Target response type ECL aptamer sensing platform and preparation method and application thereof

Abstract

The invention belongs to the technical field of electrochemical luminescence, and particularly relates to a target response type ECL aptamer sensing platform and a preparation method and application thereof. The covalent organic framework material based on imine bond connection is Btt-Tpa-COF (Btt-Tapt-COF) and Btt-Tapt-COF (Btt-Tapt-COF). According to the present invention, the Btt-Tap-COF is adopted as the efficient luminous body, and the three-dimensional DNA hydrogel is adopted as the intelligent signal switch, such that the high-sensitivity detection of the lincomycin is provided; when the target lincomycin is deleted, the DNA hydrogel can quench an ECL signal of a Btt-Tapt-COF / TEA system, and a signal closing state is formed. When the lincomycin exists, the lincomycin can be specifically combined with the aptamer sequence in the DNA hydrogel, so that the network structure of the DNA hydrogel is dissociated, and a signal opening state is formed. Therefore, an antibiotic detection method based on ECL is constructed, and the method has potential application prospects.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of electrochemiluminescence, and particularly relates to a target-responsive ECL aptamer sensing platform and a preparation method and application thereof. BACKGROUND

[0002] Covalent organic frameworks (COFs) materials have highly ordered porous structure, designable organic units and rich conjugated system, which provide a broad platform for developing high-performance functional materials. Especially in the construction of donor-acceptor (D-A) type COFs, by selecting building units with different electron affinities, a directional charge transfer path can be formed in the framework, thereby showing significant potential in the fields of photocatalysis, energy storage, photoelectrochemistry and electrochemiluminescence (ECL). Electrochemiluminescence technology has been widely used in biochemical detection, environmental monitoring and other fields due to its controllability of electrochemistry and high sensitivity of chemical luminescence. However, the traditional ECL system usually relies on the random collision between luminescent radical ions to realize charge transfer and luminescence, and the process is low in efficiency, which restricts the further improvement of ECL performance.

[0003] Although donor-acceptor COFs provide an ideal model for optimizing the ECL process, current researches are mostly focused on the electronic properties of the building units in the framework, and the key role of the chemical bonds connecting these units has not been systematically revealed. In fact, the connecting bond is not only the link to maintain the structural stability and topological characteristics of COFs, but also an important bridge to affect the charge transport behavior in the framework. Taking the common imine bond (-C=N-) as an example, the difference in electronegativity between the carbon atom and the nitrogen atom makes it itself exhibit donor-acceptor characteristics (-C + =N -). In D-A type COFs connected by imine, there may exist two charge transfer channels: one is the channel composed of the pre-designed donor and acceptor units; the other is the secondary channel introduced by the inherent dipole direction of the imine bond. The directionality (same or opposite) between the two channels will directly affect the efficiency and direction of the overall charge migration in the framework, and then determine the ECL performance of the material. However, the charge transfer mechanism of this "double donor-acceptor" system lacks in-depth understanding, especially how the intrinsic electronic structure of the connecting bond regulates the charge transfer path, which has not been systematically explained in ECL research, which limits the possibility of further improving the ECL efficiency through rational structure design.

[0004] The extensive use of antibiotics in medical, livestock and food industries has played an important role in protecting public health safety. However, the overuse and misuse of antibiotics have also led to the increasingly prominent problem of antibiotic residues in food, which further aggravates the spread and spread of multiple drug-resistant bacteria, posing a serious threat to the ecological environment and human health. Lincomycin, as a widely used lincosamide antibiotic, may cause health risks such as allergic reactions, intestinal flora imbalance and drug resistance due to its residues in animal-derived food. At present, there are various methods for detecting lincomycin, among which the ECL aptamer sensor is considered as a potential detection method due to its advantages of simple operation, rapid response and low background signal. However, the application of this type of sensor in lincomycin detection is still in its infancy, and its performance is still limited by the ECL efficiency of the signal probe and the charge transfer ability of the sensing interface. Therefore, developing ECL materials with efficient charge separation and transfer characteristics and elucidating their structure-activity relationship are of great practical significance for constructing high-sensitivity and high-selectivity lincomycin ECL sensors. SUMMARY

[0005] The purpose of the present application is to provide a target-responsive ECL aptamer sensing platform and its preparation method and application, so as to overcome the shortcomings of the prior art. Two covalent organic frameworks with different charge transfer directions are synthesized, among which the covalent organic framework material with the same charge transfer channel as the efficient luminescent material, the three-dimensional DNA hydrogel as the intelligent signal switch, and the triethylamine as the co-reactant, an electrochemiluminescence aptamer sensing platform for detecting antibiotics is constructed, which has high sensitivity and good stability, reproducibility and specificity.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] In the first aspect, the present application provides an imine bond connected covalent organic framework material, and the imine bond connected covalent organic framework material is Btt-Tpa-COF and Btt-Tapt-COF, and the structure of the repeat unit is shown in formula I and formula II, respectively: .

[0008] The present application synthesizes two imine bond connected D-A type COFs (Btt-Tpa-COF and Btt-Tapt-COF) with different charge transfer directions by changing the construction unit. Among them, Btt-Tpa-COF is composed of electron-deficient benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-tricarboxaldehyde (Btt) and electron-donating tri(4-aminophenyl)amine (Tpa), and the charge flows from the periphery electron-donating Tpa arm to the central electron-deficient Btt core, forming an inward type of charge transfer. Btt-Tapt-COF is composed of electron-donating benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-tricarboxaldehyde (Btt) and electron-deficient 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine (Tapt), and the charge flows from the central electron-donating Btt core to the peripheral electron-deficient Tapt arm, forming an outward or reverse direction of charge transfer.

