An aptamer-molecularly imprinted fluorescence covalent organic framework sensor for the determination of diethylstilbestrol and its preparation and application

Abstract

The present invention discloses an aptamer-molecularly imprinted fluorescence covalent organic framework sensor for determining diethylstilbestrol, and its preparation and application, belonging to the field of biosensing technology. The present invention uses fluorescence covalent organic framework as the fluorescence signal and carrier, and molecularly imprinted polymer and aptamer as the biological recognition elements. By utilizing the advantages of the two recognition elements, after the aptamer immobilizes diethylstilbestrol, COFs-Apt@MIPs are prepared by the sol-gel method. Then, after the sample to be measured contacts with COFs-Apt@MIPs, the fluorescence quenching is caused by the charge transfer between DES and COFs-Apt@MIPs, and the fluorescence signal intensity is obtained through the fluorescence spectrum. The fluorescence value is used to realize the quantitative detection of DES. The method of the present invention has the advantages of low detection cost, good stability, simple operation, good selectivity and high efficiency, and has great application potential in the detection of various fishery drug residues in complex food matrices.

Description

Technical Field

[0001] The present invention relates to an aptamer-molecularly imprinted fluorescence covalent organic framework sensor for determining diethylstilbestrol, and its preparation and application, belonging to the field of biosensing technology. Background Art

[0002] Diethylstilbestrol (DES), as a synthetic estrogenic substance, is often used as an estrogen regulator in the aquaculture industry, mainly for promoting the growth and development of fish. Due to long-term unreasonable application, DES is likely to remain in aquatic products and other foods and is difficult to degrade, and finally enters the human body through the food chain. A number of studies have shown that long-term exposure of humans to DES can lead to immune dysfunction, damage to normal hormone levels, and even fetal malformations. Due to its great potential hazards, DES has been classified as a class I carcinogen by the World Health Organization and has been listed in the list of drugs prohibited in foods in many countries including China.

[0003] At present, a variety of analytical methods for detecting DES have been developed, including liquid chromatography-tandem mass spectrometry (LC-MS / MS), high performance liquid chromatography (HPLC), and capillary electrophoresis methods. Although these methods are reliable, selective, and sensitive, they still have defects such as complex analytical steps, time-consuming and laborious, and low economic efficiency.

[0004] With the rapid development of modern analytical techniques, many new rapid, sensitive, and efficient technologies have emerged in the field of detection of agricultural and veterinary drug residues in foods. Biosensing is an increasingly mature technology that can obtain relevant measurable signals through the interaction between probes and specific targets. In biosensing methods, the selection and research of recognition elements have a great impact on the detection effect. As the best alternative substance for traditional recognition elements, molecularly imprinted polymers (MIPs) have advantages such as high stability, easy synthesis, and strong specificity. However, in the actual application process, macromolecules and the like present in real samples will occupy the surface space of MIPs, thereby interfering with the selective recognition of template molecules by binding sites. Moreover, in the synthesis process of MIPs, non-specific binding will also affect the detection sensitivity to complex matrices, and the recognition effect and recognition accuracy will be affected. In addition, existing aptamer-molecular imprinting detection methods often use fluorescent nanomaterials such as upconversion materials as fluorescent signals, and there are few binding and recognition sites. Summary of the Invention

[0005] [Technical Problem]

[0006] The existing molecularly imprinted polymer biosensing has poor detection effect for complex matrices; and the existing aptamer-molecular imprinting detection methods have few binding and recognition sites.

[0007] [Technical Solution]

[0008] To solve the above problems, the present invention uses a fluorescent covalent organic framework as the fluorescent signal and carrier, and molecularly imprinted polymers and aptamers as biorecognition elements. By utilizing the advantages of the two recognition elements, after the aptamer immobilizes diethylstilbestrol (DES), an aptamer-molecularly imprinted fluorescent covalent organic framework (COFs-Apt@MIPs) is prepared by the sol-gel method. The COFs-Apt@MIPs of the present invention can sensitively and synergistically determine DES through the aptamer and the molecularly imprinted polymer.

[0009] The first object of the present invention is to provide a method for preparing an aptamer-molecularly imprinted fluorescent covalent organic framework (COFs-Apt@MIPs), comprising the following steps:

[0010] (1) Preparation of TFPT-PDAN-COF:

[0011] Disperse 2,4,6-tris(4-formylphenyl)-1,3,5-triazine, 1,4-phenylenediacetonitrile, and cesium carbonate in an organic solvent. After three cycles of rapid freezing in liquid nitrogen - evacuating and introducing nitrogen - thawing, heat, centrifuge, wash, and dry to obtain TFPT-PDAN-COF powder;

[0012] (2) Preparation of amidoxime-modified TFPT-PDAN-COF-AO:

[0013] Disperse TFPT-PDAN-COF, potassium carbonate, and hydroxylamine hydrochloride in an organic solvent. Stir and heat the mixed solution system, cool, wash, and dry to obtain amidoxime-modified TFPT-PDAN-COF-AO; then disperse it in a buffer solution to obtain a buffer solution of TFPT-PDAN-COF-AO;

[0014] (3) Preparation of COFs-Apt:

[0015] Mix the buffer solution of TFPT-PDAN-COF-AO evenly with the Sulfo-SMCC solution, incubate for activation, centrifuge and collect to obtain activated TFPT-PDAN-COF-AO; then disperse the activated TFPT-PDAN-COF-AO in a buffer solution, and mix and incubate it with the DES aptamer solution, and shake overnight; centrifuge and collect the precipitate, wash to obtain COFs-Apt successfully conjugated with the aptamer; then disperse it in a buffer solution to obtain a buffer solution of COFs-Apt;

[0016] (4) Preparation of COFs-Apt@MIPs:

[0017] Mix and incubate the buffer solution of COFs-Apt with DES, and disperse it with water; then add APTES and stir for prepolymerization, and subsequently add TEOS and NH3 ·H 2 O, perform sealed stirring polymerization; centrifuge to collect the precipitate and wash it to obtain COFs-Apt@MIPs.

