Bifunctional triazine covalent organic framework material, solid-phase microextraction probe and application

Abstract

The invention discloses a bifunctional triazine covalent organic framework material, a solid-phase microextraction probe and application, and the bifunctional triazine covalent organic framework material is prepared from 2, 4, 6-tri (4-formyl-biphenyl-4-yl)-1, 3, 5-triazine, 5 '-(4-amino-3, 5-difluorophenyl)-3, 3', 5, 5 '-tetrafluoro-[1, 1': 3 ', 1'-triphenyl]-4, 4, 6-triazine-1, 3, 5-triazine-1, 3, 5 '-tetrafluoro-[1, 1': 3 ', 1'-triphenyl]-1, 3, 5 '-tetrafluoro-[1, 1': 3 ', 1 ''-triphenyl]-4, 4, 6-triazine-1, 3, 5]- The polymer is formed by polymerizing three monomers, namely 1, 4 ', 4'-diamine and diethyl phosphite. The TFP-COF disclosed by the invention is a porous material containing aromatic rings, fluorine atoms and nitrogen heteroatoms, and has a relatively large conjugated system and a plurality of recognition sites; the hydrophilicity and hydrophobicity of the COFs are adjusted through functionalization of the diethyl phosphite, the hydrophilicity is enhanced, the adsorption performance of the TFP-COF on medium and strong polarity analytes is enhanced, and then high-precision detection of the mycotoxins in a wide polarity range is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of new materials for chemical detection, and in particular to a bifunctional triazine covalent organic framework material, a solid-phase microextraction probe and applications thereof. BACKGROUND

[0002] Aflatoxins (AFs), ochratoxins (OTs) and zearalenones (ZENs) are the main mycotoxins that cause damage to the human kidney, liver and gastrointestinal tract, and have strong carcinogenicity, teratogenicity, mutagenicity and immunotoxicity. The regulatory limit of aflatoxin B1 in food is 2 µg·kg -1 , and the regulatory limit of aflatoxins AFB2, AFG1 and AFG2 is 4 µg·kg -1 . Ochratoxins can bioaccumulate in various regions of the brain and may cause neurodegeneration. ZENs are non-steroidal synthetic metabolites produced by fusarium and can accumulate as residues in animal bodies. When these residues enter the human body through the food chain, they may cause developmental defects and hormonal disorders. In addition, mycotoxins exhibit high thermal stability, making them difficult to degrade during food processing, cooking or other heat treatment processes. Therefore, it is necessary to detect mycotoxins in food and the like.

[0003] At present, the concentration of mycotoxins in complex food matrices is often low, and the sample usually needs to be extracted and purified before detection. The existing sample pretreatment methods for detecting mycotoxins in food include liquid-liquid extraction, solid-phase extraction, QuEChERS, liquid-liquid microextraction, magnetic solid-phase extraction and dispersive solid-phase microextraction. However, these sample pretreatment methods usually have the disadvantages of complicated procedures, time-consuming steps, low selectivity, obvious matrix effect and large consumption of organic solvents. Therefore, they often cannot meet the requirements of analyzing trace mycotoxins in complex matrices. Solid-phase microextraction (SPME) is an emerging sample pretreatment method that has attracted much attention due to its unique advantages such as simplicity, rapidness, cost-effectiveness and low consumption of organic solvents. In recent years, the combination of SPME and high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) has been increasingly applied to the efficient enrichment and sensitive detection of target analytes in complex matrices such as food, environmental samples and biological systems. For SPME, the extraction efficiency is largely dependent on the fiber coating. Therefore, the development of fiber coating materials with high extraction rate and enrichment efficiency is of great significance for the efficient enrichment of mycotoxins in complex matrices. SUMMARY

[0004] Therefore, the first object of the present application is to provide a bifunctional triazine covalent organic framework material, the second object is to provide a solid-phase microextraction probe, and the third object is to provide an application.

[0005] To achieve the above object, the present application adopts the following technical solutions: The bifunctionalized triazine covalent organic framework material is polymerized from 2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine, 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-terphenyl]-4,4"-diamine and diethyl phosphite, with 1,3,5-trimethylbenzene and n-butanol as solvents and with sulfamic acid as a catalyst, and is denoted as TFP-COF, and has the following structural formula: .