[0009] In a second aspect, the present application provides a preparation method of the imine bond connected covalent organic framework material of the first aspect. The benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-tricarboxaldehyde (Btt) and tri(4-aminophenyl)amine or 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine are dispersed in an organic solvent, and a catalyst is added to carry out a solvothermal reaction to prepare the imine bond connected covalent organic framework material.

[0010] Through the classical imine condensation path, the stable framework with high crystallinity, structural order and regular pore is efficiently constructed by using the reversible process of solvothermal reaction; the introduced directional charge transfer channel significantly enhances the charge separation and migration ability of the material, laying a key foundation for obtaining high-performance electrochemical luminescent materials.

[0011] In some other embodiments, the molar ratio of benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-tricarboxaldehyde to tri(4-aminophenyl)amine is 1:(1-1.2); the molar ratio of benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-tricarboxaldehyde to 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine is 1:(1-1.2).

[0012] The organic solvent is a solvent composed of o-dichlorobenzene and n-butanol, or a solvent composed of mesitylene and 1,4-dioxane.

[0013] The solvothermal reaction is a heating reaction at 115-125℃ for 2-4 days.

[0014] Specifically, the preparation method of Btt-Tpa-COF is as follows: Benzo[1,2-b:3,4-b':5,6-b'']trithiophene-2,5,8-tricarboxaldehyde (Btt) is mixed with tris(4-aminophenyl)amine (Tpa); a mixed solvent composed of o-dichlorobenzene and n-butanol is added and ultrasonically dispersed; acetic acid is then added as a catalyst; after deoxygenation treatment, the mixture is sealed and subjected to a solvothermal reaction; after the reaction is completed, the product is collected by centrifugation, washed, and vacuum dried to obtain the final product.

[0015] The preparation method of Btt-Tapt-COF is as follows: Benzo[1,2-b:3,4-b':5,6-b'']trithiophene-2,5,8-tricarboxaldehyde (Btt) is mixed with 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine (Tapt), a mixed solvent consisting of mesitylene and 1,4-dioxane is added, and ultrasonic dispersion is performed. Acetic acid is added as a catalyst. After deoxygenation treatment, the mixture is sealed and subjected to a solvothermal reaction. After the reaction is completed, the precipitate is collected by filtration, washed with organic solvent, and dried under vacuum to obtain the final product.

[0016] In some other embodiments, the molar ratio of benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-trialdehyde to tris(4-aminophenyl)amine is 1:1; the molar ratio of benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-trialdehyde to 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine is 1:1. The volume ratio of o-dichlorobenzene to n-butanol is 1:1, and the volume ratio of mesitylene to 1,4-dioxane is 1:1. The solvothermal reaction was carried out by heating at 120°C for 3 days.

[0017] Thirdly, this invention provides the application of the imine-bonded covalent organic framework material of the first aspect in electrochemiluminescence products. The electrochemiluminescence product is a target-responsive ECL aptamer sensing platform.

[0018] Fourthly, the present invention provides a target-responsive ECL aptamer sensing platform, comprising a glassy carbon electrode and a first aspect of a covalent organic framework material based on imine bonds, Au NPs, a single-stranded DNA probe, 1-hexathiol, and a DNA hydrogel, which are sequentially modified on the surface of the electrode. The DNA hydrogel is formed by polymerized DNA chain P-SA, polymerized DNA chain P-SB and aptamer chain through base complementary pairing. The polymerized DNA chains P-SA and P-SB are obtained by polymerizing DNA chains SA and SB with Acrydite end modifications in the presence of an initiator and a promoter, respectively. The nucleotide sequence of the Acrydite-modified DNA strand SA is shown in SEQ ID NO:1; The nucleotide sequence of the Acrydite-modified DNA strand SB is shown in SEQ ID NO:2; The nucleotide sequence of the single-stranded DNA probe is shown in SEQ ID NO:3; The nucleotide sequence of the aptamer chain is shown in SEQ ID NO:4.

[0019] Compared with Btt-Tpa-COF, which has reversed donor-acceptor pairs, Btt-Tapt-COF has higher ECL efficiency and a stable ECL signal, indicating that the same donor-acceptor pathway can effectively promote charge transfer to generate ECL, while the reversed donor-acceptor orientation significantly hinders charge transfer.

[0020] The target-responsive ECL aptamer sensing platform employs a three-electrode system, using a modified glassy carbon electrode as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode (containing a saturated KCl solution) as the reference electrode.

[0021] Fifthly, the present invention provides a method for preparing a target-responsive ECL aptamer sensing platform according to the fourth aspect, comprising the following steps: (1) A solution of covalent organic framework material based on imine bond linkage is dropped onto the surface of the pretreated glassy carbon electrode and dried to obtain a glassy carbon electrode modified with covalent organic framework material; (2) Gold nanoparticles, single-stranded DNA probes and 1-hexylthiol were sequentially modified onto the electrode obtained in step (1); (3) The DNA hydrogel and the electrode modified in step (2) are incubated together at room temperature to obtain the sensing platform.

[0022] In some other embodiments, in step (1), the concentration of the covalent organic framework material solution based on imine bonds is 0.8-1.2 mg / mL; the pretreatment of the glassy carbon electrode includes: polishing the electrode surface with alumina polishing powder, then ultrasonically cleaning it sequentially with ultrapure water, ethanol and ultrapure water, and finally drying it. In step (2), before modifying the single-stranded DNA probe, the single-stranded DNA probe is co-incubated with tris(2-carboxyethyl)phosphine hydrochloride in TE buffer at room temperature to reduce its disulfide bonds; the cDNA is activated by tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and then passed through a gold... Thiol bonds self-assembled and immobilized on the surface of the modified electrode Au NPs / Btt-Tapt-COF / GCE. After each modification step, the electrode surface was gently rinsed with 0.1 M phosphate buffer solution (pH 7.4).

