Bionic nanofluid sensor-based method for detecting trace amount of patulin

Abstract

The invention relates to a method for detecting trace patulin based on a bionic nanofluid sensor, and belongs to the field of detection. The preparation method comprises the following steps: modifying an anodic aluminum oxide (AAO) nano-channel membrane; growing a covalent organic framework (COFs) in situ on the surface of the AAO nano-channel film from bottom to top, and preparing a covalent organic framework nano-channel with the aperture smaller than 2 nm; gold ions grow in situ in a COFs pore channel through a post-synthesis method, and a sub-nano-channel metal covalent organic framework (Au-COF) nano-channel is prepared; the aptamer (Apt) is anchored on the surface of the Au-COF through a covalent coupling strategy, and the Apt / Au-COF / AAO nanofluid sensor is prepared. According to the invention, Au-COF is used as a nano channel, Apt is used as a recognition unit, the sensor integrates specific recognition, signal amplification and rapid detection, and high-selectivity and high-sensitivity analysis of trace patulin (PAT) in a complex food matrix is realized.

Description

Technical Field

[0001] The present application relates to a method for ultra-sensitive and highly selective detection of trace amounts of patulin in complex food matrices based on a biomimetic nanofluidic sensor, belonging to the technical field of trace mycotoxin detection and nanochannel analysis. Background Art

[0002] Patulin (PAT) is a mycotoxin widely found in fruits, particularly apples and their derivatives. PAT can cause significant histopathological changes in several vital organs, including the liver and kidneys. The Codex Alimentarius Commission has established a maximum limit of 50 μg / L for PAT in food. Therefore, a real-time, sensitive, and highly selective detection technology is needed for the detection of trace levels of PAT in food to minimize the risk of trace toxins posing a food safety risk.

[0003] Currently, food contaminant detection technologies are relatively mature, but in practice, they primarily rely on cumbersome instrumentation, such as gas chromatography-tandem mass spectrometry, high-performance liquid chromatography-tandem mass spectrometry, and enzyme-linked immunosorbent assays (ELISAs). While these methods offer high accuracy, they are typically expensive and time-consuming, limiting their application and preventing rapid, on-site detection of food contaminants. Therefore, the development of simple, highly specific, and ultrasensitive PAT methods remains a pressing need.

[0004] Nanofluidics studies the transport behavior of substances within nanoscale channels. As a groundbreaking, highly sensitive analytical method, nanofluidic sensor detection technology derives its core advantage from the unique physicochemical response mechanism induced by the nanoconfinement effect (such as the regulation of ion transport kinetics and the reconstruction of surface charge distribution). It can capture the dynamic concentration changes or specific binding signals of trace targets (such as single molecules and heavy metal ions) in the fluid in real time through molecular-scale interactions. Such sensors are constructed based on precisely designed nanomaterials (such as AAO and graphene). With their nanoscale pore structure and high specific surface area, they exhibit single-molecule resolution and femtomolar detection limits. In the field of food safety, this technology can accurately identify targets in complex food matrices through functionalized surface modifications (such as aptamers and molecularly imprinted polymers), such as the direct detection of trace melamine in milk and organophosphorus pesticide residues on the surface of fruits and vegetables. Its anti-interference ability and label-free detection characteristics are significantly better than traditional chromatography or immunoassays. Although AAO nanofluid-based sensing systems have made great progress, their specific surface area is relatively low and the functionalized sites are limited. This makes it difficult to effectively and specifically modify the channel surface, resulting in insufficient selectivity for target pollutants. In addition, constructing ordered artificial solid-state channels with sub-nanometer pores remains a significant technical challenge. Sub-nanometer pore ordered structures are crucial for improving channel performance, but current preparation technologies make it difficult to precisely control the size, shape, and arrangement of nanochannels.

[0005] In recent years, covalent organic frameworks (COFs) have emerged as a new class of porous crystalline frameworks composed of organic monomers linked by covalent bonds. They offer advantages such as high stability, structural designability, high specific surface area, ease of post-modification with functional groups, and excellent biocompatibility. Furthermore, their structural units can be functionalized to expose more active sites for specific interactions with pollutants, leading to their widespread application in analytical detection and adsorption. However, COFs are composed entirely of light elements, which imposes several inherent limitations on their physical and chemical properties. For example, low density and weak interlayer molecular forces can lead to loose packing during the fabrication of 2D COF membranes or films. Furthermore, high hydrophobicity is incompatible with surface compatibility in materials chemistry and biocompatibility in biological systems. Furthermore, metal-free COFs are often not sufficiently functionally complex to meet the requirements of modern science. The role of metals in their structures is gaining increasing attention in areas such as gas adsorption and separation, heterogeneous catalysis, and electrochemical energy storage.