[0006] The TFP-COF prepared by the present application is a porous material containing aromatic rings, fluorine atoms and nitrogen atoms, has a large conjugated system and multiple recognition sites, can adsorb mycotoxins through π-π, C-H…π and hydrophobic interaction, C-F…π interaction force and the like, can realize high-throughput adsorption and enrichment of trace or even ultra-trace mycotoxins in complex food matrices, and can realize high-precision detection of mycotoxins.

[0007] Preferably, the bifunctionalized triazine covalent organic framework material is prepared by the following steps: accurately weighing 2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine, 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-terphenyl]-4,4"-diamine and diethyl phosphite, then adding 1,3,5-trimethylbenzene, n-butanol and sulfamic acid, and ultrasonically mixing them uniformly; rapidly freezing and degassing the reaction system in a liquid nitrogen environment, sealing the reaction container and placing it in a high-temperature environment for reaction; after the reaction is completed, washing with water, ethanol and tetrahydrofuran in sequence, and vacuum drying to obtain the TFP-COF. The three-component synthesis strategy is adopted to construct the TFP-COF with fluorine atoms and phosphite functional groups in two directions through Kabachnik-Fields reaction in one step, and the preparation method is simple and conducive to later scale-up production.

[0008] More preferably, the molar ratio of 2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine, 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-terphenyl]-4,4"-diamine and diethyl phosphite is 1:1:3.

[0009] In actual preparation, the temperature of the high-temperature environment is 130-150°C, more preferably 140°C.

[0010] The application further provides a solid phase microextraction probe comprising a coating layer, wherein the coating layer is a bifunctionalized triazine covalent organic framework material.

[0011] The application further provides application of the solid phase microextraction probe in detection of trace or ultratrace mycotoxins in food, wherein the coating layer of the solid phase microextraction probe is the TFP-COF.

[0012] Preferably, after enrichment and extraction, the target analyte is eluted for 10-20 min by using an elution solvent, wherein the elution solvent is a mixture of acetone, formic acid and water, the volume percentage of water in the elution solvent is 1%, the volume percentage of acetone is 87%-89%, and the rest is formic acid.

[0013] Preferably, after elution, the eluent is detected by using UPLC-MS / MS, and the liquid chromatography condition of UPLC-MS / MS is as follows: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile, gradient elution is adopted, and the flow rate is 0.3 mL / min.

[0014] Compared with the prior art, the application has the following advantages: The TFP-COF of the application is a porous material containing aromatic rings, fluorine atoms and nitrogen atoms itself, has a large conjugated system and multiple recognition sites; the functionalization of diethyl phosphite adjusts the hydrophilicity and hydrophobicity of the COF, and enhances the hydrophilicity of the TFP-COF, which is beneficial to enhancing the adsorption performance of the TFP-COF on medium and strong polar analytes. In actual adsorption, the material of the application can adsorb mycotoxin molecules through π-π, C-H…π and hydrophobic interaction. In addition, the F, N and O atoms in the TFP-COF can form pseudo hydrogen bonds such as C-H…F, C-H…N and C-H…O with C-H in the mycotoxin molecules, further promoting adsorption; the C-F functional group in the TFP-COF can also interact with the aromatic ring in the mycotoxin molecules to produce C-F…π interaction force. In summary, the TFP-COF has conjugated systems, fluorine groups and phosphate groups, and has multiple binding sites, can realize high-throughput adsorption and enrichment of ten kinds of mycotoxins with a wide range of polarity, and further realize high-precision detection of ten kinds of ultra-trace mycotoxins in complex matrices, has practical application value and application prospect, and helps food safety. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a preparation flow chart of the TFP-COF described in the application.

[0016] Figure 2 is a characterization of the application Figure 1 . Figure 2 (a) is an FT-IR spectrum, (b) is a 13 CNMR spectrum.

[0017] Figure 3 is a characterization of the application Figure 2 . Figure 3 (b) is a full spectrum of TF-COF and TFP-COF, (a) is a N 1s high-resolution spectrum of TF-COF and TFP-COF, (c) and (d) correspond to the O 1s high-resolution spectrum and P 2p high-resolution spectrum of TFP-COF, respectively.