[0018] In one embodiment of the present invention, the mass ratio of 1,4-benzenedicarbonitrile to 2,4,6-tris(4-formylphenyl)-1,3,5-triazine in step (1) is 1:(1.5 - 1.7), further preferably 1:1.6; the mass ratio of 2,4,6-tris(4-formylphenyl)-1,3,5-triazine to cesium carbonate is 1:(4 - 6), further preferably 1:5.

[0019] In one embodiment of the present invention, the organic solvent in step (1) is 1,4-dioxane, and the dosage ratio of 2,4,6-tris(4-formylphenyl)-1,3,5-triazine to the organic solvent is (24 - 26) mg:2 mL.

[0020] In one embodiment of the present invention, the heating in step (1) is carried out at 110 - 120 °C for 92 - 96 h, further preferably at 120 °C for 72 h.

[0021] In one embodiment of the present invention, the drying in step (1) is vacuum drying, specifically carried out under vacuum at 60 - 120 °C.

[0022] In one embodiment of the present invention, the mass ratio of TFPT-PDAN-COF, potassium carbonate and hydroxylamine hydrochloride in step (2) is 1:(2.5 - 3):(2 - 14).

[0023] In one embodiment of the present invention, the organic solvent in step (2) is anhydrous methanol, and the dosage ratio of TFPT-PDAN-COF to the organic solvent is 1 mg:(1 - 2) mL.

[0024] In one embodiment of the present invention, the heating in step (2) is carried out at 60 - 80 °C for 12 - 24 h, further preferably at 80 °C for 24 h.

[0025] In one embodiment of the present invention, the washing in step (2) is carried out by washing with water several times; the drying is vacuum drying at a temperature of 75 - 85 °C.

[0026] In one embodiment of the present invention, the buffer solution in step (2) is HEPES buffer solution with a concentration of 10 mmol / L and pH = 7.2.

[0027] In one embodiment of the present invention, in the buffer solution of TFPT-PDAN-COF-AO in step (2), the dosage ratio of TFPT-PDAN-COF-AO to the buffer is 1 mg:(1 - 2) mL.

[0028] In one embodiment of the present invention, in step (3), the concentration of the Sulfo-SMCC solution is 1 - 4 mg / mL, and more preferably 2 mg / mL.

[0029] In one embodiment of the present invention, in step (3), the volume ratio of the buffer solution of TFPT-PDAN-COF-AO, the Sulfo-SMCC solution and the DES aptamer solution is 7:3:(0.6 - 1.4).

[0030] In one embodiment of the present invention, in step (3), the concentration of the DES aptamer solution is 1.5 - 2.5 nmol.

[0031] In one embodiment of the present invention, the incubation activation in step (3) is carried out at 15 - 37 °C for 2 h, and more preferably at 25 °C for 2 h.

[0032] In one embodiment of the present invention, the sequence of the DES aptamer in step (3) is 5’-GGCGATGGGGTAGGGGGTGTGGAGGGGCCGGACGGAGGGG-3’.

[0033] In one embodiment of the present invention, the buffer in step (3) is HEPES buffer with a concentration of 10 mmol / L and pH = 7.2.

[0034] In one embodiment of the present invention, in step (4), the dosage ratio of the buffer solution of COFs-Apt to DES is 1 mL:(4 - 6) mg.

[0035] In one embodiment of the present invention, the mixed incubation in step (4) is carried out at 25 °C for 0.5 - 2 h, and more preferably for 1 h.

[0036] In one embodiment of the present invention, in step (4), the dosage ratio of DES to APTES, TEOS, NH 3 ·H 2 O is 5.4 mg:23 μL:(10 - 60) μL:50 μL.

[0037] In one embodiment of the present invention, the pre-polymerization in step (4) is carried out by stirring at 25 °C for 5 h; the sealed stirring is carried out for 12 h; the washing is carried out by washing the precipitate several times with a methanol / acetic acid mixture (9:1, v / v), and washing away the excess acetic acid with methanol, so as to completely remove the template molecules.

[0038] The second object of the present invention is COFs-Apt@MIPs prepared by the method of the present invention.

[0039] The third object of the present invention is to provide a method for detecting diethylstilbestrol (DES), and the method uses the COFs-Apt@MIPs of the present invention.