[0023] In step (3), the DNA hydrogel is prepared as follows: Acrydite-terminated DNA chains SA and SB are added to a stock solution containing acrylamide monomer. After deoxygenation, ammonium persulfate initiator and N,N,N',N'-tetramethylenediamine promoter are added, and polymerization is carried out at 35-40℃ for 10-20 minutes to obtain polymerized DNA chains P-SA and P-SB. Then, P-SA, P-SB and aptamer chains are mixed at a molar ratio of (2-2.2):1:1 and incubated at room temperature to form a DNA hydrogel. Excess P-SA chains in the DNA hydrogel effectively hybridize with single-stranded DNA probes on the electrode surface, fixing the DNA hydrogel to the electrode surface. Finally, the modified electrode is incubated with different concentrations of lincomycin at 4℃ to allow lincomycin to specifically bind to the aptamer sequence in the DNA hydrogel. The stock solution includes 10 mM Tris, 1 mM EDTA (pH=8.0), 200 mM NaCl, and 4% acrylamide.

[0024] In a sixth aspect, the present invention provides the application of the target-responsive ECL aptamer sensing platform of the fourth aspect in antibiotic detection, wherein the antibiotics include lincosamide antibiotics, specifically, lincomycin.

[0025] In a seventh aspect, the present invention provides a method for detecting lincomycin, wherein the target-responsive ECL aptamer sensing platform of the fourth aspect is placed in a phosphate buffer solution containing triethylamine as a co-reactant and a sample solution to be tested, and an electrochemiluminescence test is performed; wherein the potential range of the electrochemiluminescence test is 0-1.4 V, the scan rate is 0.2-0.4 V / s, and the photomultiplier tube voltage is 700-900 V.

[0026] In some other embodiments, the concentration of the triethylamine phosphate buffer solution is 10-30 mM; the test sample solution is an animal-derived food, specifically, meat, milk and eggs.

[0027] This ECL aptamer sensor achieves highly sensitive and specific detection of lincomycin, with a detection limit as low as 0.10 pg / mL and a wide linear response range of 0.0001 to 10 ng / mL.

[0028] The beneficial effects of this invention are: (1) This invention successfully synthesized two novel donor-acceptor covalent organic framework (COF) materials, Btt-Tpa-COF and Btt-Tapt-COF, which have high crystallinity and stable chemical properties, laying the material foundation for constructing a high-performance electrochemiluminescence (ECL) sensing platform.

[0029] (2) This invention reveals for the first time the key influence of charge transfer directionality on the electrochemiluminescence performance of COF materials. COFs with unidirectional charge transfer channels can effectively promote charge separation and migration, and can serve as highly efficient anodic co-reactant-dependent ECL luminescent materials. Based on the above COF materials, a signal "on" circuit was constructed. close The "open" type ECL aptamer sensing system is designed to achieve sensitive and specific responses to target analytes and has been successfully applied to the highly selective detection of lincomycin.

[0030] (3) A DNA hydrogel with a three-dimensional network structure is introduced into the sensing system. Its good biocompatibility, structural stability, programmability and stimulus response characteristics enable efficient quenching and recovery of COF electrochemiluminescence signals, which significantly improves signal contrast and detection reliability.

[0031] (4) The established ECL detection method exhibits excellent performance in terms of sensitivity, selectivity, reproducibility, and stability, demonstrating good analytical performance and reliability. The sensor maintains high accuracy and stability even in complex real-world samples, showcasing significant practical application potential and promotional value in the field of food safety monitoring. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 The above are the synthesis flowchart and characterization diagram of Btt-Tpa-COF in Example 1 of the present invention, wherein A is the synthesis flowchart of Btt-Tpa-COF, B is the powder X-ray diffraction (PXRD) of Btt-Tpa-COF, and C is the AA stacking model diagram of Btt-Tpa-COF. Figure 2 The diagram shows the synthesis flowchart and characterization diagram of Btt-Tapt-COF in Embodiment 2 of the present invention, wherein A is the synthesis flowchart of Btt-Tapt-COF, B is the PXRD pattern of Btt-Tapt-COF, and C is the AA stacking model diagram of Btt-Tapt-COF. Figure 3The Fourier transform infrared (FT-IR) spectra of benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-trialdehyde, tris(4-aminophenyl)amine, 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine, Btt-Tpa-COF, and Btt-Tapt-COF in Example 3 of the present invention are shown. Curve a is benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-trialdehyde, curve b is tris(4-aminophenyl)amine, curve c is 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine, curve d is Btt-Tpa-COF, and curve e is Btt-Tapt-COF. Figure 4 Figure 4 shows the construction and characterization of the Btt-Tpa-COF / TEA and Btt-Tapt-COF / TEA systems in Example 4 of this invention. Figure A shows the ECL intensity-potential curves and corresponding cyclic voltammetry (CV) curves of the bare electrode (GCE), Btt-Tpa-COF, and Btt-Tapt-COF in 0.1 M phosphate buffer solution; Figure B shows the ECL intensity-potential curves and corresponding CV curves of the bare GCE, Btt-Tpa-COF, and Btt-Tapt-COF in 20 mM triethylamine; Figure C shows the ECL intensity-time curves of Btt-Tpa-COF and Btt-Tapt-COF in 20 mM triethylamine; Figure D shows the possible ECL reaction mechanism of the Btt-Tapt-COF / triethylamine system. Figure 5 This is a schematic diagram of the construction and principle of the ECL aptamer sensing platform for lincomycin detection constructed in Example 5 of the present invention, wherein A is a construction diagram of the Btt-Tapt-COF / TEA system and B is a schematic diagram of the principle of lincomycin detection. Figure 6 This is a feasibility analysis diagram of the sensing platform for detecting ECL aptamers for lincomycin constructed in Embodiment 6 of the present invention, where A is the measured CV diagram and B is the impedance spectrum (EIS) diagram. Figure 7 This is an ECL response diagram of the ECL aptamer sensing platform constructed in Example 7 of the present invention to different concentrations of lincomycin; Figure 8 This is a calibration curve of ECL signal versus the logarithm of lincomycin concentration in Example 8 of the present invention; Figure 9 The diagrams show the specificity, stability, and reproducibility of the ECL aptamer sensing platform constructed in Embodiment 9 of the present invention, where A is the specificity diagram, B is the stability diagram, and C is the reproducibility diagram. Detailed Implementation