[0006] The targeted introduction of metal ions into porous COFs to form metal covalent organic frameworks (MCOFs) may help address these issues. MCOFs can bridge the gap between MOFs and COFs, exhibiting a balanced combination of crystallinity, porosity, stability, and tunability, resulting in complementary properties between the two materials. Post-synthetic metallization, a common approach to synthesizing MCOFs, involves pre-engineering a COF with metal ion coordination sites and then incorporating the metal ions into the COF framework. The periodic integration of active metal sites within the extended framework effectively prevents metal site aggregation. Compared to metal-free COFs, MCOFs retain the inherent advantages of COFs while providing additional metal active sites and cationic versatility, making them an ideal platform for high-performance nanofluidic sensing. For example, the highly thermally sensitive Ln-COFs luminescent thermometer developed by Kaczmarek and the two-dimensional Ni-COFs gas sensor reported by Meng, which achieved ppb-level detection limits, demonstrate the breakthrough potential of MCOFs in sensor development. Among MCOFs, gold-COFs (Au-COFs) have attracted considerable attention due to their exceptional stability, significant biofunctionality, and high active site density. Currently, there are no reports on trace toxin detection systems based on the integration of Au-COFs and nanofluidic sensors in the fields of environmental monitoring and food safety. Given the significant advantages of Au-COFs in terms of specific surface area, structural tunability, and molecular recognition performance, as well as the technological potential of nanofluidic sensors in terms of sensitivity and response speed, the collaborative construction of a novel detection platform based on the two has significant scientific value and application prospects. Therefore, there is an urgent need to establish new methods for trace toxin detection in food samples based on the Au-COFs-nanofluidic sensor integrated system to overcome the limitations of existing detection technologies in terms of sensitivity, selectivity, and field applicability, and to provide innovative analytical tools for food safety assurance. Summary of the Invention

[0007] Aptamers (Apts), with their high thermal stability, low dissociation constant, and programmable modification properties, have become core recognition elements for trace PAT detection in food. Au-COFs, with their highly ordered nanopores, interfacial biocompatibility, and the complexity and synergy between different metals, provide an ideal platform for the directional immobilization and conformational regulation of Apts. Apt is anchored to the Au-COFs surface through a covalent coupling strategy. While maintaining the stability of Apt's tertiary structure, its confined spatial preconcentration effect can also be utilized to convert its binding to PAT into an electrochemical signal that can be amplified and recorded. Apt and Au-COFs, through a synergistic mechanism of molecular recognition and signal amplification, have constructed a novel biosensor platform. The coupled confined enrichment effect and conformational responsiveness significantly enhance the detection performance of the biosensor, enabling efficient capture and specific identification of trace PATs in the presence of interference from complex food matrices, providing a highly sensitive, interference-resistant, on-site rapid screening solution for food safety.

[0008] In order to solve the above problems, the present application provides a metal covalent organic framework biomimetic nanofluidic sensor based on aptamer functionalization, and based on this, provides a method for sensitive and selective detection of trace patulin. First, COF / AAO membrane is synthesized by a bottom-up method, and then Au-COFs membrane is prepared by a post-synthesis method and used as a nanochannel; by using aptamers covalently bonded to the surface of Au-COFs as recognition units, a new nanofluidic sensing platform is constructed and applied to the detection of trace patulin in food matrices.

[0009] The first aspect of the present application provides a method for preparing a biomimetic nanofluid sensor for detecting trace amounts of patulin, characterized in that the method comprises the following steps: Step S11: modifying an anodic aluminum oxide film (AAO) with an amino-functionalized solution, washing, and then baking at a high temperature to obtain an amino-functionalized AAO film; the amino-functionalized solution is an acetone solution of 3-aminopropyltrimethoxysilane; Step S12: dissolving a triamine monomer and a trialdehyde monomer in an organic solvent, and then immersing the amino-functionalized AAO membrane obtained in step S11 in the organic solvent, reacting and washing to obtain an amorphous polymer / 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 trialdehyde monomer is selected from at least one of 1,4,6-trihydroxybenzene-1,3,5-tricarboxaldehyde, pyromellitic acid trimesic acid, and 2,4,6-tris(4-formaldehyde phenyl)-1,3,5-triazine; Step S13: preparing the reaction solvent required for the solvothermal reaction of crystalline COFs to obtain solution A, and then immersing the amorphous COF / AAO membrane obtained in step S12 in solution A again for reaction, and then washing and drying to obtain a crystalline COF / AAO membrane; The solution A is a mixture of 1,4-dioxane, mesitylene and acetic acid or a mixture of 2-dichlorobenzene, n-butanol and acetic acid; Step S14: reacting the crystalline COF / AAO film obtained in step S13 with a chloroauric acid solution, and washing to obtain an Au-COF / AAO film; Step S15: reacting the Au-COF / AAO membrane prepared in step S14 with an aptamer buffer solution, and washing to obtain an Apt / Au-COF / AAO nanochannel; the aptamer is SH-5′-GGC CCG CCA ACC CGC ATC ATC TAC ACTGAT ATT TTA CCT T-3′; Step S16: incubating the patulin solution with the Apt / Au-COF / AAO nanochannel, and then rinsing to obtain the Apt / Au-COF / AAO-PAT bionic nanofluid sensor.