[0040] In one embodiment of the present invention, the method for detecting diethylstilbestrol (DES) is that after the sample to be tested is contacted with COFs-Apt@MIPs, the fluorescence quenching is caused by the charge transfer between DES and COFs-Apt@MIPs, and the fluorescence signal intensity is obtained through the fluorescence spectrum, and the quantitative detection of DES is realized by using the fluorescence value;

[0041] Specifically, it includes the following steps:

[0042] (1) Treatment of the sample to be tested:

[0043] Add an acetonitrile-acetone solution to the homogenate of the sample to be tested and oscillate, centrifuge to collect the supernatant, dry it with nitrogen, and then re-dissolve it with chloroform; then continue to oscillate and centrifuge after adding a NaOH solution; then add a phosphoric acid solution and oscillate; then extract, oscillate and centrifuge to separate the upper organic phase; finally, re-dissolve the collected residue to obtain the treated sample to be tested;

[0044] (2) Detection:

[0045] After incubating the treated sample to be tested at 25 °C for 30 min, measure the fluorescence spectrum with an emission wavelength range of 400 - 700 nm at an excitation wavelength of 365 nm to obtain the fluorescence intensity at 540 nm;

[0046] (3) Calculate the content or concentration of diethylstilbestrol (DES):

[0047] Substitute the fluorescence intensity at 540 nm into the standard working curve to calculate the concentration of DES in the sample to be tested.

[0048] In one embodiment of the present invention, the method for making the standard working curve in step (3) is as follows:

[0049] To 1 mL of 1 mg / L LCOFs-Apt@MIPs ethanol solution, 1 mL of DES solution with different concentrations (specifically 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 mg / L) was added. After incubation at 25 °C for 30 min, the fluorescence spectrum with an emission wavelength range of 400 - 700 nm was measured at an excitation wavelength of 365 nm. The standard working curve was plotted with the fluorescence intensity at 540 nm and the DES concentration.

[0050] In one embodiment of the present invention, in step (1), the dosage ratio of the sample to be measured, acetonitrile-acetone solution, chloroform, NaOH solution, and phosphoric acid solution is 2 g: 6 mL: 0.5 mL: 2 mL: 200 μL; in the acetonitrile-acetone solution, V 乙腈 :V 丙酮 = 4:1; the concentration of the NaOH solution is 2 mol / L; the concentration of the phosphoric acid solution is 6 mol / L; the extraction is carried out with acetonitrile.

[0051] In one embodiment of the present invention, the sample to be measured in step (2) is aquatic products, including commercially available crucian carp, etc.

[0052] In one embodiment of the present invention, the standard working curve in step (3) is F 0 / F = 0.03783C + 1.01745 (R 2 = 0.9983), the LOD value is 0.23 mg / L, (3σ / s, n = 3); where F 0 is the fluorescence intensity of COFs-Apt@MIPs without adding DES, and F is the fluorescence intensity of COFs-Apt@MIPs after incubation with DES.

[0053] [Beneficial effects]

[0054] (1) The fluorescent covalent organic framework (TFPT-PDAN-COF) described in the present invention is synthesized by Knoevenagel condensation reaction. The sp 2 carbon-carbon double bond conjugate structure has strong stability, which is beneficial for long-term storage at room temperature. It can not only be used as a fluorescence signal but also as a fixed and supporting skeleton for molecularly imprinted materials, overcoming the defect that the recognition sites of conventional molecularly imprinted polymer materials are prone to collapse and ensuring the accuracy of recognition.

[0055] (2) The amidoxime group-modified TFPT-PDAN-COF-AO described in the present invention contains a large number of amino groups, which can provide coupling sites for aptamers.

[0056] (3) In the COFs-Apt@MIPs of the present invention, the molecularly imprinted polymer can provide a protective layer for the aptamer, and the aptamer can reduce the non-specific recognition of the molecularly imprinted polymer, and then co-recognize DES through the cooperation of the aptamer and the molecularly imprinted polymer.

[0057] (4) The method for detecting diethylstilbestrol (DES) of the present invention uses a fluorescent covalent organic framework as a fluorescent signal and detects DES by a fluorescence quenching method; it has the advantages of convenient operation, high selectivity, etc., and co-existing banned fishery drugs, heavy metal ions and structural analogs do not interfere with the determination; it overcomes the deficiencies of time-consuming and expensive instrumental methods such as high performance liquid chromatography. Brief Description of the Drawings

[0058] Figure 1 SEM images of TFPT-PDAN-COF (A) and TFPT-PDAN-COF-AO (B).

[0059] Figure 2 Fluorescence intensities of TFPT-PDAN-COF-AO at different times (A), pH values (B) and temperatures (C).

[0060] Figure 3 SEM image of COFs-Apt@NIPs.

[0061] Figure 4 Optimization of the synthesis conditions of COFs-Apt@MIPs; where A is the optimization of the addition amount of TFPT-PDAN-COF-AO; B is the optimization of the dosage of the DES aptamer solution; C is the optimization of the addition amount of the cross-linking agent TEOS.

[0062] Figure 5 Feasibility verification of the detection of DES by COFs-Apt@MIPs.

[0063] Figure 6 Fluorescence spectra of COFs-Apt@MIPs after incubation with different concentrations of DES (A); linear curve between relative fluorescence intensity and DES concentration (B); fluorescence spectra of COFs-Apt@NIPs after incubation with different concentrations of DES (C).

[0064] Figure 7 Reusability performance of COFs-Apt@MIPs.