[0034] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions are not specified in the embodiments; they are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all components used are commercially available conventional products. The sequences used are shown in Table 1: Table 1. Sequences used in the embodiments Name Sequence (5'-3') Number SA' CAT CGA CCA CAT CAC GCG SB' GCG CGA CTC ACC GTA TCG SA Acrydite-CAT CGA CCA CAT CAC GCG SEQ ID NO: 1 SB Acrydite-GCG CGA CTC ACC GTA TCG SEQ ID NO: 2 cDNA CGC GTG ATG TGG TCG ATG TTT-(CH2)6-SH SEQ ID NO: 3 aptamer CGC GTG ATG TGG TCG ATG CGA TAC GGT GAG TCG CGC CAC GGC TAC ACA CGT CTC AGC GA SEQ ID NO: 4 As mentioned earlier, the current research on ECL aptamer sensors for lincomycin detection faces the following key challenges: First, the charge transfer efficiency of traditional ECL systems is limited; second, the role of linkage bonds (such as imine bonds) in charge transfer in COFs is not fully understood, and the ECL enhancement mechanism has not been revealed from the perspective of "dual donor-acceptor" channel synergy; and third, there is still a lack of efficient and stable luminescent material systems for high-performance ECL aptamer sensors for detecting antibiotic residues such as lincomycin.

[0035] This invention synthesized two imine-linked DA-type COFs (Btt-Tpa-COF and Btt-Tapt-COF) with different charge transfer directions by changing the building blocks. Compared with Btt-Tpa-COF, which has a reverse donor-acceptor pair, Btt-Tapt-COF showed an 8.94-fold increase in ECL efficiency and a stable ECL signal, indicating that the same donor-acceptor pathway can effectively promote charge transfer to generate ECL, while the reverse donor-acceptor direction significantly hinders charge transfer. As a proof of concept, we successfully constructed a target-responsive ECL aptamer sensing platform, using Btt-Tapt-COF as a highly efficient luminescent agent and a three-dimensional DNA hydrogel as a smart signal switch, for high-sensitivity detection of lincomycin. This ECL aptamer sensor achieved highly sensitive and specific detection of lincomycin, with a detection limit as low as 0.10 pg / mL and a wide linear response range of 0.0001 to 10 ng / mL. This work reveals the impact of charge transfer direction modulation on ECL performance and provides a promising option for meeting the demand for sensitive detection of antibiotics in food safety and environmental monitoring.

[0036] The solution of the present invention will be described below with reference to specific embodiments: Example 1 This embodiment provides a method for preparing and characterizing Btt-Tpa-COF. The preparation process of Btt-Tpa-COF is as follows: Figure 1As shown in A, specifically, benzo[1,2-b:3,4-b':5,6-b'']trithiophene-2,5,8-tricarboxaldehyde (Btt, 66.1 mg, 0.2 mmol) and tris(4-aminophenyl)amine (Tpa, 58.07 mg, 0.2 mmol) were added to a 10 mL heat-resistant glass tube. Then, anhydrous o-dichlorobenzene (o-DCB, 3 mL) and anhydrous n-butanol (3 mL) were added to prepare a mixture. After sonicating the mixture for 10 minutes, acetic acid (6 M, 0.3 mL) was added to the tube. After three freeze-evacuation-thawing cycles, the heat-resistant glass tube was sealed and heated at 120 °C for 3 days. After the reaction mixture cooled to room temperature, the precipitate was collected by centrifugation. It was then washed several times with N,N-dimethylformamide, methanol, and ethanol, and dried under vacuum at 120 °C to obtain a dark red powder, labeled Btt-Tpa-COF, with the following structural formula: .

[0037] The crystallinity of Btt-Tpa-COF was characterized using powder X-ray diffraction (PXRD) patterns, such as... Figure 1 As shown in Figure B, the experimental PXRD pattern of Btt-Tpa-COF exhibits strong diffraction peaks at 5.16°, 9.02°, 10.12°, and 25.72°, corresponding to the (100), (110), (200), and (001) crystal planes, respectively. Furthermore, the experimental PXRD pattern of Btt-Tpa-COF is consistent with the AA stacking model. After Pawley refinement, the cell parameters are obtained as follows: a = 19.82 Å, b = 19.82 Å, c = 3.99 Å, and α = β =90°, γ = 120° ( R p = 7.99%, R wp = 10.62%). For example... Figure 1 As shown in C, Btt-Tpa-COF has a crystalline hexagonal structure with an AA stacking mode, thus forming a layered framework structure.