[0018] Figure 4 is a characterization of the application Figure 3 . Figure 4 (a) is an XRD diffraction spectrum of TF-COF and TFP-COF; (b) is a PXRD diffraction spectrum of TFP-COF after soaking in different solvents.

[0019] Figure 5 is a characterization of the application Figure 4 . Figure 5 (a) is the water contact angle of TF-COF; (b) is the water contact angle of TFP-COF.

[0020] Figure 6 is a characterization of the present invention Figure 5 . Figure 6 Fig. 1 is a characterization of the present invention, wherein (a)-(b) are SEM characterization images of TFP-COF; (c) is a SEM characterization image of TF-COF; (d) is a SEM characterization image of stainless steel wire; (e)-(f) are SEM characterization images of TFP-COF probe.

[0021] Figure 7 is a characterization of the present invention Figure 6 . Figure 7 Fig. 2 is a characterization of the present invention, wherein (a) is a TEM image of TFP-COF; (b)-(e) are EDS mapping images of TFP-COF.

[0022] Figure 8 is a flow chart of detection steps in Example 2 of the present invention.

[0023] Figure 9 is a spiked spectrum and a probe repeatability chart of Example 2 of the present invention. Figure 9 Fig. 3 is a characterization of the present invention, wherein (a) is a liquid chromatogram of spiked wheat flour, and peaks 1-10 in (a) correspond to fungal toxins AFG2, AFG1, AFB2, AFB1, β-ZOL, β-ZAL, α-ZOL, α-ZAL, ZAN and OTC in turn; (b) is a repeatability of TFP-COF; (c) is a spiked recovery rate and corresponding RSDs.

[0024] Figure 10 is a comparison chart between different probes in Example 2.

[0025] Figure 11 is an adsorption principle of the TFP-COF probe of the present invention for different fungal toxins Figure 1 . Figure 11 Fig. 5 is an adsorption principle of the TFP-COF probe of the present invention for different fungal toxins, wherein (a)-(d) are fungal toxins AFB1, AFB2, AFG1 and AFG2 in turn.

[0026] Figure 12 is an adsorption principle of the TFP-COF probe of the present invention for different fungal toxins Figure 2 . Figure 12 Fig. 6 is an adsorption principle of the TFP-COF probe of the present invention for different fungal toxins, wherein (f)-(j) are fungal toxins OTC, ZAN, ɑ-ZAL, β-ZAL, ɑ-ZOL and β-ZOL in turn. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below with reference to the accompanying drawings, and the embodiments are implemented on the premise of the technical solution of the present invention, and detailed embodiments are given, but the protection scope of the present invention is not limited to the following embodiments.

[0028] It should be noted that the reagents used in the present application are all commercially available reagents, and the equipment used is all conventional laboratory equipment. Among them, the comonomer of the present application and its corresponding abbreviation are as follows: the abbreviation of diethyl phosphite is DP, the abbreviation of 2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine is TFPAB, and the abbreviation of 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-terphenyl]-4,4"-diamine is F-TAM; the physicochemical properties of ten mycotoxins are shown in Table 1.

[0029] Table 1 Physicochemical properties of ten mycotoxins in the present application Example 1 Bifunctionalized triazine covalent organic framework material and probe according to the present application First, the bifunctionalized triazine covalent organic framework material according to the present application is polymerized from 2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine, 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-terphenyl]-4,4"-diamine and diethyl phosphite. It can be known that the preparation method is as follows: Figure 1 Into a Pyrex glass tube, 2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine (49.6 mg, 0.08 mmol), 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-terphenyl]-4,4"-diamine (36.8 mg, 0.08 mmol), diethyl phosphite (62 μL, 0.24 mmol), sulfamic acid (5 mg), 1,3,5-trimethylbenzene (1.7 mL) and n-BuOH (0.3 mL) were added, and ultrasonic treatment was performed for 30 min to mix the raw materials uniformly, then rapid freezing was performed under liquid nitrogen conditions, followed by thawing and degassing, and three cycles of degassing and sealing were performed, and reaction was performed at 140°C for 4 days; after completion, the reaction product was washed with water, ethanol and tetrahydrofuran in sequence, and vacuum drying was performed at 80°C for 12 h, to obtain 80.4 mg of deep yellow TFP-COF, and the structural formula of TFP-COF is as follows: .