[0065] Figure 8Specific analysis of COFs-Apt@MIPs; where A is the effect of a single structural analogue on COFs-Apt@MIPs; B is the effect of the coexistence of a structural analogue and DES on COFs-Apt@MIPs; C is the effect of metal ions on COFs-Apt@MIPs; D is the effect of other fishery drugs on COFs-Apt@MIPs. Detailed implementation

[0066] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0067] Unless otherwise specified, the solvent of the solution in the embodiments is water.

[0068] The sequence of the DES aptamer used in the embodiment is 5’-GGCGATGGGGTAGGGGGTGTGGAGGGGCCGGACGGAGGGG-3’.

[0069] Example 1

[0070] A method for preparing aptamer-molecularly imprinted fluorescent covalent organic framework (COFs-Apt@MIPs) includes the following steps:

[0071] (1) Preparation of fluorescent covalent organic framework (TFPT-PDAN-COF):

[0072] Accurately weigh 25.58 mg of 2,4,6-tris(4-formylphenyl)-1,3,5-triazine and 15.99 mg of 1,4-phenylenediacetonitrile into a 25 mL thick-walled reaction tube. Then add 2 mL of 1,4-dioxane and 127.90 mg of cesium carbonate, and mix evenly by ultrasound to obtain a mixed solution. After that, subject the mixed solution to three cycles of rapid freezing with liquid nitrogen - vacuum evacuation and nitrogen purging - thawing to remove oxygen and moisture in the reaction system, and then seal it with nitrogen. After the reaction tube returns to room temperature, heat it at 120 °C for 72 h. After the reaction is completed, centrifuge to obtain a yellow precipitate, and wash it several times with tetrahydrofuran and water in turn. Finally, dry it overnight in a vacuum drying oven at 80 °C to obtain a yellow powder with bright fluorescence, namely: TFPT-PDAN-COF powder;

[0073] (2) Preparation of amidoxime-functionalized fluorescent covalent organic framework (TFPT-PDAN-COF-AO):

[0074] Weigh 50 mg of TFPT-PDAN-COF into a 100 mL round-bottom flask, and successively add 138.2 mg of potassium carbonate and 694.90 mg of hydroxylamine hydrochloride thereto. Finally, add 50 mL of anhydrous methanol, and ultrasonically treat to disperse evenly to obtain a mixed solution. Stir the mixed solution at 80 °C for 24 h. After cooling to room temperature, wash it several times with ultrapure water, and finally dry it overnight in a vacuum drying oven at 80 °C to obtain amidoxime group-modified TFPT-PDAN-COF-AO. Then, disperse and dissolve 1 mg of the dried TFPT-PDAN-COF-AO in 1 mL of HEPES buffer solution (10 mmol / L, pH = 7.2) to obtain a buffer solution of TFPT-PDAN-COF-AO for standby.

[0075] (3) Preparation of aptamer-coupled (COFs-Apt) of post-modified fluorescent covalent organic frameworks:

[0076] Mix 700 μL of the buffer solution of TFPT-PDAN-COF-AO (1.5 mg of TFPT-PDAN-COF-AO) with 300 μL of a Sulfo-SMCC solution with a concentration of 2 mg / mL evenly, incubate and activate on a shaker at 25 °C for 2 h, centrifuge at 10000 r / min for 10 min, and collect the activated TFPT-PDAN-COF-AO. Wash the precipitate several times with HEPES buffer solution to elute the excess Sulfo-SMCC, and finally redisperse it in 1 mL of HEPES buffer solution. Then, add 100 μL of a DES aptamer solution with a concentration of 2 nmol to the buffer solution of the activated TFPT-PDAN-COF-AO, incubate at 25 °C, and shake overnight. Centrifuge to collect the precipitate, and wash the precipitate with ultrapure water multiple times to remove the uncoupled aptamer to obtain COFs-Apt with successfully coupled aptamer. Then, disperse it with 1 mL of buffer solution to obtain a buffer solution of COFs-Apt;

[0077] (4) Preparation of COFs-Apt@MIPs:

[0078] Incubate 1 mL of the buffer solution of COFs-Apt with 5.4 mg of DES in a shaker at 25 °C for 1 h for the capture of DES. Then, transfer the system to a 25 mL round-bottom flask, disperse it with 5 mL of ultrapure water, add 23 μL of APTES, and stir at 25 °C for 5 h for prepolymerization. Subsequently, add 40 μL of TEOS and 50 μL of NH 3 ·H 2O was sealed and stirred for polymerization at 25 °C for 12 h. After the polymerization was completed, ultrapure water was added and the precipitate was collected by centrifugation. The precipitate was washed several times with a methanol / acetic acid mixture (9:1, v / v), and the excess acetic acid was washed away with methanol to completely remove the template molecules, obtaining COFs-Apt@MIPs.