[0038] Example 2 This embodiment provides a method for preparing and characterizing Btt-Tapt-COF. The preparation process of Btt-Tapt-COF is as follows: Figure 2As shown in A, specifically, benzo[1,2-b:3,4-b':5,6-b'']trithiophene-2,5,8-tricarboxaldehyde (Btt, 66.1 mg, 0.2 mmol) and 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine (Tapt, 70.8 mg, 0.2 mmol) were added to a 10 mL heat-resistant glass tube. Then, 3 mL of mesitylene and 3 mL of 1,4-dioxane were added and the mixture was sonicated to obtain a uniform dispersion. Acetic acid (6 M, 0.5 mL) was then added. After three freeze-evacuation-thawing cycles for degassing, the tube was sealed and heated at 120 °C for 3 days. The resulting precipitate was collected by filtration and washed with tetrahydrofuran and acetone. Finally, it was vacuum dried at 80 °C for 24 h to obtain a yellow powder, labeled Btt-Tapt-COF, with the following structural formula: .

[0039] The crystallinity of Btt-Tapt-COF was characterized using PXRD patterns, such as... Figure 2 As shown in Figure B, the diffraction peaks of Btt-Tapt-COF at 4.56°, 7.16°, 7.94°, and 27.16° correspond to the (100), (110), (200), and (001) crystal planes, respectively. Furthermore, the experimental PXRD pattern of Btt-Tapt-COF is essentially consistent with the PXRD pattern of the AA stacked model. The cell parameters of Btt-Tapt-COF are... a = 21.98 Å, b = 21.98 Å, c = 3.49 Å, and α = β = 90°, γ = 120° ( R p = 10.79%, R wp = 15.79%). For example... Figure 2 As shown in C, Btt-Tapt-COF has a crystalline hexagonal structure with an AA stacking pattern, thus forming a layered framework structure.

[0040] Example 3 This embodiment provides Fourier transform infrared spectral characterization of Btt precursor, Tpa precursor, Tapt precursor, Btt-Tpa-COF, and Btt-Tapt-COF.

[0041] The chemical structures of Btt-Tpa-COF and Btt-Tapt-COF were characterized using Fourier transform infrared spectroscopy (FT-IR). Figure 3As shown, Btt-Tpa-COF at 1621 cm⁻¹ -1 A new stretching vibration band (curve d) is exhibited at this point, which is assigned to the C=N bond. Simultaneously, the C=O group in the Btt precursor shows a stretching vibration band at 1664 cm⁻¹. -1 The characteristic peak at (curve a) and the NH bond in the Tpa precursor at 1103 cm⁻¹ -1 The characteristic peaks at 1620 cm⁻¹ (curves b and c) are significantly weakened. For Btt-Tapt-COF, the characteristic peaks at 1620 cm⁻¹ are significantly weakened. -1 A characteristic stretching vibration peak of the C=N bond appears at this point (curve e). Furthermore, Btt-Tapt-COF shows a peak at 814 cm⁻¹. -1 1366 cm -1 and 1506 cm -1 The structure exhibits typical triazine ring vibrational bands; however, Btt-Tpa-COF lacks a triazine structure and therefore shows no triazine ring vibrational signal, directly confirming the topological differences at the molecular level. These results demonstrate that the method of this invention successfully prepared a framework structure with highly crystalline imine bonds.

[0042] Example 4 This embodiment provides the construction and characterization of the Btt-Tpa-COF / TEA and Btt-Tapt-COF / TEA systems.

[0043] Before modifying the glassy carbon electrode (GCE), the surface of the GCE was wiped with toilet paper soaked in ultrapure water. Then, the GCE was polished to a mirror finish with 1.0, 0.3 and 0.05 μm α-Al2O3 powder, respectively. The polished electrode was then ultrasonically treated for 5 minutes in ultrapure water, ethanol and ultrapure water, respectively, and finally dried with nitrogen gas.

[0044] The two COFs (Btt-Tpa-COF and Btt-Tapt-COF) prepared in Examples 1-2 were dispersed in N,N-dimethylformamide to prepare homogeneous solutions of 1 mg / mL. 10 μL of each COF solution was dropped onto the pre-treated bare GCE surface and allowed to dry naturally at room temperature to obtain the working electrode. Furthermore, a platinum wire was used as the counter electrode, and an Ag / AgCl electrode (containing saturated KCl) was used as the reference electrode. All three electrodes were placed together in a phosphate buffer solution containing the co-reactant triethylamine (TEA) for cyclic voltammetry (CV) and electrochemiluminescence (ECL) measurements.

[0045] Figure 4Figure A shows the ECL intensity-potential and CV curves of the constructed GCE, Btt-Tpa-COF / GCE, and Btt-Tapt-COF / GCE systems in phosphate buffer solution. Both Btt-Tpa-COF / GCE and Btt-Tapt-COF / GCE exhibit weak peak currents and ECL signals, indicating that they have low interfacial electron transfer efficiency.