[0010] In one embodiment, step S11 specifically comprises: ultrasonically cleaning the anodic aluminum oxide film in ethanol and water respectively, soaking it in dilute hydrochloric acid, and freeze-drying it; modifying the dried anodic aluminum oxide film with an amino functional solution; and baking it at high temperature after washing; Step S12 specifically comprises dissolving triamine monomer and trialdehyde monomer in an organic solvent under ultrasonic conditions, then immersing the amino-functionalized AAO membrane in the organic solvent, heating for reaction, and then washing to obtain an amorphous polymer / AAO membrane; In step S13, the reaction time is 72 hours and the reaction temperature is 60°C; In step S14, the chloroauric acid solution is a methanol solution containing 10 mM chloroauric acid; In step S15, the aptamer is activated, the buffer pH is 7.4, the reaction time is 10 minutes, and the reaction temperature is room temperature; In step S16, the buffer solution has a pH of 7.2 and the incubation time is 45 minutes.

[0011] In one embodiment, 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 more combinations of tetrahydrofuran, n-butanol and acetic acid; And / or, in step S13, the solution A is mesitylene, 1,4-dioxane and acetic acid in a volume ratio of 1:1:0.1.

[0012] In one embodiment, step S11: ultrasonically clean the anodized aluminum oxide membrane in ethanol and water respectively, then soak it in dilute hydrochloric acid, and finally dry it with a vacuum freeze dryer. The cleaned AAO membrane is modified with an amino functional solution, washed, and then baked at high temperature to obtain an amino functionalized AAO membrane.

[0013] In one embodiment, in step S11, the anodized aluminum oxide film is ultrasonically cleaned in ethanol and water for 5 minutes each.

[0014] In one embodiment, in step S11, the concentration of the dilute hydrochloric acid is 5 wt % and the soaking time is 1 min.

[0015] In one embodiment, in step S11, the freeze-drying time is 24 hours.

[0016] In one embodiment, in step S11, the baking temperature is 120° C. and the baking time is 2 h.

[0017] In one embodiment, in step S11, the concentration of the acetone solution of 3-aminopropyltrimethoxysilane is 15 wt %.

[0018] In one embodiment, in step S11, the pore density of the anodized aluminum film is about 2.8×10 10 cm -2 .

[0019] In one embodiment, in step S12, the triamine monomer and the trialdehyde monomer are 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and trimesic acid aldehyde, respectively; 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and trimesic acid aldehyde 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 polymer / AAO membrane.

[0020] In one embodiment, in step S12, the molar ratio of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine to trimesaldehyde is 1:1.

[0021] In one embodiment, the concentration of the 2,4,6-tris(4-aminophenyl)-1,3,5-triazine is 0.002-0.02 mol / L.

[0022] In one embodiment, the concentration of trimesaldehyde is 0.002-0.02 mol / L.

[0023] In one embodiment, the molar concentration of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine is 0.02 mmol.

[0024] In one embodiment, the molar concentration of trimesaldehyde is 0.02 mmol.

[0025] In one embodiment, the 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and trimesic acid aldehyde are dissolved in tetrahydrofuran / n-butanol (1:3, v / v) at a molar ratio of 1:1.

[0026] In one embodiment, 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 h; In one embodiment, in step S12, washing is performed by washing with anhydrous ethanol three times.

[0027] In one embodiment, in step S13, the solution A comprises 1,4-dioxane, mesitylene, and acetic acid, wherein the volume ratio of the three is 1:1:0.1.

[0028] In one embodiment, the acetic acid concentration is 6 mol / L.

[0029] In one embodiment, in step S13, the washing and drying are performed using tetrahydrofuran and ethanol, which can effectively remove unreacted monomers, and the mixture is dried in a vacuum drying oven at 60° C. for 12 h.

[0030] In one embodiment, in step S14, the reaction conditions are 25° C. and 120 rpm on a shaker for 4 h.

[0031] In one embodiment, in step S14, the washing conditions are washing with anhydrous ethanol and deionized water three times each, storing in PBS buffer solution and placing in a 4°C refrigerator until use.

[0032] In one embodiment, in step S15, the aptamer buffer solution is a PBS buffer solution containing the aptamer.