[0079] The obtained TFPT-PDAN-COF, TFPT-PDAN-COF-AO, COFs-Apt, and COFs-Apt@MIPs were subjected to performance tests, and the test results are as follows:

[0080] ① Preparation and post-modification characterization of fluorescent covalent organic framework (TFPT-PDAN-COF)

[0081] Figure 1 are the SEM images of TFPT-PDAN-COF (A) and TFPT-PDAN-COF-AO (B). From Figure 1 it can be seen that the clear surface morphology of TFPT-PDAN-COF observed at the 2-μm scale is composed of many relatively thick strip structures arranged, and there are voids between the thick strips; compared with TFPT-PDAN-COF, the surface morphology of TFPT-PDAN-COF-AO does not show significant changes, proving that the post-synthesis modification has no negative impact on the morphology and integrity of the COFs framework, and the porosity can also be well maintained. The relevant characteristic functional groups of TFPT-PDAN-COF and TFPT-PDAN-COF-AO were characterized by FT-IR spectroscopy. By comparing the changes in functional groups between the monomer and TFPT-PDAN-COF, the successful synthesis of TFPT-PDAN-COF can be further confirmed. Compared with TFPT, the characteristic peak signals at 2731 cm -1 and 1702 cm -1 in TFPT-PDAN-COF are significantly weakened, which respectively correspond to the stretching vibration peaks of C-H and C=O functional groups. The new characteristic peak appearing at 2217 cm -1 in TFPT-PDAN-COF is the same as the characteristic peak of the -CN functional group in PDAN, further verifying that PDNA was successfully combined and introduced with the corresponding functional groups during the synthesis of TFPT-PDAN-COF. The two characteristic peaks appearing at 3368 cm -1 and 3288 cm -1 in TFPT-PDAN-COF-AO modified with amidoxime group both correspond to the stretching vibration of the amino group.

[0082] Figure 2 are the fluorescence intensities of TFPT-PDAN-COF-AO at different times (A), pH values (B), and temperatures (C). From Figure 2It can be seen that the fluorescence intensity of TFPT-PDAN-COF-AO is stable at different times, pH values, and temperatures.

[0083] ② Characterization of the aptamer functionalization of the post-modified fluorescent covalent organic framework

[0084] The relevant experiments were characterized by an ultraviolet-visible spectrophotometer. After incubating TFPT-PDAN-COF-AO with the DES aptamer, the supernatant of the reaction was collected to measure the ultraviolet-visible absorption spectrum, and it was compared with the aptamer of the same concentration as a control. The DES aptamer has an obvious ultraviolet-visible absorption peak at 254 nm, while no obvious characteristic peak of the DES aptamer appears in the supernatant of the reaction between TFPT-PDAN-COF-AO and the aptamer.

[0085] ③ Preparation and characterization of COFs-Apt@MIPs

[0086] Figure 3 Figure Figure 3 It can be seen that the imprinted layer can uniformly coat COFs-Apt to form an obvious silicon spherical structure. The FT-IR spectra of COFs-Apt@MIPs and COFs-Apt@NIPs are similar, and characteristic peaks of the molecular imprinted layer such as 1028 cm -1 and 790 cm -1 appear, corresponding to the anti / symmetric stretching vibration peaks of Si-O-Si, respectively. This can fully prove the successful synthesis of COFs-Apt@MIPs and COFs-Apt@NIPs.

[0087] Example 2

[0088] The addition amount of COFs, the addition amount of aptamers, and the thickness of MIPs will all affect the fluorescence sensing detection effect of COFs-Apt@MIPs. Therefore, the synthesis conditions of COFs-Apt@MIPs are optimized from these three parts respectively. The intrinsic fluorescence intensity value (F) of the fluorescent material and the fluorescence quenching efficiency (F 0 / F) before and after reacting with the target are important indicators reflecting the sensing detection performance. Therefore, these two indicators are selected to evaluate the synthesis effect of COFs-Apt@MIPs.

[0089] (1) Adjust the addition amount of TFPT-PDAN-COF-AO:

[0090] Adjust the addition amount of TFPT-PDAN-COF-AO in the buffer solution of TFPT-PDAN-COF-AO in step (2) of Example 1 to 0.20 mg - 2.00 mg, and keep the others the same as in Example 1 to prepare COFs-Apt@MIPs.

[0091] After that, the obtained COFs-Apt@MIPs and DES were mixed to make the final concentration of DES 10 mg / L, incubated at 25 °C for 2 h, and the fluorescence intensities before and after incubation were measured.

[0092] The results are as Figure 4 shown in A.

[0093] As can be seen from Figure 4 A: When the addition amount of TFPT-PDAN-COF-AO was below 1.50 mg, the original intensity of COFs-Apt@MIPs increased with the increase of the addition amount of TP-COF-AO. When the addition amount of TFPT-PDAN-COF-AO exceeded 1.5 mg, the fluorescence quenching effect began to decrease. Therefore, the optimal addition amount of TFPT-PDAN-COF-AO was 1.5 mg.

[0094] (2) Adjust the addition amount of DES aptamer:

[0095] The dosages of the DES aptamer solution in step (3) of Example 1 were adjusted to 60, 80, 100, 120, and 140 μL, and the others were kept the same as in Example 1 to prepare COFs-Apt@MIPs.

[0096] After that, the obtained COFs-Apt@MIPs and DES were mixed to make the final concentration of DES 10 mg / L, incubated at 25 °C for 2 h, and the fluorescence intensities before and after incubation were measured.

[0097] The results are as Figure 4 shown in B.

[0098] As can be seen from Figure 4 B: With the increase of the aptamer addition amount, the fluorescence quenching effect first increased and then decreased, and the aptamer addition amount with the best fluorescence response effect was 100 μL.