[0046] Figure 4 In the figure, B represents the ECL intensity-potential and CV curves of the constructed GCE, Btt-Tpa-COF / GCE, and Btt-Tapt-COF / GCE in 20 mM TEA solution. The bare GCE showed an oxidation onset potential of +0.55 V for TEA. After modification with Btt-Tpa-COF and Btt-Tapt-COF, the onset potentials of the Btt-Tpa-COF / TEA and Btt-Tapt-COF / TEA systems shifted negatively by 0.07 V relative to the bare GCE, indicating that these two COFs have excellent catalytic activity for TEA. Furthermore, the peak current of the Btt-Tapt-COF / TEA system was higher than that of the Btt-Tpa-COF / TEA system, indicating that Btt-Tapt-COF has a stronger catalytic ability for TEA. Importantly, the ECL intensity of the Btt-Tapt-COF / TEA system (17243 au) is 118.1 times that of Btt-Tpa-COF / TEA (146 a.u.) and 9.52 times that of bare GCE / TEA (1812 au), indicating that a co-directional charge transfer mechanism promotes the catalytic oxidation of TEA. The potential range was 0 to +2.0 V, and the scan rate was 0.1 V / s.

[0047] Figure 4 In the figure, C represents the ECL-time curves of the constructed Btt-Tpa-COF / GCE and Btt-Tapt-COF / GCE systems tested in 20 mM TEA solution. Under the test conditions of a potential range of 0 to +1.4 V, a scan rate of 0.3 V / s, and a photomultiplier tube voltage of 800 V, the average ECL intensity of the Btt-Tapt-COF / TEA system (17759 au) is 9.24 times (1921 au) that of the Btt-Tpa-COF / TEA system. Furthermore, the ECL emission of the Btt-Tapt-COF / TEA system exhibits extremely high stability over 18 consecutive scan cycles, with a relative standard deviation (RSD) of 0.16%, which is superior to that of the Btt-Tpa-COF / TEA system (RSD = 26.6%).

[0048] Figure 4D in the figure represents the possible ECL reaction mechanism of the constructed Btt-Tapt-COF / TEA system.

[0049] Example 5 This embodiment provides a method for constructing an ECL aptamer sensor based on high-luminescence Btt-Tapt-COF and a method for detecting lincomycin.

[0050] Figure 5 This diagram illustrates the principle of constructing and detecting lincomycin based on the Btt-Tapt-COF / TEA system provided by this method. Btt-Tapt-COF, Au NPs, single-stranded DNA probes (cDNA), and 1-hexathiol (HT) are sequentially modified onto a GCE. Then, a pre-prepared DNA hydrogel is coupled to the electrode. Figure 5 (A) The DNA hydrogel acts as an ECL quencher, in which case the ECL signal is turned off. When the target lincomycin is present, its binding to the aptamer in the DNA hydrogel causes the bridge between P-SA and P-SB to break, triggering DNA hydrogel dissociation. At this point, the ECL signal is turned on. Figure 5 (B) Specifically, it includes the following steps: (1) Btt-Tapt-COF / GCE was obtained according to Example 4; (2) Activation of single-stranded DNA: Single-stranded DNA probe (cDNA, 20 μL, 10 μM), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 20 μL, 1000 μM) and 60 μL of 1 The disulfide-bonded oligonucleotides were incubated in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) at room temperature for 1 hour to reduce the disulfide bonded oligonucleotides. (3) Blocking of non-specific sites: The modified electrode was placed in 15 μL of 1-hexanethiol (HT, 1 mM) solution and incubated for 30 minutes to block non-specific recognition sites; (4) Preparation of DNA hydrogels: Acrydite-modified DNA strands SA (100 μM, 4 μL) and SB (100 μM, 2 μL) were added to a stock solution with a total volume of 96 μL. The stock solution contained 10 mM Tris, 1 mM EDTA (pH=8.0), 200 mM NaCl, and 4% acrylamide. Nitrogen gas was then purged for 15 minutes to remove oxygen. Then, freshly prepared initiator ammonium persulfate (1.5% v / v, 0.1 g / mL) and promoter N,N,N',N'-tetramethylenediamine (2% v / v) were rapidly added to the SA and SB solutions. The mixture was then reacted in a vacuum drying oven at 37°C for 15 minutes to obtain polymerized chains: P-SA and P-SB. Then, P-SA (4 μM, 100 μL), P-SB (2 μM, 100 μL), and aptamer strand (100 μM, 2 μL) were mixed and incubated at room temperature for 30 minutes to form a DNA hydrogel. (5) Crosslinking process: The freshly prepared DNA hydrogel (20 μL) was incubated at room temperature for 2 hours on the HT / cDNA / Au NPs / Btt-Tapt-COF / GCE electrode to promote the effective coupling of excess SA in the DNA hydrogel with cDNA to achieve the fixation of DNA hydrogel on the electrode. (6) Construction of ECL aptamer sensor: Btt-Tapt-COF / GCE was obtained according to the above steps. 15 μL of Au NPs was modified on the surface of Btt-Tapt-COF / GCE and dried at 37℃. Then, cDNA probe (20 μL, 2 μM) was added to the surface of Au NPs / Btt-Tapt-COF / GCE and incubated overnight at 4℃. Then, the modified electrode cDNA / Au NPs / Btt-Tapt-COF / GCE was incubated with HT solution (15 μL, 1 mM) at room temperature for 30 minutes. The newly prepared DNA hydrogel in step (4) was incubated on the HT / cDNA / AuNPs / Btt-Tapt-COF / GCE modified electrode surface at room temperature for 2 hours. Finally, different concentrations of lincomycin were incubated. (7) Electrode cleaning: When assembling the ECL aptamer sensor, the surface of the modified electrode should be rinsed with 0.1 M phosphate buffer solution (pH 7.4) after each modification to remove impurities and unreacted substances; (8) Signal detection: The modified electrode obtained in step (6) was subjected to ECL testing using a three-electrode system. The test conditions were: 10 mM TEA solution, potential range from 0 to +1.4 V, scan rate of 0.3 V / s, and photomultiplier tube voltage of 800 V.

