Covalent organic framework and quantum dot composite material as well as preparation and application thereof

Abstract

The invention discloses a covalent organic framework (at) quantum dot composite material as well as preparation and application thereof. The covalent organic framework (at) quantum dot composite material comprises a two-dimensional sheet-shaped covalent organic framework material MCA and S-BNQDs quantum dots packaged in the two-dimensional sheet-shaped covalent organic framework material MCA, the two-dimensional sheet-shaped covalent organic framework material MCA is used as a carrier of the S-BNQDs, so that the trigger potential of the S-BNQDs is reduced, the ECL intensity and the luminescence stability are greatly improved, the performance of the sensor is improved, the signal amplification effect is achieved, and meanwhile the detection sensitivity is improved.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical technologies, and particularly to a covalent organic framework @ quantum dot composite material and its preparation and application. Background Art

[0002] Pesticides play an important role in controlling crop pests and weeds and improving crop yield and quality. Among them, malathion (C 10 H 19 O6S2P, Malathion, abbreviated as Mal) is one of the most common commercialized organophosphorus insecticides and is widely used as a protective agent for grain storage and crop pest control. Although it has significant economic benefits in agricultural production. To avoid more pesticide poisonings, it is crucial to explore and develop highly sensitive sensors for detecting malathion.

[0003] Based on quantum dots (QDs), electrochemiluminescence (ECL) analysis technology provides a new platform for the development of pesticide residue analysis in the environment due to its advantages such as high sensitivity and strong specificity. The development of ECL analysis technology is inseparable from the design and preparation of new luminescence systems, and efficient signal probes are crucial for improving the analytical performance of sensors. Using nanomaterials as signal amplifiers is of particular significance in the design of biosensors because of their large specific surface area, strong electron transport ability, easy labeling, and excellent biocompatibility. Porous materials, such as metal-organic frameworks (MOF), hydrogen-bonded organic frameworks (HOF), and covalent organic frameworks (COF), are used as carriers for fixing ECL luminophores because they have good ordered porosity and high surface area, which can significantly increase the number of fixed luminophores and promote the electrochemical excitation of luminophores. However, the above materials do not perform well in detecting malathion, such as stability, sensitivity, etc.

[0004] Therefore, it is necessary to provide a novel COF-based electrochemiluminescence biosensor to achieve ultrasensitive detection of malathion. Summary of the Invention

[0005] In view of this, the present application provides a covalent organic framework @ quantum dot composite material and its preparation and application, aiming to solve the problem of how to improve the detection sensitivity of COF-based electrochemiluminescence biosensors to malathion.

[0006] To achieve the above technical purpose, the present application adopts the following technical solutions: In the first aspect, the present application provides a covalent organic framework @ quantum dot composite material, including a two-dimensional sheet-like covalent organic framework material MCA and S-BNQDs quantum dots encapsulated inside the two-dimensional sheet-like covalent organic framework material MCA.

[0007] Second aspect, the present application provides a method for preparing a covalent organic framework @ quantum dot composite material, including the following steps: Dissolve cyanuric acid and melamine in a solvent, then add the S-BNQDs quantum dot solution, and stir and react to obtain the covalent organic framework @ quantum dot composite material.

[0008] Preferably, after the stirring reaction, the following steps are further included: filtering the mixture after the stirring reaction by suction, then washing and drying in vacuum.

[0009] Preferably, the solvent is dimethyl sulfoxide; the volume ratio of dimethyl sulfoxide to the S-BNQDs solution is 1-2:1; the molar ratio of cyanuric acid to melamine is 1:1-1.2. V(dimethyl sulfoxide): V(S-BNQDs)=30~60 mL: 30 mL, and dimethyl sulfoxide is an analytical pure organic solvent, and S-BNQDs is prepared as a liquid product.

[0010] Preferably, the temperature of the stirring reaction is 20-25 °C, and the time of the stirring reaction is 60-72 h.

[0011] Third aspect, the present application provides an ECL-RET aptamer biosensor containing a covalent organic framework @ quantum dot composite material.