[0033] In one embodiment, in step S15, the aptamer buffer solution includes a PBS buffer solution containing a NaCl solution and a MgCl2 solution.

[0034] In one embodiment, in step S15, the aptamer buffer solution includes a 2.5 μM aptamer solution.

[0035] In one embodiment, in step S15, the PBS buffer solution has a concentration of 10 mM and a pH of 7.4; the NaCl solution has a concentration of 500 mM and the MgCl2 solution has a concentration of 1 mM.

[0036] In one embodiment, in step S15, the crystalline Au-COF / AAO membrane and the aptamer buffer solution are reacted in a three-necked flask; the reaction conditions are: vacuuming for 2 hours, adding the solution under vacuum, and reacting for 5 hours on a shaker at 25°C and 100 rpm.

[0037] In one embodiment, in step S15, the rinsing step is performed 3-6 times with a 10 mM PBS buffer solution at pH 7.4, and the rinsing step is performed to remove the incompletely reacted aptamers.

[0038] In one embodiment, the aptamer is activated by using 10 mM TCEP at 25°C for 30 minutes.

[0039] In one embodiment, in step S16, the incubation time is ≥ 45 min; In one embodiment, in step S16, the washing step uses 0.1 mM PBS buffer solution to wash 3-6 times.

[0040] In one embodiment, the wash is performed with a 0.1 mM PBS buffer solution at pH 7.2.

[0041] In one embodiment, in step S16, the concentration of the aptamer solution is 2.5 μmol / L.

[0042] In one embodiment, in step S16, the incubation time is ≥ 45 min; In one embodiment, in step S16, the washing step uses 0.1 mM PBS buffer solution to wash 3-6 times.

[0043] The second object of the present invention is to provide a bionic nanofluid sensor for detecting trace amounts of patulin prepared by the above preparation method.

[0044] A third object of the present invention is to provide a device for detecting patulin, comprising a circulation cell, a first electrode, a second electrode, the Apt / Au-COF / AAO-PAT bionic nanofluid sensor according to any one of claims 1 to 4, an electrolyte, and an ammeter; the Apt / Au-COF / AAO-PAT bionic nanofluid sensor is installed in the circulation cell, and the Apt / Au-COF / AAO-PAT bionic nanofluid sensor 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.

[0045] In one embodiment, the first electrode is an Ag / AgCl electrode.

[0046] In one embodiment, the second electrode is an Ag / AgCl electrode.

[0047] A fourth object of the present invention is to provide a method for detecting trace amounts of patulin based on the above-mentioned patulin detection device, comprising: The Apt / Au-COF / AAO-PAT bionic nanofluid sensor is placed in the middle of the circulation pool, and electrolyte is added to both sides of the Apt / Au-COF / AAO-PAT bionic nanofluid sensor. Voltage is applied, and the measured concentration value is obtained according to the electrical signal.

[0048] In one embodiment, the electrolyte in the electrolyte is selected from at least one of potassium chloride, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium phosphate; The transmembrane potential on both sides of the Apt / Au-COF / AAO-PAT nanofluid sensor is -1 V to -1 V.

[0049] In one embodiment, the scan rate is 10 mV / s.

[0050] In one embodiment, the electrolyte in the electrolyte solution is phosphate buffered saline.

[0051] In one embodiment, the concentration of the electrolyte is 0.1 mmol / L.

[0052] In one embodiment, the pH in the flow cell is 7.2.

[0053] In one embodiment, the material of the flow cell is tetrafluoroethylene or quartz glass.

[0054] In one embodiment, the limit of detection is no more than 3.83 fg / mL; The detection range is 0.05 pg / mL - 1000 pg / mL.

[0055] In one embodiment, when applied to actual sample testing, the sample preparation method is as follows: 0, 0.6, 3, and 6 ng / mL of patulin standard solutions are added to the food to be tested, respectively. After adding the standard solutions, the mixture is thoroughly stirred and then the pH value is adjusted to 7.2 with 10 mM PBS buffer solution; each sample is then filtered using a 0.22 μm pore size filter membrane to remove particulate impurities; finally, the resulting solution is diluted to the desired concentration and stored in a refrigerator at 4°C until testing; The food to be tested includes one or more combinations of apple juice, apple cider vinegar, tomato juice, grape juice, kiwi juice or pear juice.

[0056] In one embodiment, each food sample contains 5 mL of juice.

[0057] In one embodiment, the filtrate is diluted 60-fold using a volumetric flask.