[0051] Example 6 This embodiment provides a feasibility analysis of detecting lincomycin using an ECL sensor based on Btt-Tapt-COF.

[0052] To demonstrate the feasibility of this analytical method, a 5 mM [Fe(CN)6] solution containing 0.1 M KCl was prepared. 3- / 4- The results were validated by CV and impedance spectroscopy (EIS) analysis measured in solution.

[0053] Figure 6 In the figure, A represents the CV analysis constructed using this method. A pair of reversible redox peaks were observed in the bare GCE (curve a). Compared to the bare electrode, Btt-Tapt-COF / GCE showed a reduced peak current, attributed to the poor conductivity of Btt-Tapt-COF (curve b). When Au NPs with excellent conductivity were assembled onto the Btt-Tapt-COF / GCE surface, the peak current increased (curve c). With the stepwise assembly of cDNA (curve d), HT (curve e), and DNA hydrogel (curve f), the current of the modified electrode gradually decreased. Upon introduction of lincomycin, the lincomycin aptamer specifically recognized lincomycin and formed a target-aptamer complex, leading to hydrogel dissociation and consequently an increase in peak current (curve g).

[0054] Figure 6 B in the figure represents the EIS analysis constructed using this method. The bare GCE exhibits a small semicircle, indicating the electron transfer resistance of the bare GCE (…). R et The coefficient of variation is lower (curve a). Compared to pure GCE, Btt-Tapt-COF / GCE exhibits greater [performance / performance]. R et (Curve b), this is attributed to the poor conductivity of Btt-Tapt-COF. After introducing Au NPs with excellent conductivity, R et The value decreases (curve c). cDNA is attached to the electrode via a gold-sulfur bond (curve d), due to the negatively charged DNA strands [Fe(CN)6]. 3- / 4- The blocking effect of electron transfer R et The value increased. Subsequently, after blocking the nonspecific binding sites using HT, R et The value further increases (curve e), which is attributed to the hindrance of electron transfer by HT. Subsequently, due to the hindrance of electron transfer by the DNA hydrogel, the corresponding... R et The value increased (curve f). Finally, it was observed during incubation with lincomycin. R etThe decrease in value is attributed to the dissociation of the DNA hydrogel (curve g). The CV results are consistent with the EIS results, indicating the successful assembly of the ECL aptamer sensor.

[0055] Example 7 This embodiment provides the ECL response of an ECL sensing system to different concentrations of lincomycin.

[0056] Figure 7 The response of the ECL sensing signal provided by this invention to different concentrations of lincomycin was investigated. Lincomycin solutions were diluted in deionized water at concentrations of 0.0001, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 ng / mL to obtain lincomycin solutions of different concentrations. Under optimal conditions, the ECL signal gradually increased with increasing lincomycin concentration.

[0057] Example 8 This embodiment provides a calibration curve of ECL signal versus the logarithm of lincomycin concentration.

[0058] Figure 8 The calibration curve of the ECL sensor signal versus the logarithm of lincomycin concentration provided by this invention is shown. As the lincomycin concentration increases, the corresponding ECL signal gradually strengthens, indicating that the detection method of this invention can achieve quantitative detection of lincomycin. Furthermore, within the concentration range of 100 fg / mL to 10 ng / mL, a good linear relationship is shown between the ECL signal and the logarithm of the lincomycin concentration, with the linear equation being: I ECL = 2754.5 lg C + 12369 ( R 2 = 0.9982). The detection limit was calculated to be 0.10 pg / mL.

[0059] Example 9 This embodiment provides the specificity, stability, and reproducibility of the constructed ECL aptamer sensor. Figure 9 In this paper, A represents the specificity of the method for lincomycin detection. Amoxicillin (AMO), potassium penicillin (POT-P), kanamycin (KAN), ciprofloxacin (CIP), ofloxacin (OFL), and roxithromycin (ROX) were used as interfering agents to evaluate the specificity of this ECL aptamer sensor. The concentration of lincomycin was 10 ng / mL, and the concentration of the other interfering agents was 100 ng / mL. The results showed that the signals produced by all interfering agents were almost identical to those of the control group. However, in the presence of lincomycin, a significant enhancement of the ECL signal was observed, indicating that the detection method of this invention is less affected by other interfering agents and has high selectivity.

[0060] Figure 9 In the figure, B represents the stability of the ECL aptamer sensing platform provided by the method of the present invention. After 12 consecutive scan cycles, the ECL signals responding to different concentrations of lincomycin (0.01, 0.1, and 0.5 ng / mL) showed negligible changes, with relative standard deviations of 2.14%, 1.60%, and 4.17%, respectively, indicating that the ECL aptamer sensor constructed by the method of the present invention has excellent stability.

[0061] Figure 9 In this diagram, C represents the reproducibility of the ECL aptamer sensing platform provided by the method of this invention. Five detection systems from the same batch (intra-batch detection) and different batches (inter-batch detection) were used to determine 0.1 ng / mL lincomycin, with relative standard deviations of 2.06% and 0.99%, respectively, indicating that the ECL aptamer sensor constructed by the method of this invention has excellent reproducibility.

[0062] Example 10 This embodiment demonstrates the practical applicability and reliability of the constructed ECL aptamer sensor.

[0063] To evaluate the practical applicability and reliability of the ECL aptamer sensor designed using the method of this invention, the method was applied to the detection of lincomycin in milk samples. Spiking recovery experiments were performed using treated milk samples, with lincomycin concentrations of 0.1, 1, and 10 ng / mL added, respectively. The detection results are shown in Table 2.