[0030] Second, the TFP-COF obtained by copolymerization of 2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine and 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-terphenyl]-4,4"-diamine is used as a comparison. ​

[0031] The specific preparation method of TF-COF is as follows: 2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine (49.6 mg, 0.08 mmol), 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-triphenyl]-4,4"-diamine (36.8 mg, 0.08 mmol), HAc (9M, 0.01 mL), and thiol / n-butanol (1.7 / 0.3 mL, v / v) are added to a Pyrex glass tube, sonicated for 20 min, rapidly frozen at 77 K (liquid nitrogen bath), and degassed by three freeze-thaw cycles; then heated to 140°C for 4 days; after the reaction is completed, the mixture is washed successively with acetone, tetrahydrofuran, and ethanol, and dried under vacuum at 80°C for 12 h to obtain solid powder TF-COF.

[0032] III. The solid-phase microextraction probe described in this invention The solid-phase microextraction probe (i.e., TFP-COF probe) described in this invention is a TFP-COF prepared in Example 1 that is cured on a stainless steel wire. The specific preparation method includes the following: Cut several 5 cm long and 500 μm diameter stainless steel wires (i.e., SSW), wash them sequentially with ultrapure water, ethanol and acetone, and then air dry them at room temperature for later use; grind TFP-COF and pass it through a 100-mesh sieve for later use. The SSW was vertically immersed in a silicone sealant solution (composed of 1g of neutral silicone sealant and 2 mL of cyclohexane) for 30 seconds, then slowly and uniformly pulled out of the solution. Immediately after being pulled out, it was inserted into TFP-COF powder and gently rotated in one direction for 60 seconds. The SSW was then gently rolled on weighing paper to obtain a uniform coating. It was then cured in an oven at 150°C for 30 minutes to remove volatile cyclohexane. This process was repeated several times, and finally cured at 150°C for 3 hours to successfully prepare the TFP-COF probe. The coating length on the probe was controlled to be 1 cm.

[0033] Similarly, the TF-COF probe can be obtained using the method described above.

[0034] IV. Characterization of Materials and Probes 1. FT-IR spectra of TF-COF, TFP-COF and their precursors, as shown in the figure. Figure 2 As shown in Figure (a), the TFPAB contains 1701 cm. -1 The characteristic peak of -C=O at 2428 cm⁻¹ weakened, while the characteristic peak of -C=N appeared in TF-COF, indicating that TF-COF was successfully synthesized via the Schiff base reaction; DP at 2428 cm⁻¹ -1The typical pH vibration peak at 1258 cm⁻¹ disappeared after polymerization, but appeared in TFP-COF. -1 1045 cm -1 and 2929-2958 cm -1 The absorption peaks at these locations can be attributed to the characteristic stretching vibrations of P=O, POC, and CH, respectively, confirming that the present invention successfully introduced α-aminophosphonate into TFP-COF through the MCRs strategy.

[0035] 2. Solid-state drives of TF-COF and TFP-COF 13 CNMR see Figure 2 (b) In the TF-COF NMR spectrum, the resonance signal at 164 ppm can be attributed to the carbon atom in the imine bond, the signals at 127 and 108 ppm to the carbon atoms of different phenyl groups in TF-COF, and the 13C signals at 167 and 153 ppm to the carbon atoms in the triazine ring and Ar-CF, respectively. The NMR peaks at 18 and 48 ppm can be attributed to the saturated carbon in the α-aminophosphonate, respectively, and the signal at 56 ppm to the carbon atom in the CP bond, further proving the formation of the α-aminophosphonate group.

[0036] 3. XPS characterization spectra of TF-COF and TFP-COF are shown in [reference needed]. Figure 3 .Depend on Figure 3 As can be seen, compared with the XPS full spectrum of TF-COF, the prominent peak at 133 eV in TFP-COF corresponds to P 2p, confirming the presence of P element in TFP-COF. The high-resolution N 1s spectrum of TF-COF can be decomposed into two peaks, attributed to C=N (398.7 eV) and -NH2 (400.5 eV), respectively. The high-resolution N 1s spectrum of TFP-COF shows a prominent peak at 399.7 eV, attributed to PCN; in the high-resolution O 1s spectrum of TFP-COF, the two peaks at 532.1 and 533.6 eV are attributed to P=O and COP, respectively; in the high-resolution P 2p spectrum of TFP-COF, the three peaks at 132.7, 133.5, and 134.2 eV are attributed to PC, PO, and P=O, respectively. The XPS characterization results further confirm the formation of α-aminophosphonate groups.