[0058] Beneficial effects The present application provides a biomimetic nanofluidic sensor for ultra-sensitive and highly selective detection of trace patulin. COFs membranes are synthesized on the surface of an AAO membrane by a bottom-up method. This method can conveniently and controllably obtain a COF / AAO membrane. Gold nanoparticles are then grown in situ within the COF pores by a post-modification method to prepare an Au-COF / AAO membrane, which, as a nanochannel, has good surface functionalization efficiency and ion current performance. Highly specific aptamers are prepared as recognition units. When the target substance PAT is present, the specific binding of the aptamer triggers its conformational transition, which in turn causes changes in the surface charge density and effective pore size of the nanochannel, resulting in significant changes in the ion current. The sensor integrates specific recognition, signal amplification, and rapid detection, achieving highly selective and sensitive detection of trace patulin in food. This application innovatively constructs a biomimetic nanofluidic sensor that achieves ultra-sensitive and highly selective detection of trace patulin in food through a multi-level structural design and synergistic mechanism.

[0059] 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 strong anti-interference properties, high sensitivity, ease of operation, low cost, and economical efficiency. It enables high-throughput and highly selective detection of trace patulin, and has broad application prospects in fields such as medicine, food safety, and environmental science.

[0060] The detection method and device based on the present application can achieve a wide detection range and low detection limit for PAT; the detection limit is as low as 3.83 fg / mL and shows a good linear relationship in the range of 0.05 pg / mL to 1000 pg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Schematic diagram of the method for ultra-sensitive and highly selective detection of trace patulin based on biomimetic nanofluidic sensors in this application: a is the fabrication of the Apt / Au-COF / AAO nanofluidic sensor; b is the sensing mechanism of the nanofluidic sensor; Figure 2 1 is an SEM image of the COF / AAO film prepared in Example 1 and a DES image of the Au-COF / AAO prepared by the post-synthesis method; a is an SEM image of the surface and cross section of the AAO-NH2 and COF / AAO films; b is an SEM elemental map of Au-COF; Figure 3 1 are XPS spectra of the Au-COF / AAO film prepared in Example 1: a is the full XPS spectra of the COF / AAO film and the Au-COF / AAO film; b is the high-resolution XPS spectra of Au 4f in the Au-COF / AAO film; Figure 4 Figures 1 and 2 are XPS spectra and Zeta potential diagrams of the Apt / Au-COF / AAO membranes prepared in Example 1: (a) P 2p XPS spectra of Au-COF / AAO and Apt / Au-COF / AAO; (b) Zeta potentials of AAO, AAO-NH2, COF / AAO, Au-COF / AAO, and Apt / Au-COF / AAO at a pH of 7.4. Figure 5 Schematic diagram of the current-voltage curves for detecting different concentrations of PAT: a is the IV curve of the Apt / Au-COF / AAO nanofluid sensor at different PAT concentrations; b is the linear relationship between the current increase rate and the logarithm of the PAT concentration (0.05-1000 pg / mL); Figure 6Figure 1 is a schematic diagram of the current-voltage curves for the specific detection of PAT in the presence of different mycotoxins. (a) Specific detection of PAT by the Apt / Au-COF / AAO biomimetic nanofluidic sensor; (b) The effect of the presence of other mycotoxins (1000 pg / mL) in PBS buffer (pH 7.2) on the detection of PAT (100 pg / mL) (1, PAT + AFB1; 2, PAT + AFB2; 3, PAT + AFG2; 4, PAT + OTA; 5, PAT + DON; 6, PAT + ZEN; and 7, PAT + AFB1 + AFB2 + AFG2 + OTA + DON + ZEN). DETAILED DESCRIPTION

[0062] 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; Sources: In the examples of the present application, the AAO membrane used was purchased from Hefei Puyuan Nanotechnology Co., Ltd.; fungal toxins such as PAT, AFB1, AFB2, AFG2, OTA, ZEN, and DON were all from Shanghai Anpu Laboratory Technology Co., Ltd.; the PAT aptamer was purchased from Shanghai Sangon Bioengineering Co., Ltd.; and the electrochemical testing system consisted of a 2450 SourceMeter and a version 2.06 data acquisition system (Keithley, USA).

[0063] Example 1: Preparation of Au-COF / AAO membrane The anodic aluminum oxide membrane was ultrasonically cleaned in ethanol and water respectively, then immersed in 5wt% dilute hydrochloric acid for 1 min and freeze-dried for 24 h. The dried anodic aluminum oxide membrane was modified with 15wt% 3-aminopropyltrimethoxysilane in acetone solution; ultrasonically cleaned in ethanol and water for 3 min each, and then baked at high temperature (baking temperature: 120 ℃, time: 2 h) after washing to obtain amino-functionalized AAO membrane (AAO-NH2).

[0064] In order to achieve in situ uniform growth of Au-COF on AAO-NH2 film, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (0.02 mol / L) and 1,3,5-trisaldehyde (0.02 mol / L) (molar concentration ratio 1:1) were dissolved in a mixed solution of tetrahydrofuran and n-butanol (1:3, v / v, 2 mL) under ultrasonic conditions; then, the amino-functionalized AAO membrane was immersed in the above solution and reacted at 60 °C for 7 h to obtain an amorphous COF / AAO membrane.