[0064] Table 2 Recovery rate of lincomycin in milk samples (n=3) Sample Added (ng / mL) Measured (ng / mL) Recovery (%) RSD (%, n=3) 1 0.1 0.1030 103.0 4.34 2 1 0.9791 97.91 3.36 3 10 9.726 97.26 3.00 As shown in Table 2, the recovery rate of lincomycin detected by this method is between 97.26% and 103.0%, and the relative standard deviation is between 3.00% and 4.34%, indicating that the ECL aptamer sensor proposed in this invention has good application prospects in the fields of food safety and public health.

[0065] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A covalent organic framework material based on imine bonds, characterized in that, The covalent organic framework materials based on imine bonds are Btt-Tpa-COF and Btt-Tapt-COF, whose repeating structural units are shown in Equation I and Equation II, respectively: 。 2. The method for preparing the covalent organic framework material based on imine bonds as described in claim 1, characterized in that, Benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-trialdehyde (Btt) was dispersed in an organic solvent with tris(4-aminophenyl)amine or 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine, and a catalyst was added to carry out a solvothermal reaction to obtain a covalent organic framework material based on imine bonds.

3. The method for preparing a covalent organic framework material based on imine bonds according to claim 2, characterized in that, The molar ratio of benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-trialdehyde to tris(4-aminophenyl)amine is 1:(1-1.2); the molar ratio of benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-trialdehyde to 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine is 1:(1-1.2). The organic solvent is a solvent composed of o-dichlorobenzene and n-butanol or a solvent composed of mesitylene and 1,4-dioxane; The solvothermal reaction is carried out by heating at 115-125℃ for 2-4 days.

4. The method for preparing a covalent organic framework material based on imine bonds according to claim 3, characterized in that, The molar ratio of benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-trialdehyde to tris(4-aminophenyl)amine is 1:1; the molar ratio of benzo[1,2-b:3,4-b':5,6-b']trithiophene-2,5,8-trialdehyde to 4,4',4''-(1,3,5-triazine-2,4,6-triyl)triphenylamine is 1:

1. The volume ratio of o-dichlorobenzene to n-butanol is 1:1, and the volume ratio of mesitylene to 1,4-dioxane is 1:

1. The solvothermal reaction was carried out by heating at 120°C for 3 days.

5. The application of the covalent organic framework material based on imine bond linkage as described in claim 1 in electrochemiluminescence products.

6. A target-responsive ECL aptamer sensing platform, characterized in that, The invention comprises a glassy carbon electrode and its surface sequentially modified with the imine-linked covalent organic framework material, Au NPs, single-stranded DNA probes, 1-hexathiol and DNA hydrogel as described in claim 1; The DNA hydrogel is formed by polymerized DNA chain P-SA, polymerized DNA chain P-SB and aptamer chain through base complementary pairing. The polymerized DNA chains P-SA and P-SB are obtained by polymerizing DNA chains SA and SB with Acrydite end modifications in the presence of an initiator and a promoter, respectively. The nucleotide sequence of the Acrydite-modified DNA strand SA is shown in SEQ ID NO:1; The nucleotide sequence of the Acrydite-modified DNA strand SB is shown in SEQ ID NO:2; The nucleotide sequence of the single-stranded DNA probe is shown in SEQ ID NO:3; The nucleotide sequence of the aptamer chain is shown in SEQ ID NO:

4.

7. A method for preparing the target-responsive ECL aptamer sensing platform according to claim 6, characterized in that, Includes the following steps: (1) A solution of covalent organic framework material based on imine bond linkage is dropped onto the surface of the pretreated glassy carbon electrode and dried to obtain a glassy carbon electrode modified with covalent organic framework material; (2) Gold nanoparticles, single-stranded DNA probes and 1-hexathiol were sequentially modified onto the electrode obtained in step (1); (3) The DNA hydrogel and the electrode modified in step (2) are incubated together at room temperature to obtain the sensing platform.

8. The method for preparing the target-responsive ECL aptamer sensing platform according to claim 7, characterized in that, In step (1), the concentration of the covalent organic framework material solution based on imine bond linkage is 0.8-1.2 mg / mL; the pretreatment of the glassy carbon electrode includes: polishing the electrode surface with alumina polishing powder, then ultrasonically cleaning it with ultrapure water, ethanol and ultrapure water in sequence, and finally drying it. In step (2), before modifying the single-stranded DNA probe, the single-stranded DNA probe is first co-incubated with tris(2-carboxyethyl)phosphine hydrochloride in TE buffer solution at room temperature to reduce its disulfide bonds. In step (3), the DNA hydrogel is prepared as follows: Acrydite-terminated DNA chains SA and SB are added to a stock solution containing acrylamide monomer. After deoxygenation, ammonium persulfate initiator and N,N,N',N'-tetramethylenediamine promoter are added. The polymerization reaction is carried out at 35-40℃ for 10-20 minutes to obtain polymerized DNA chains P-SA and P-SB. Then, P-SA, P-SB and aptamer chains are mixed with each other at a molar ratio of (2-2.2):1:1 and incubated at room temperature to form a DNA hydrogel.

9. The application of the target-responsive ECL aptamer sensing platform of claim 6 in antibiotic detection, characterized in that, The antibiotics include lincosamide antibiotics.

10. A method for detecting lincomycin, characterized in that, The target-responsive ECL aptamer sensing platform of claim 6 was placed in a phosphate buffer solution containing triethylamine as a co-reactant and the sample solution to be tested for electrochemiluminescence testing; wherein the potential range of the electrochemiluminescence test was 0-1.4 V, the scan rate was 0.2-0.4 V / s, and the photomultiplier tube voltage was 700-900 V.

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