[0037] 4. The crystal structure of α-aminophosphonate-grafted TFP-COF was determined using PXRD characterization. Figure 4As shown in Figure (a), the diffraction peaks of TFP-COF were significantly enhanced after the introduction of α-aminophosphonate, indicating that the MCR strategy improved the crystallinity of the prepared TFP-COF. PXRD analysis was performed after immersing TFP-COF in different solvents (ACN, MeOH, EtOH, THF, acetone, and boiling water) for 24 h, and the results are shown in Figure (b). Figure 4 Figure (b) shows that TFP-COF still retains the PXRD diffraction peaks after treatment with different solvents, indicating that TFP-COF has good chemical stability.

[0038] 5. The water contact angle results for TF-COF and TFP-COF are shown in [the table below]. Figure 5 .Depend on Figure 5 As shown in (a), the water contact angle of TF-COF is 125°, indicating that the material is hydrophobic; the water contact angle of TFP-COF is 97°. After TFP-COF was successfully modified with α-aminophosphonate, its water contact angle decreased significantly, indicating that the hydrophilicity of TFP-COF increased, which is beneficial for the adsorption of moderately polar fungal toxins by TFP-COF.

[0039] 6. SEM results for TF-COF and TFP-COF are shown in [reference needed]. Figure 6 .Depend on Figure 6 As shown in (a)-(c), TFP-COF exhibits a hollow tubular morphology similar to TF-COF, with a cross-sectional diameter of approximately 200 nm. The SEM results of the TFP-COF probe are shown in [Figure number missing]. Figure 6 In (d)-(f), the results show that TFP-COF is uniformly coated on the SSW surface, with a probe diameter of approximately 886 μm and a coating thickness of approximately 192 μm.

[0040] 7. The elemental composition of TFP-COF was further analyzed and confirmed using energy-dispersive X-ray spectroscopy (EDS mapping). The results are shown in [Figure number missing]. Figure 7 .Depend on Figure 7 It can be seen that the hollow tubular structure of TFP-COF shows that C, N, F and P elements are uniformly distributed in TFP-COF, indicating that α-aminophosphonate has been successfully modified onto the surface of TFP-COF.

[0041] In summary, α-aminophosphonates were successfully modified onto the surface of TFP-COF, exhibiting good chemical stability and significantly increased hydrophilicity compared to TF-COF, laying the foundation for its adsorption of fungal toxins.

[0042] Example 2: A method for detecting (ultra) trace amounts of mycotoxins in food. I. This invention, based on TFP-COF probes and UPLC-MS / MS technology, constructs a method for detecting trace or ultra-trace mycotoxins in food. Combined with...Figure 8 The method includes the following detection contents: Firstly, the salt concentration (i.e. sodium chloride) of the actual sample or the standard working solution is adjusted to 15% (w / v), and the TFP-COF probe prepared in Example 2 is immersed in the sample solution or the standard working solution, and is oscillated and adsorbed at room temperature for 50 min, and the shaking speed is 900 rpm; Secondly, after the adsorption is completed, the solution on the TFP-COF probe is removed by using a dust-free paper, and the adsorbed probe is eluted by using an elution solvent at room temperature for 10 min, and is filtered to remove impurities; wherein, if the eluate cannot be detected immediately after the filtration, the eluate is stored at -20°C for standby; the elution solvent is preferably a mixed aqueous solution of acetone and formic acid, wherein the volume fraction of acetone is 87%, the volume fraction of water is 12%, and the volume fraction of formic acid is 1%; Thirdly, the eluate is analyzed by using UPLC-MS / MS, wherein the UPLC-MS / MS analysis is performed by using an ultra-high performance liquid chromatograph (Shimadzu, Japan) and an AB Sciex 7500 triple quadrupole mass spectrometer (AB Sciex 7500, USA), and a BEH C18 high performance liquid chromatography column (2.1 mm × 100 mm, 1.7 μm, Waters, USA) is used to separate 10 kinds of mycotoxins at 40°C; wherein, the liquid chromatography conditions are as follows: the mobile phase A is a formic acid aqueous solution with a formic acid volume fraction of 0.1%, the mobile phase B is 100% acetonitrile, the flow rate is 0.3 mL / min, the injection amount is 5 μL, and the gradient elution program is shown in Table 2.