[0099] (3) Adjust the addition amount of crosslinking agent TEOS

[0100] The addition amount of the crosslinking agent TEOS in step (4) of Example 1 was adjusted to 10 μL - 60 μL, and the others were kept the same as in Example 1 to prepare COFs-Apt@MIPs.

[0101] After that, the obtained COFs-Apt@MIPs and DES were mixed to make the final concentration of DES 10 mg / L, incubated at 25 °C for 2 h, and the fluorescence intensities before and after incubation were measured.

[0102] The results are as Figure 4 shown in C.

[0103] As can be seen fromFigure 4 It can be seen from Figure C that an increase in the addition amount of TEOS will lead to a gradual decrease in the fluorescence intensity of COFs-Apt@MIPs. Moreover, the fluorescence quenching efficiency of COFs-Apt@MIPs shows a trend of first increasing and then decreasing with the increase in the addition amount of TEOS. Therefore, the optimal addition amount of TEOS is 40 μL.

[0104] Comparative Example 1

[0105] Omit the addition of DES in step (4) of Example 1, and keep the others the same as in Example 1 to obtain COFs-Apt@NIPs without molecular imprinting.

[0106] Comparative Example 2

[0107] Omit the addition of the DES aptamer solution in step (3) of Example 1, and keep the others the same as in Example 1 to obtain COFs@MIPs without aptamers.

[0108] Detection performance of the sensor in Example 3

[0109] The fluorescence sensing performance of COFs-Apt@MIPs is achieved based on the synergistic recognition of DES by the molecular imprinting layer and the aptamer.

[0110] (1) Molecular imprinting and aptamer

[0111] To verify the fluorescence sensing performance of COFs-Apt@MIPs, the sensors of Example 1, Comparative Example 1, and Comparative Example 2 were mixed with DES so that the final concentration of DES was 10 mg / L, and incubated at 25 °C for 2 h, and the changes in fluorescence signals of the three were compared.

[0112] The results are as Figure 5 .

[0113] From Figure 5 It can be seen that the fluorescence intensities of COFs-Apt@MIPs and COFs-Apt@NIPs are similar before incubation with DES. When the two sensors are incubated with DES for a certain time respectively, the fluorescence of COFs-Apt@MIPs is significantly reduced compared with that of COFs-Apt@NIPs. This shows that COFs-Apt@MIPs can achieve the best recognition and sensing performance under the synergistic action of the aptamer and MIPs. Although COFs-Apt@NIPs have the DES aptamer, since NIPs are non-imprinted layers polymerized in the absence of template molecules, they will hinder the passage of DES through the silicon layer and thus it is difficult to bind to the aptamer. Therefore, the successful synthesis of MIPs is an important condition for achieving selective recognition and detection.

[0114] (2) Concentration of DES detected

[0115] To 1 mL of an ethanol solution of aptamer-molecularly imprinted fluorescent covalent organic frameworks (COFs-Apt@MIPs) at 1 mg / mL, 1 mL of DES solution with different concentrations (0, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 mg / L) was added. After incubation for 30 min, the fluorescence intensity at an emission wavelength of 540 nm was measured at an excitation wavelength of 365 nm.

[0116] Meanwhile, COFs-Apt@NIPs was used as a comparison.

[0117] As Figure 6 shown.

[0118] It can be seen from Figure 6 that as the concentration of DES increases, the fluorescence intensity of COFs-Apt@MIPs decreases. However, since COFs-Apt@NIPs lacks sites that can specifically recognize DES, its fluorescence intensity is not sensitive to changes in the concentration of DES. For DES with a final concentration range of 0.4 mg / L - 25.6 mg / L, the content of DES has a linear relationship with the relative fluorescence intensity (F 0 / F) of COFs-Apt@MIPs (where F 0 is the fluorescence intensity of COFs-Apt@MIPs without adding DES, and F is the fluorescence intensity of COFs-Apt@MIPs after incubation with DES), and the linear equation is F 0 / F = 0.03783C + 1.01745 (R 2 = 0.9983), and the LOD value is 0.23 mg / L, (3σ / s, n = 3).

[0119] By comparing with the reported DES detection methods, the COFs-Apt@MIPs fluorescence sensor constructed in the present invention has a relatively wide linear range and a low detection limit.

[0120] (3) Reusability

[0121] The sensor COFs-Apt@MIPs in Example 1 was used through 5 adsorption-elution cycles.

[0122] The results are as Figure 7 .

[0123] It can be seen from Figure 7It can be seen that after 5 adsorption-elution cycles, the fluorescence intensity of COFs-Apt@MIPs decreased slightly compared with the initial value, but the fluorescence signal response to DES still maintained a good effect. It is proved that during the process of adsorption-elution-re-adsorption of COFs-Apt@MIPs, the imprinting sites can basically maintain their original performance, and the aptamer also maintains good high affinity under the double protection of COFs and the imprinting layer. This result shows that the constructed COFs-Apt@MIPs fluorescence sensor has the potential for multiple reuse.

[0124] (4) Specific recognition ability

[0125] Using structural analogs as interferents, the fluorescence quenching of four substances, namely nonylphenol (NP), estradiol (E2), bisphenol A (BPA), and hexestrol (HEX), on COFs-Apt@MIPs and COFs-Apt@NIPs was investigated.

