / AAO Nanofluid Sensor

[0101] To immobilize the aptamer, the COF / AAO membrane prepared in Example 1 was placed in a three-necked flask and evacuated for 2 hours. The flask was then gently shaken in Tris-HCl (10 mM, pH 8.0) containing 500 mM NaCl, 1 mM MgCl2, and 1.5 μM Apt for 5 hours. The membrane was then rinsed with Tris-HCl (10 mM, pH 7.0) buffer solution to remove the unreacted aptamer and finally dried to obtain the Apt / COF / AAO nanofluid sensor.

[0102] The prepared Apt / COF / AAO nanofluid sensor was characterized by XPS and Zeta potential. Figure 3 XPS spectra of different AAO films: (a) Si 2p; (b) N 1s; (c) P 2p; (d) Zeta potential of AAO, AAO-NH2, COF, COF / AAO and Apt / COF / AAO. Figure 3 a and Figure 3 b The XPS spectrum showed N1s and Si2p peaks different from those of AAO, which confirmed the successful modification of APTES. Figure 3 The decrease in electronegativity of AAO-NH2 in d also confirms the successful modification of APTES. In addition, the presence of a large amount of triamino monomers in COF, Figure 3 The spectrum in Figure (b) shows N1s with a higher electronegativity than that of AAO-NH2. Since COF is negatively charged, its electronegativity increases compared to AAO-NH2, demonstrating the successful formation of COF on the AAO-NH2 membrane. The aptamer is an oligonucleotide sequence containing the characteristic element P in its structure. Figure (c) shows a distinct P 2p peak on Apt / COF / AAO compared to AAO-NH2 and COF / AAO, confirming the successful grafting of Apt onto COF / AAO. Similarly, due to the electronegativity of the bases in Apt, the electronegativity of Apt / COF / AAO increases after coupling with Apt, as shown in Figure (d). These experimental results confirm the successful construction of the Apt / COF / AAO nanofluidic sensor.

[0103] Example 3: Detection of AFB1 at different concentrations

[0104] The Apt / COF / AAO sensor obtained in Example 2 was placed in different concentrations of AFB1 buffer solution for incubation, and then rinsed with Tris-HCl (10 mM, pH 7.0) buffer solution after 40 minutes to obtain an AFB1-bound Apt / COF / AAO sensor, referred to as Apt / COF / AAO-AFB1 sensor. The obtained Apt / COF / AAO-AFB1 sensor was installed in the middle of a polytetrafluoroethylene flow cell, and a current-voltage (IV) curve test was performed. The transmembrane potential was set to -1 V to 1 V, the scan rate was 10 mV / s, and the sodium chloride (10 -5 mol / L) as the electrolyte.

[0105] The results are as follows Figure 4 As shown, Figure 4 a is the IV curve of the Apt / COF / AAO sensor and different concentrations of AFB1. The transmembrane ion current changes significantly with the increase of AFB1 concentration. For example, at 1V, the transmembrane ion current gradually decreases with the increase of AFB1 concentration. This is because the covalent recognition of Apt and AFB1 will cause the surface charge of the nanochannel to change, resulting in a specific change in the ion current, which is directly reflected in the decrease of the ion current. Therefore, according to this principle, the detection of trace amounts of AFB1 in food samples can be achieved. In the range of 1pg / mL to 500pg / mL, the (I0-I) / I0 value has a good linear relationship with the logarithm of the AFB1 concentration ( Figure 4 b), the coefficient of determination was 0.9958, and the limit of detection was 0.11 pg / mL. These results indicate that the constructed Apt / COF / AAO nanofluidic sensor has a wide detection range and low detection limit for AFB1.

[0106] Example 4: Selective detection of trace amounts of AFB1 by nanofluidic sensors in the presence of different mycotoxins

[0107] Different fungal toxins include: AFB2, AFG2, OTA, DON, and ZEN; the Apt / COF / AAO sensors obtained in Example 2 were placed in buffer solutions containing different fungal toxins for incubation, and after 40 minutes, rinsed with Tris-HCl (10 mM, pH 7.0) buffer solution to obtain Apt / COF / AAO-AFB1, Apt / COF / AAO-AFB2, Apt / COF / AAO-AFG2, Apt / COF / AAO-OTA, Apt / COF / AAO-DON and Apt / COF / AAO-ZEN sensors, respectively; the obtained different types of sensors were installed in the middle of a polytetrafluoroethylene flow cell, and a current-voltage (IV) test was performed, with the transmembrane potential set to -1 V to 1 V, the scan rate to 10 mV / s, and sodium chloride as the electrolyte.