[0065] The obtained amorphous polymer / AAO membrane was washed with ethanol and then immersed in the reaction solvent (1.4-dioxane / mesitylene / 6 M acetic acid, 1:1:0.1, v / v / v) again. After reacting at 60 °C for 3 days, a 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.

[0066] Au-COFs were prepared via a post-synthesis strategy. The prepared crystalline COF / AAO membrane was immersed in a 5 mL methanol solution containing 10 mM chloroauric acid and shaken at 120 rpm for 4 hours. After the reaction, the membrane was repeatedly washed with methanol and deionized water to remove unbound gold ions and then freeze-dried to obtain the Au-COF / AAO membrane.

[0067] Test example: The pore density of the anodic aluminum oxide membrane is 2.8×10 10 cm -2 , the pore size is 30nm. Figure 2 As can be seen in Figure a, the surface of the prepared COF membrane is smooth and evenly covers all channels, with a thickness of approximately 58 nanometers. Preliminary analysis of the synthesized Au-COFs by X-ray energy spectrum confirmed the high density distribution of gold ions in the Au-COF. Further XPS characterization of the synthesized Au-COF / AAO membrane revealed that Figure 3 It can be seen that after Au³⁺ coordination, the Au-COF / AAO film has a higher lattice strength at 82.3 eV (Au 4f 7 / 2)和 85.9 eV(Au 4f5 / 2 ) showed obvious gold 4f characteristic peaks, which strongly confirmed that Au³⁺ was successfully loaded into the COF framework.

[0068] Example 2: Preparation of Apt / Au-COF / AAO biomimetic nanofluid sensor To immobilize the aptamer, the Au-COF / AAO membrane prepared in Example 1 was placed in a three-necked flask and evacuated for 2 hours. Simultaneously, a thiol aptamer (100 µM) was activated with 10 mM TCEP at 25°C for 30 minutes. Subsequently, a PBS buffer solution (10 mM, pH 7.4) containing 2.5 µM of the reduced aptamer was added to the flask and incubated at room temperature for 10 minutes. After repeated washing with deionized water, an Apt / Au-COF / AAO biomimetic nanofluidic sensor was prepared. The aptamer was SH-5′-GGC CCG CCA ACC CGC ATC ATC TAC ACT GAT ATT TTA CCT T-3′. The aptamer PBS buffer solution consisted of a PBS buffer solution containing 500 mM NaCl and 1 mM MgCl₂.

[0069] The Apt / Au-COF / AAO nanochannels obtained in Example 2 were placed in 0.01-5000 pg / mL PAT buffer solutions for incubation. After incubation for 45 min, the samples were rinsed three times with PBS buffer solution (0.1 mM, pH 7.2) to obtain PAT-bound Apt / Au-COF / AAO sensors, referred to as Apt / Au-COF / AAO-PAT sensors.

[0070] Test example: The prepared biomimetic Apt / Au-COF / AAO nanochannel was characterized by XPS and Zeta potential. Since the aptamer is an oligonucleotide sequence and contains the characteristic element P in its structure, Figure 4 It can be seen from the XPS spectrum of a that compared with the Au-COF / AAO membrane, the Apt / Au-COF / AAO nanochannel has an obvious P 2p peak, which confirms the successful grafting of the aptamer on the Au-COF / AAO membrane. In addition, since COF is negatively charged, Figure 4 The Zeta spectrum of b shows an increase in electronegativity compared to amino-functionalized AAO, demonstrating the successful formation of COF on the amino-functionalized AAO membrane. Using a post-synthesis strategy, Au³⁺ was in situ loaded into the COF framework. The presence of high-density Au³⁺ resulted in a lower electronegativity of the Au-COF compared to the COF. Similarly, due to the electronegativity of the base in the aptamer, the electronegativity of the Apt / Au-COF / AAO membrane increased after covalently attaching the ligand via Au-S bonds. These experimental results confirm the successful construction of the Apt / Au-COF / AAO-PAT biomimetic nanofluidic sensor.

[0071] Example 3: Detection of PAT at different concentrations The obtained Apt / Au-COF / AAO-PAT sensor was installed in the middle of the flow cell and the 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 PBS buffer solution (0.1 mM) was used as the electrolyte.