[0126] To 1 mL of 1 mg / mL aptamer-molecularly imprinted fluorescent covalent organic framework (COFs-Apt@MIPs) ethanol solution, 1 mL of 5 mg / L nonylphenol (NP), estradiol (E2), bisphenol A (BPA), and hexestrol (HEX) solutions were added respectively. After incubation at 25 °C for 30 min, the fluorescence intensity at an emission wavelength of 540 nm was measured at an excitation wavelength of 365 nm.

[0127] At the same time, COFs-Apt@NIPs was used as a comparison.

[0128] The results are as Figure 8 shown in A.

[0129] As can be seen from Figure 8 A: The fluorescence quenching effects of the structural analogs of DES on COFs-Apt@MIPs and COFs-Apt@NIPs are very close, and both show weak fluorescence intensity changes. However, the fluorescence quenching effect of DES on COFs-Apt@NIPs is similar to that of other substances, thus proving the non-specific recognition property of COFs-Apt@NIPs.

[0130] Since there are often multiple substances coexisting in the actual samples to be measured, the competitive binding experiments of COFs-Apt@MIPs and COFs-Apt@NIPs were further investigated when the structural analogs and DES coexist.

[0131] To 1 mL of 1 mg / mL ethanol solution of aptamer-molecularly imprinted fluorescent covalent organic frameworks (COFs-Apt@MIPs), 1 mL of 5 mg / L nonylphenol (NP), estradiol (E2), bisphenol A (BPA), and hexestrol (HEX) solutions were added respectively. Subsequently, DES was added respectively to make the final concentration of DES 10 mg / L. After incubation at 25 °C for 30 min, the fluorescence intensity at the emission wavelength of 540 nm was measured at the excitation wavelength of 365 nm.

[0132] The results are as Figure 8 shown in

[0133] As can be seen from Figure 8 Figure B: When only DES was present, COFs-Apt@MIPs showed good fluorescence sensing performance. When the subsequent interfering substances and DES were present simultaneously, it did not affect the change in the fluorescence signal of COFs-Apt@MIPs, proving that COFs-Apt@MIPs could still maintain its signal response ability of specific recognition in the mixed system.

[0134] Subsequently, the effects of other fishery drugs and metal ions that may exist in aquatic products on the detection performance of COFs-Apt@MIPs were also explored.

[0135] To 1 mL of 1 mg / mL ethanol solution of aptamer-molecularly imprinted fluorescent covalent organic frameworks (COFs-Apt@MIPs), 1 mL of 5 mg / L oxytetracycline (OXY), enrofloxacin (ENR), sulfamethazine (SM 2 ), trimethoprim (TMP) solutions, sodium chloride (NaCl), magnesium chloride (MgCl 2 ), calcium chloride (CaC l2 ), copper chloride (CuCl 2 ), zinc chloride (ZnCl 2 ), lithium chloride (LiCl), potassium chloride (KCl) solutions were added respectively. After incubation at 25 °C for 30 min, the fluorescence intensity at the emission wavelength of 540 nm was measured at the excitation wavelength of 365 nm.

[0136] As Figure 8 shown in Figures C and D.

[0137] As can be seen from Figure 8 Figures C and D: The relative fluorescence intensity of COFs-Apt@MIPs proved that it was not affected by the presence of such substances.

[0138] The above results can fully prove the excellent anti-interference ability of the dual recognition element of COFs-Apt@MIPs.

[0139] Example 4 Spike Recovery

[0140] To explore the practical application potential of COFs-Apt@MIPs, a spike recovery experiment was conducted on fish samples purchased from the market.

[0141] Crucian carp was purchased from a local supermarket, minced, and homogenized with a tissue homogenizer to obtain a homogenized sample.

[0142] 1 mL of DES solutions with different concentrations (specifically 0.5, 1, and 10 mg / L) were added to 2 ± 0.05 g of the homogenized sample. Subsequently, through 6 mL of an acetonitrile-acetone solution (the ratio of the two was V 乙腈 :V 丙酮 = 4:1), after shaking the spiked homogenate for 2 min, centrifuging at 4000 r / min (15 °C) for 10 min, collecting the supernatant, and drying it with nitrogen under a 60 °C water bath. The residue after drying was collected, redissolved with 0.5 mL of chloroform, and shaken.

[0143] Subsequently, continue to shake for 30 s and then centrifuge by adding 2 mL of NaOH solution (2 mol / L); add 200 μL of phosphoric acid solution (6 mol / L) to the supernatant and shake for 5 s.

[0144] Subsequently, extraction treatment was carried out with acetonitrile. After adding acetonitrile and shaking for 2 min, centrifuging at room temperature for 10 min, separating the upper organic phase, and drying it with nitrogen under a 60 °C water bath to obtain the treated sample to be measured; the collected residue was redissolved with acetonitrile and stored for later use.

[0145] The treated sample to be measured and the purchased fish were directly used to test DES in them with a commercial ELISA kit and the COFs-Apt@MIPs of Example 1; the test conditions for COFs-Apt@MIPs were: after incubating at 25 °C for 30 min, measuring the fluorescence spectrum with an emission wavelength range of 400 - 700 nm at an excitation wavelength of 365 nm to obtain the fluorescence intensity at 540 nm; substituting the fluorescence intensity into the standard curve F 0 / F = 0.03783C + 1.01745 (R 2 = 0.9983) to obtain the DES concentration.