[0108] Figure 5 Figure 2 shows the current-voltage curves for the specific detection of AFB1 in the presence of different mycotoxins. It is clear from the figure that the prepared Apt / COF / AAO nanofluid sensor has high specificity and selectivity for AFB1, indicating that the prepared Apt / COF / AAO nanofluid sensor can achieve selective detection of trace amounts of AFB1.

[0109] Example 5: Detection of trace amounts of AFB1 in actual food samples using nanofluidic sensors

[0110] As shown in Table 1, in order to evaluate the feasibility of the Apt / COF / AAO nanofluidic sensor in complex food matrices, the sensor was used to detect AFB1 in wheat, rice, peanuts, and corn. The wheat, rice, peanut, and corn samples to be tested were respectively taken and crushed by a wall crusher to obtain sample powders; 1g of the above sample powders before and after spiked were added to 4mL ACN / water (8:2, v / v) solution and sonicated for 30 minutes. Then, the supernatant was collected by centrifugation at 10,000 rpm for 10 minutes and washed with Tris-HCl buffer solution (10 mmol L -1 The pH of the supernatant was adjusted to 7.4 with 0.22 μm filter. The filtrate was diluted 40 times with a 5 mL volumetric flask for subsequent analysis. Three different concentrations of AFB1 (1.6 μg kg -1 , 8.0 μg kg -1 or 16 μg kg -1 ), and after incubation with Apt / COF / AAO sensors, the recovery of AFB1 after addition ranged from 86.9% to 102.5%. In addition, trace amounts of AFB1 were detected in peanut and corn-2 food samples, with concentrations of 0.36 ± 0.10 and 0.26 ± 0.10 μg kg, respectively. -1 The results showed that the prepared nanofluidic sensor can achieve sensitive detection of trace amounts of AFB1 in complex food samples.

[0111] Table 1 Determination and recovery of AFB1 in food samples wheat, rice, peanut and corn

[0112]

[0113] This application uses COFs as nanochannels. Due to their advantages such as large specific surface area, rich hydrogen bonding sites, and high intrinsic order of nanochannels, the combination of COFs and AAO membranes not only provides more binding sites for subsequent aptamer modification, but also greatly reduces the size of the nanochannel, significantly improving the ion selectivity of the nanochannel. Aptamers, as recognition units of AFB1, can highly specifically identify trace aflatoxin B1 in food matrices. When the aptamer interacts with AFB1, the hydrogen bonding interaction between the molecules causes a change in the surface charge of the nanochannel, which is reflected in different current change rates (I-I0) / I0, thereby achieving selective and sensitive detection of trace AFB1 in food. The research on the use of aptamer-functionalized 2D COFs combined with the surface of AAO nanochannels to construct an efficient electrochemical sensor to detect trace AFB1 in food matrices is an innovative combination strategy that is expected to bring breakthrough progress to the field of food safety testing, by improving detection sensitivity and selectivity, and achieving accurate monitoring of harmful substances such as AFB1. It has broad application prospects in fields such as food testing and environmental science.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a covalent organic framework nanochannel membrane based on aptamer functionalization, characterized in that: The following steps are involved: Step S11: modifying the anodic aluminum oxide film with an amino functional solution, washing it, and then baking it at a high temperature to obtain an amino functional AAO film; Step S12: dissolving the triamine monomer and the dialdehyde monomer in an organic solvent, and then immersing the amino-functionalized AAO membrane prepared in step S11, heating for reaction, and then washing to obtain an amorphous COF / AAO membrane; Step S13: preparing a reaction solvent required for the solvothermal reaction of COF to obtain solution B, and then immersing the amorphous COF / AAO membrane obtained in step S12 in solution B again for reaction, and then washing and drying to obtain a COF / AAO membrane; The solution B is a mixture of acetonitrile and acetic acid; Step S14: reacting the COF / AAO membrane obtained in step S13 with an aptamer buffer solution, and washing to obtain an Apt / COF / AAO membrane; The triamine monomer is selected from at least one of 1,3,5-tris(4-aminophenyl)benzene, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine, tris(4-aminophenyl)amine, and 1,3,5-benzenetricarboxylic acid hydrazide; The dialdehyde monomer is selected from at least one of 2,5-divinylterephthalaldehyde, terephthalaldehyde, and 2,5-dimethoxybenzene-1,4-dicarbaldehyde; The aptamer is CHO-5′-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCGCTA GGC CCA CA-3′; The aptamer buffer solution is a Tris-HCl buffer solution containing the aptamer.