[0072] The results are as follows Figure 5 As shown, Figure 5 Figure a shows the IV curve of the Apt / Au-COF / AAO biomimetic nanofluidic sensor at different PAT concentrations. For example, at 1V, the transmembrane ion current gradually increases with increasing PAT concentration. This is because the specific recognition of Apt and PAT causes an increase in the surface charge density of the nanochannel and an increase in the effective diameter, resulting in a significant change in the transmembrane ion current, which is directly reflected in an increase in the ion current. Therefore, based on this principle, the detection of trace amounts of PAT in complex food matrices can be achieved. In the range of 0.05 pg / mL to 1000 pg / mL, the (I-I0) / I0 value shows a good linear relationship with the logarithm of the PAT concentration ( Figure 5 b), the coefficient of determination was 0.9973, and the detection limit was as low as 3.83 fg / mL. These results demonstrate that the constructed Apt / Au-COF / AAO biomimetic nanofluidic sensor achieves ultrasensitive detection of trace amounts of PAT.

[0073] Example 4: Selective detection of trace PAT by nanofluidic sensors in the presence of different mycotoxins Different mycotoxins include: AFB1, AFB2, AFG2, OTA, DON, and ZEN; the Apt / Au-COF / AAO biomimetic sensors obtained in Example 2 were placed in buffer solutions containing different mycotoxins for incubation, and rinsed with PBS buffer solution (0.1 mM, pH 7.2) after 45 minutes to obtain different types of mycotoxin sensors; the obtained mycotoxin sensors were subjected to the same steps as in Example 3 for current-voltage (IV) testing.

[0074] Figure 6 a is a schematic diagram of (I-I0) / I0 for selective detection of PAT in the presence of different fungal toxins. It can be clearly seen from the figure that the prepared Apt / Au-COF / AAO biomimetic nanofluid sensor has high specific selectivity for PAT. This shows that the prepared Apt / Au-COF / AAO biomimetic nanofluid sensor can achieve selective detection of trace amounts of PAT. In addition, to further test the anti-interference ability of the prepared biomimetic nanofluid sensor, an anti-interference experiment was conducted. Figure 6As shown in Figure b, the biomimetic nanofluidic sensor was incubated with a series of PBS buffers containing 1000 pg / mL interfering mycotoxins (a concentration 10 times that of PAT) for 45 minutes, and then the current-voltage (IV) characteristics were tested. The experimental data showed that the current increase (I-I0) / I0 of each test group was significantly different, fully confirming that the constructed Apt / Au-COF / AAO biomimetic nanofluidic sensing system has excellent resistance to matrix interference. This result not only verifies the synergistic effect between the molecular recognition probe and Au-COFs, but also demonstrates that the sensing platform has high selectivity and detection reliability in the analysis of complex food matrices, providing important experimental basis for the practical detection of trace toxins.

[0075] This application innovatively uses Au-COFs as nanochannels, and its structural advantages are mainly reflected in three aspects: excellent interface functionalization efficiency, controllable confinement effect, and the multifunctional properties of metal components. By integrating Au-COFs with AAO membranes, this biomimetic nanofluid sensing platform achieves dual performance optimization - on the one hand, the structural stability of the AAO membrane and the sub-nanometer channels of Au-COFs form a synergistic effect, significantly enhancing the interaction efficiency between the probe molecule and the target; on the other hand, the higher interface functionalization efficiency of Au-COFs provides a large number of active sites for the high-density grafting of aptamers, thereby achieving significant amplification of the sensing signal. This coupling design of molecular recognition elements and confinement structures improves the sensor in both detection sensitivity and signal amplification.

[0076] Based on the molecular recognition properties of aptamers, this sensor can specifically recognize trace amounts of patulin in food matrices. When PAT binds specifically to the aptamer, conformational changes in the aptamer trigger changes in the surface charge density and effective diameter of the nanochannel. This change is converted into a quantifiable (I-I0) / I0 signal output via current-voltage (IV) characteristics, enabling highly selective and ultrasensitive detection of trace amounts of PAT in food. This innovative structural design strategy, utilizing aptamer-functionalized Au-COFs grown in situ on the surface of AAO nanochannels, provides a new technological path for food safety testing. By constructing a biomimetic sensing interface with the dual functions of confinement enhancement and molecular recognition, it not only overcomes the sensitivity and selectivity limitations of existing detection technologies but also provides a scalable platform for the precise monitoring of harmful substances in food. This approach shows broad application prospects in the rapid detection of trace toxins, the elimination of interference from complex matrices, and the development of portable detection devices. It has important theoretical and practical significance for promoting the intelligent and miniaturized development of food safety testing technologies.