[0043] Table 2 Gradient elution program of the mobile phase of ten kinds of mycotoxins in the application Secondly, the reliability analysis of the method for detecting trace or ultra-trace mycotoxins in food constructed in the application 1. Linear range, limit of quantification and limit of detection of mycotoxins When the standard curve of mycotoxins is drawn, the standard working solution is usually prepared by using a blank sample matrix, for example, when the detection object is wheat flour, the blank wheat flour is used as the matrix to prepare the standard solution, and the specific process is as follows: Firstly, the mixed standard solution of AFB1, AFB2, AFG1, AFG2, OTC, ZAN, α-ZAL, β-ZAL, α-ZOL and β-ZOL is prepared by using acetonitrile, and the concentration of each mycotoxin is 500 ng·mL -1 ; Second step, 1 g of blank wheat flour is added into 5 mL of acetonitrile-water-formic acid (84:15:1, v / v), vortexed for 5 min, ultrasonicated for 30 min, centrifuged at 10000 rpm for 5 min; the supernatant is transferred into a centrifuge tube, then evaporated to dryness under a gentle stream of nitrogen, and redissolved with ultrapure water to obtain an ultrapure water solution of the blank wheat flour matrix; Third step, the mixed standard mother liquor is diluted with the ultrapure water of the second step to obtain standard working solutions with a certain concentration gradient: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 5, 7, 10, 20, 30, 40, 50 μg kg -1 ; Fourth step, the above standard working solutions are analyzed and detected according to the method constructed in the application, and the results are shown in Table 3.

[0044] Table 3 Standard curve, detection limit and quantification limit of the detection method constructed in the application As shown in Table 3, in the concentration range of 0.01-50.0 μg·kg -1 , the R values of the 10 kinds of mycotoxins are all greater than 0.99, the detection limit and the quantification limit of the mycotoxins are 0.002-0.180 μg·kg -1 and 0.005-0.600 μg·kg -1 respectively, indicating that the method constructed in the application can be used for high-precision detection of the 10 kinds of mycotoxins.

[0045] 2, repeatability and reproducibility of the detection method of the application A mixed working solution with a concentration of 0.5 μg·kg -1 is added into the ultrapure water redissolved solution of the blank wheat flour, and actual sample working solutions with a spiked concentration of 0.5 μg·kg -1 are determined in parallel for 3 days and 3 times within one day to obtain the intra-day and inter-day precision, and the results are shown in Table 3. As shown in Table 3, the intra-day RSD of the application is 0.7-6.6%, the inter-day RSD is 1.2-5.4%, and the reproducibility between probes is 1.7%-6.5%, further proving that the method constructed in the application based on the TFP-COF probe has good repeatability and reproducibility.

[0046] 3, spiked verification The actual samples are obtained by treating the wheat flour according to the treatment method of the first item, and the mixed standard working solution is added into the actual samples respectively to perform spiked recovery test, and the concentrations of the low, medium and high levels of the mixed standard working solution are 0.5 μg·kg -1 , 2 μg·kg -1 and 10 μg·kg -1, and the results are shown in Table 4 and Figure 9 .

[0047] Table 4. Results of the spiking recovery test of the detection method of the application for mycotoxins in wheat flour As can be seen from the table, the spiking recovery rate of the detection method of the application is in the range of 71.77%-103.02%, and the RSD is 0.23%-13.38%, indicating that the method has high accuracy and can be used for reliable detection of mycotoxins in food matrices.