2. The method for preparing a covalent organic framework nanochannel membrane based on aptamer functionalization according to claim 1, characterized in that: Step S11 specifically comprises: ultrasonically cleaning the anodic aluminum oxide film in ethanol and water respectively, soaking the film in dilute hydrochloric acid, and drying the film in a freeze dryer; modifying the cleaned anodic aluminum oxide film with an amino functional solution; Step S12 specifically comprises dissolving the triamine monomer and the dialdehyde monomer in an organic solvent under ultrasonic conditions, then immersing the amino-functionalized AAO membrane therein, heating for reaction, and then washing to obtain an amorphous COF / AAO membrane.

3. The method for preparing a covalent organic framework nanochannel membrane based on aptamer functionalization according to claim 1, characterized in that: In step S11, the amino functionalization solution is a methanol solution of 3-aminopropyltrimethoxysilane; And / or, in step S11, the pore size of the anodized aluminum oxide membrane is 30±5 nm and the thickness is 60±5 nm; And / or, in step S12, the organic solvent is selected from one or a combination of tetrahydrofuran, n-butanol and acetic acid; And / or, in step S13, the solution B comprises acetonitrile and acetic acid in a volume ratio of 50:

7.

4. A covalent organic framework nanochannel membrane based on aptamer functionalization prepared by the preparation method according to any one of claims 1 to 3.

5. A method for detecting aflatoxin B1 based on the aptamer-functionalized covalent organic framework nanochannel membrane according to claim 4, characterized in that: include: Step S21: incubating the aflatoxin B1 solution to be tested with the Apt / COF / AAO membrane, and then rinsing to obtain the Apt / COF / AAO-AFB1 membrane; Step S22: The Apt / COF / AAO-AFB1 membrane obtained in step S21 is placed in the middle of the flow cell, electrolyte is added to both sides of the Apt / COF / AAO-AFB1 membrane, voltage is applied, and the concentration value to be measured is obtained based on the electrical signal.

6. The method for detecting aflatoxin B1 based on aptamer-functionalized covalent organic framework nanochannel membrane according to claim 5, characterized in that: In the step S21, the incubation time is ≥ 40 min; In step S21, the rinsing step is performed using a Tris-HCl buffer solution for 3 to 6 times; And / or, in step S22, the electrolyte in the electrolyte is selected from at least one of potassium chloride, sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate; And / or, in step S22, the transmembrane potential on both sides of the Apt / COF / AAO-AFB1 membrane is -1 V to 1 V.

7. The method for detecting aflatoxin B1 based on aptamer-functionalized covalent organic framework nanochannel membrane according to claim 5, characterized in that: The detection limit does not exceed 0.11 pg / mL; The detection range is 1 pg / mL - 500 pg / mL.

8. The method for detecting aflatoxin B1 based on aptamer-functionalized covalent organic framework nanochannel membrane according to claim 5, characterized in that: When applied to actual sample detection, the preparation method of the aflatoxin B1 solution to be tested is: S20, crush the food sample to be tested by a wall-breaking machine, weigh the sample powder and add it to the solvent, then add a certain concentration of AFB1 solution, sonicate, centrifuge and extract, collect the supernatant, adjust the pH, filter, and dilute for later use; The actual sample is selected from one or a combination of wheat, corn, peanuts and rice.

9. A detection device based on the aflatoxin B1 detection method according to any one of claims 5 to 8, characterized in that: The aflatoxin B1 detection device includes a flow cell, a first electrode, a second electrode, an Apt / COF / AAO membrane, an electrolyte, and an ammeter; the Apt / COF / AAO membrane is installed in the flow cell, and the Apt / COF / AAO membrane divides the flow cell into a first cell body and a second cell body; the first electrode is fixed in the first cell body, and the second electrode is fixed in the second cell body; The first electrode is connected to a power source, the second electrode is connected to a power source, and the ammeter is arranged in a loop formed by the first electrode and the second electrode.

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