[0077] 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 detecting trace amounts of patulin based on a biomimetic nanofluid sensor, characterized in that: The method comprises: placing an Apt / Au-COF / AAO-PAT bionic nanofluid sensor in the middle of a flow cell, adding electrolytes to both sides of the Apt / Au-COF / AAO-PAT bionic nanofluid sensor, applying voltage, and obtaining a concentration value to be measured based on an electrical signal; The preparation method of the Apt / Au-COF / AAO-PAT biomimetic nanofluid sensor comprises the following steps: Step S11: modifying the anodic aluminum oxide film with an amino functionalization solution, washing it, and then baking it at a high temperature to obtain amino functionalized AAO nanochannels; the amino functionalization solution is an acetone solution of 3-aminopropyltrimethoxysilane; Step S12: dissolving a triamine monomer and a trialdehyde monomer in an organic solvent, and then immersing the amino-functionalized AAO membrane obtained in step S11 in the organic solvent, reacting and washing to obtain an amorphous polymer / 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 trialdehyde monomer is selected from at least one of 1,4,6-trihydroxybenzene-1,3,5-tricarboxaldehyde, pyromellitic acid trimesic acid, and 2,4,6-tris(4-formaldehyde phenyl)-1,3,5-triazine; Step S13: soaking the amorphous polymer / AAO membrane nanochannel obtained in step S12 in solution A for reaction, washing and drying to obtain a crystalline COF / AAO membrane; the solution A is a mixed solution of mesitylene, 1,4-dioxane and acetic acid or a mixed solution of 2-dichlorobenzene, n-butanol and acetic acid; Step S14: reacting the crystalline COF / AAO film synthesized in step S13 with a chloroauric acid solution, and washing to obtain an Au-COF / AAO film; Step S15: incubating the Au-COF / AAO membrane prepared in step S14 with an aptamer buffer solution, and washing to obtain an Apt / Au-COF / AAO nanochannel; the aptamer is SH-5′-GGC CCG CCA ACC CGC ATC ATC TAC ACTGAT ATT TTA CCT T-3′; Step S16: incubating the patulin solution with the Apt / Au-COF / AAO nanochannel, and then rinsing to obtain the Apt / Au-COF / AAO-PAT bionic nanofluid sensor.

2. The method according to claim 1, characterized in that Step S11 specifically comprises: ultrasonically cleaning the anodic aluminum oxide film in ethanol and water respectively, soaking it in dilute hydrochloric acid, and freeze-drying it; modifying the dried anodic aluminum oxide film with an amino functional solution; and baking it at high temperature after washing; Step S12 specifically comprises dissolving triamine monomer and trialdehyde monomer in an organic solvent under ultrasonic conditions, then immersing the amino-functionalized AAO membrane in the organic solvent, heating for reaction, and then washing to obtain an amorphous polymer / AAO membrane; In step S13, the reaction time is 7 hours and the reaction temperature is 60°C; In step S14, the chloroauric acid solution is a methanol solution containing 10 mM chloroauric acid; In step S15, the aptamer is activated, the buffer pH is 7.4, the reaction time is 10 minutes, and the reaction temperature is room temperature; In step S16, the buffer solution has a pH of 7.2 and the incubation time is 45-60 minutes.

3. The method according to claim 1, characterized in that 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 A comprises mesitylene, 1,4-dioxane and acetic acid in a volume ratio of 1:1:0.1 or one of 1,2-dichlorobenzene, n-butanol and acetic acid in a volume ratio of 1:1:0.1; And / or, in step S16, the washing step uses 0.1 mM PBS buffer solution to wash 3-6 times.

4. The method according to claim 1, characterized in that The electrolyte in the electrolyte is selected from at least one of potassium chloride, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium phosphate; The transmembrane potential on both sides of the Apt / Au-COF / AAO-PAT nanofluid sensor is -1 V to -1 V.

5. The method for detecting trace amounts of patulin based on a biomimetic nanofluid sensor according to claim 1, characterized in that: The detection limit does not exceed 3.83 fg / mL; The detection range is 0.05 pg / mL - 1000 pg / mL.

6. The method for detecting trace amounts of patulin based on a biomimetic nanofluid sensor according to claim 1, characterized in that: For actual sample testing, the sample preparation method is as follows: add 0, 0.6, 3, and 6 ng / mL of patulin standard solution to the food to be tested. After adding the standard solution, the mixture is thoroughly stirred and then the pH value is adjusted to 7.2 with 10 mM PBS buffer solution. Each sample is then filtered using a 0.22 μm pore size filter membrane to remove particulate impurities. Finally, the resulting solution is diluted to the desired concentration and stored in a refrigerator at 4°C until testing. The food to be tested includes one or more combinations of apple juice, apple cider vinegar, tomato juice, grape juice, kiwi juice and pear juice.

7. A device for detecting patulin, characterized in that: The patulin detection device comprises a flow cell, a first electrode, a second electrode, the Apt / Au-COF / AAO-PAT bionic nanofluid sensor according to any one of claims 1 to 6, an electrolyte, and an ammeter; the Apt / Au-COF / AAO-PAT bionic nanofluid sensor is installed in the flow cell, and the Apt / Au-COF / AAO-PAT bionic nanofluid sensor 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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