[0048] 4. Reproducibility of the probe of the application The application investigated the reproducibility of the TFP-COF probe, i.e. the TFP-COF probe of the application was repeatedly used for extraction of mycotoxins, and the extraction conditions were the same each time; after each extraction, the probe was soaked and cleaned with acetonitrile and elution solvent, and after the probe was dried, the next extraction was performed, and the results of repeated use are shown in Figure 9 (b). As can be seen from Figure 9 (b), under the same extraction conditions, after 10 times of repeated use, the enrichment efficiency of the TFP-COF probe did not decrease significantly, indicating that the TFP-COF probe of the application has good reproducibility.

[0049] 5. Commercial value of the TFP-COF probe of the application In order to evaluate the commercial potential of the TFP-COF probe, the application compared it with commercial fibers hydrophilic-lipophilic balance HLB, octadecylsilane C18, TF-COF and TFP-COF, i.e. under the same extraction conditions, the sample solution spiked with 3 μg kg -1 of mycotoxins was extracted by using the TFP-COF probe of the application and commercial fibers respectively, and then eluted and detected and analyzed by UPLC-MS / MS under the same conditions, and the results are shown in Figure 10 .

[0050] As can be seen from Figure 10 , the extraction performance of the TFP-COF probe in the application is significantly better than that of the TF-COF which is not modified by phosphorous acid; for strongly polar mycotoxins, the extraction performance of the TFP-COF is better than that of HLB and C18; for weakly polar mycotoxins, the extraction performance of the TFP-COF is comparable to that of HLB and C18. Overall, compared with TF-COF, commercial materials HLB and C18, the TFP-COF coated fiber has better enrichment efficiency.

[0051] In summary, the method for detecting trace or ultra-trace mycotoxins in food based on the TFP-COF probe of the application not only has a good linear range, but also has high sensitivity, high accuracy and good reproducibility, and can be used for high-precision detection of up to 10 kinds of mycotoxins in food.

[0052] Thirdly, the adsorption mechanism of the TFP-COF probe for mycotoxins The TFP-COF of the present application is a porous material containing aromatic rings, fluorine atoms and nitrogen atoms, has a large conjugated system and multiple recognition sites; the functionalization of diethyl phosphite adjusts the hydrophilicity and hydrophobicity of the COF, and enhances the hydrophilicity of the TFP-COF, which is conducive to enhancing the adsorption performance of the TFP-COF for medium and strong polar analytes. At the same time, the structure of mycotoxins contains a large number of oxygen atoms and aromatic rings, which can produce synergistic effects such as hydrophobic interaction, π-π interaction, p-π interaction and hydrogen bond interaction with TFP-COF. In order to further verify the adsorption mechanism between the adsorbent and the analyte, we carried out visualization analysis of non-covalent interaction (NCI), and the results are shown in Figures 11-12 .

[0053] The NCI visualization results show that there are multiple interactions between the molecular structure of TFP-COF and the mycotoxin molecules. Since both TFP-COF and mycotoxin molecules have a conjugated system, they can be attracted to each other through π-π, C-H…π and hydrophobic interaction, thereby promoting the occurrence of adsorption behavior. In addition, the F, N and O atoms in TFP-COF can form pseudo-hydrogen bonds such as C-H…F, C-H…N and C-H…O with C-H in mycotoxin molecules. At the same time, the C-F functional group in TFP-COF can also interact with the aromatic ring in mycotoxin molecules to produce C-F…π interaction force. The results show that the conjugated system, fluorine group and phosphate group of the TFP-COF structure can achieve high-throughput adsorption enrichment of 10 kinds of mycotoxins with a wide range of polarity.

[0054] Example 3 Application of the TFP-COF probe of the present application in actual samples (i.e. wheat flour) 90 samples of wheat flour collected from different cities in Henan Province were detected and analyzed by the method constructed in the present application, wherein the pretreatment method of the wheat flour was as follows: 1 g of wheat flour was added to 5 mL of acetonitrile-water-formic acid (84:15:1, v / v), vortexed for 5 min, ultrasonicated for 30 min, and centrifuged at 10000 rpm for 5 min; the supernatant was transferred to a centrifuge tube, then evaporated to dryness under a gentle stream of nitrogen, and redissolved with ultrapure water, and the ion strength of the redissolved solution was adjusted to 15%, and then extracted, eluted and detected according to the first item of the present example, and the results are shown in Table 5.