[0146] The results are as follows:

[0147] The recovery rate range of DES determined by COFs-Apt@MIPs was 98.68% - 101.97%, and the relative standard deviation (RSD) was all below 3.05%.

[0148] The ELISA experimental results were close to the measurement results of the present invention, further proving the reliability of the measurement results of the method of the present invention.

[0149] Although the present invention has been disclosed above in its preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for preparing aptamer-molecularly imprinted fluorescent covalent organic frameworks (COFs-Apt@MIPs), characterized in that, it includes the following steps: (1) Preparation of TFPT-PDAN-COF: Disperse 2,4,6-tris(4-formylphenyl)-1,3,5-triazine, 1,4-phenylenediacetonitrile and cesium carbonate in an organic solvent, and perform three cycles of rapid freezing with liquid nitrogen - evacuating and purging with nitrogen - thawing, heating, centrifuging, washing, and drying to obtain TFPT-PDAN-COF powder; (2) Preparation of amidoxime group-modified TFPT-PDAN-COF-AO: Disperse TFPT-PDAN-COF, potassium carbonate and hydroxylamine hydrochloride in an organic solvent, stir and heat the mixed solution system, cool, wash, and dry to obtain amidoxime group-modified TFPT-PDAN-COF-AO; then disperse it with a buffer solution to obtain a buffer solution of TFPT-PDAN-COF-AO; (3) Preparation of COFs-Apt: Mix the buffer solution of TFPT-PDAN-COF-AO with Sulfo-SMCC solution evenly, incubate for activation, centrifuge and collect to obtain activated TFPT-PDAN-COF-AO; then disperse the activated TFPT-PDAN-COF-AO in a buffer solution, and then mix and incubate with the DES aptamer solution, shake overnight; centrifuge and collect the precipitate, wash to obtain COFs-Apt successfully conjugated with the aptamer; then disperse it with a buffer solution to obtain a buffer solution of COFs-Apt; (4) Preparation of COFs-Apt@MIPs: The buffer solution of COFs-Apt was mixed and incubated with DES and dispersed in water; then APTES was added and stirred for prepolymerization, and subsequently TEOS and NH 3 ·H 2 O were added, and the mixture was sealed and stirred for polymerization; the precipitate was collected by centrifugation, washed, and COFs-Apt@MIPs were obtained.

2. According to the method described in claim 1, characterized in that, the mass ratio of 1,4-phenylenediacetonitrile to 2,4,6-tris(4-formylphenyl)-1,3,5-triazine described in step (1) is 1:(1.5 - 1.7); the mass ratio of 2,4,6-tris(4-formylphenyl)-1,3,5-triazine to cesium carbonate is 1:(4 - 6).

3. According to the method described in claim 1, characterized in that, the mass ratio of TFPT-PDAN-COF, potassium carbonate and hydroxylamine hydrochloride described in step (2) is 1:(2.5 - 3):(2 - 14).

4. According to the method described in claim 1, characterized in that, the volume ratio of the buffer solution of TFPT-PDAN-COF-AO, Sulfo-SMCC solution and DES aptamer in step (3) is 7:3:0.6 - 1.

4.

5. According to the method described in claim 1, characterized in that, the sequence of the DES aptamer in step (3) is 5’-GGCGATGGGGTAGGGGGTGTGGAGGGGCCGGACGGAGGGG-3’.

6. According to the method described in claim 1, characterized in that, The dosage ratio of DES, APTES, TEOS, and NH 3 ·H 2 O in step (4) is 5.4 mg : 23 μL : 10 - 60 μL : 50 μL.

7. COFs-Apt@MIPs prepared by the method described in any one of claims 1 - 6.

8. A method for detecting diethylstilbestrol (DES), characterized in that, The method described above uses the COFs-Apt@MIPs described in claim 7.

9. The method according to claim 8, characterized in that the method for detecting diethylstilbestrol (DES) is that after the sample to be tested is contacted with COFs-Apt@MIPs, the fluorescence quenching is caused by the charge transfer between DES and COFs-Apt@MIPs, and the fluorescence signal intensity is obtained through the fluorescence spectrum, and the quantitative detection of DES is realized by using the fluorescence value.

10. The method according to claim 8, characterized in that the method for detecting diethylstilbestrol (DES) specifically includes the following steps: (1) Treatment of the sample to be tested: Add acetonitrile-acetone solution to the homogenate of the sample to be tested, shake, centrifuge to collect the supernatant, dry it with nitrogen, and then dissolve it in chloroform; then continue to shake and centrifuge after adding NaOH solution; then add phosphoric acid solution and shake; then extract, shake and centrifuge to separate the upper organic phase; finally dissolve the collected residue to obtain the treated sample to be tested; (2) Detection: After incubating the treated sample to be tested at 25 °C for 30 min, measure the fluorescence spectrum with an emission wavelength range of 400-700 nm at an excitation wavelength of 365 nm to obtain the fluorescence intensity at 540 nm; (3) Calculate the content or concentration of diethylstilbestrol (DES): Substitute the fluorescence intensity at 540 nm into the standard working curve to calculate the concentration of DES in the sample to be tested.

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