[0055] Table 5 Detection of mycotoxins in wheat flour From table 5, in 90 parts of wheat flour, the detection rate of aflatoxin B1 is the highest, followed by aflatoxin B2 and ochratoxin C; the maximum detection of aflatoxin B1 is 4.19 μg·kg -1 , the maximum of aflatoxin B2 is 4.86 μg·kg -1 , the maximum of ochratoxin C is 1.62 μg·kg -1 ; the detection rate of zearalenone metabolites is lower. In addition, although many samples detect aflatoxin B1, the pollution level of AFB1 in all positive samples does not exceed the maximum limit value (5 μg·kg -1 ) specified in GB 2761-2017; in addition, through evaluation, the pollution levels of zearalenone compounds and OTC in 90 parts of wheat flour are all in the safe level, but it is still necessary to continuously monitor the wheat and its products.

[0056] From the above, the TFP-COF of the present application contains aromatic ring, fluorine atom and nitrogen atom itself, and the hydrophilicity is improved after phosphite group modification, so that the TFP-COF of the present application has more binding sites, so it can synergize with mycotoxins through hydrophobic interaction, π-π interaction, p-π interaction and the like, and then realize high-precision detection of mycotoxins with wide polarity range, can be used for quantitative detection of ultra-trace mycotoxins in complex matrix samples, and has practical application prospect.

Claims

1. A bifunctionalized triazine covalent organic framework material, characterized in that: It is polymerized from three monomers—2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine, 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-triphenyl]-4,4"-diamine, and diethyl phosphite—using 1,3,5-trimethylbenzene and n-butanol as solvents and aminosulfonic acid as a catalyst. It is denoted as TFP-COF, and its structural formula is as follows: 。 2. The bifunctionalized triazine covalent organic framework material according to claim 1, characterized in that: The TFP-COF was prepared as follows: 2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine, 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-triphenyl]-4,4"-diamine and diethyl phosphite were accurately weighed, and then 1,3,5-trimethylbenzene, n-butanol and aminosulfonic acid were added and ultrasonically mixed evenly; the reaction system was rapidly degassed by freezing in liquid nitrogen, and the reaction vessel was sealed and placed in a high-temperature environment for reaction; after the reaction was completed, the mixture was washed repeatedly with water, ethanol and tetrahydrofuran, and dried under vacuum to obtain TFP-COF.

3. The bifunctionalized triazine covalent organic framework material according to claim 2, characterized in that: The molar ratio of 2,4,6-tris(4-formyl-biphenyl-4-yl)-1,3,5-triazine, 5'-(4-amino-3,5-difluorophenyl)-3,3",5,5"-tetrafluoro-[1,1':3',1"-triphenyl]-4,4"-diamine and diethyl phosphite is 1:1:

3.

4. The bifunctionalized triazine covalent organic framework material according to claim 2, characterized in that: The temperature of the high-temperature environment is 130℃~150℃.

5. A solid-phase microextraction probe, comprising a coating, characterized in that: The coating uses the bifunctionalized triazine covalent organic framework material as described in any one of claims 1-4.

6. The application of the solid-phase microextraction probe according to claim 5 in the detection of trace or ultra-trace mycotoxins in food, wherein, The application involves using a solid-phase microextraction probe to enrich and extract trace or ultra-trace mycotoxins in food. The enrichment and extraction conditions are: extraction time ≥30 min, pH 2.0–10.0, and NaCl concentration 15%.

7. The application according to claim 6, characterized in that: The fungal toxins include AFB1, AFB2, AFG1, AFG2, OTC, ZAN, α-ZAL, β-ZAL, α-ZOL, and β-ZOL.

8. The application according to claim 6, characterized in that: After enrichment extraction, the mixture is eluted for 10 to 20 minutes with an elution solvent consisting of acetone, formic acid, and water. The volume percentage of water in the elution solvent is 1%, the volume percentage of acetone is 87% to 89%, and the remainder is formic acid.

9. The application according to claim 6, characterized in that: After elution, the eluent was analyzed by UPLC-MS / MS. The UPLC-MS / MS conditions were as follows: mobile phase A was an aqueous solution of formic acid with a volume fraction of 0.1%; mobile phase B was acetonitrile, and gradient elution was used.

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