[0012] Fourth aspect, the present application provides a method for preparing an ECL-RET aptamer biosensor, including the following steps: S1. Coat the covalent organic framework @ quantum dot composite material on the surface of a pretreated glassy carbon electrode, and dry it under an infrared lamp to obtain the S-BNQD-MCA working electrode; S2. Drop the Mal-Apt aptamer solution onto the surface of the S-BNQD-MCA working electrode, incubate and wash it, then drop the BSA blocking solution and incubate and wash it again to obtain the S-BNQD-MCA / Mal-Apt / BSA working electrode; S3. Drop the quenching probe cDNA-Au@NH2-UiO-66 onto the surface of the S-BNQD-MCA / Mal-Apt / BSA working electrode, incubate and wash it to obtain the ECL-RET aptamer biosensor.

[0013] Preferably, the pretreatment method of the Mal-Apt aptamer is as follows: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to the Mal-Apt solution modified with a carboxyl group at the 5' end, and react with shaking at room temperature to obtain the Mal-Apt aptamer; The sequence of 5'-carboxyl modified Mal-Apt is: 5′-COOH-ATC CGT CAC ACC TGC TCT TAT ACACAA TTG TTT TTC TCT TAA CTT CTT GAC TGC TGG TGT TGG CT-3′ (SEQ ID NO.1).

[0014] Preferably, the preparation method of the quenching probe cDNA-Au@NH2-UiO-66 is as follows: After mixing tris(2-carboxyethyl)phosphine hydrochloride with thiol-modified cDNA, incubating and diluting, a mixed solution is obtained. Mix Au@NH2-UiO-66 with the mixed solution and incubate with shaking at room temperature to obtain the quenching probe cDNA-Au@NH2-UiO-66; The sequence of thiol-modified cDNA is: 5′-AGC CAA CAC CAG CAG TCA AGA AGT TAA GAG AAAAAC AAT TGT GTA TAA GAG CAG GTG TGA CGG AT-(CH2)6-HS-SH-3′ (SEQ ID NO.2).

[0015] Fifthly, the present application provides a method for detecting Mal based on an ECL-RET aptamer biosensor, comprising the following steps: K1. Drop different concentrations of the target Mal onto the ECL-RET aptamer biosensor, and after incubation, obtain the sensor after incubating the target; K2. Place the sensor after incubating the target in an ECL detection solution, use the modified glassy carbon electrode as the working electrode, the platinum sheet electrode as the counter electrode, and the saturated Ag / AgCl electrode as the reference electrode to detect the ECL intensity of different concentrations of the target Mal, and draw a standard curve; K3. Detect the ECL intensity of the Mal to be detected, and calculate the concentration of the Mal to be detected according to the standard curve.

[0016] The beneficial effects of the present application are as follows: The technical solution of the present invention uses the ECL-RET technology to prepare the luminescent material S-BNQD-MCA by encapsulating sulfur-doped boron nitride quantum dots with a covalent organic framework material, uses Au@NH2-UIO-66 as a double quencher to prepare an electrochemiluminescence aptasensor, and realizes the ultrasensitive detection of malathion through an "on-off" detection mode. Compared with traditional malathion detection methods, the method of the present invention avoids the problem of expensive equipment, and has high sensitivity, strong specificity, fast response speed, and simple operation.

[0017] Compared with single S-BNQDs, by using two-dimensional sheet-like covalent organic framework material MCA as the carrier of S-BNQDs, not only the triggering potential of S-BNQDs is reduced, but also the ECL intensity and luminescence stability are greatly improved, thereby improving the sensor performance to achieve signal amplification, and at the same time improving the detection sensitivity. And the operation is simple, the raw materials are cheap, with low toxicity and environmental friendliness.

[0018] By preparing AuNPs and UIO-66-NH2 through the preparation method of mixing at room temperature and stirring vigorously, AuNPs are evenly distributed on the surface of octahedral UIO-66-NH2, and the double quencher Au@NH2-UIO-66 is prepared. The preparation method is simple and can be prepared in large quantities. Description of the Drawings

[0019] Figure 1 SEM images of MCA prepared under conditions of 80 °C and 25 °C Figure 1 (a)SEM image of MCA prepared under the conditions of Comparative Example 4 Figure 1 (b)SEM image of MCA prepared at room temperature of 25 °C Figure 2 Identification diagrams of S-BNQDs and S-BNQD-MCA in Example 1 Figure 2 (a)TEM image of S-BNQDs Figure 2 (b)SEM image of S-BNQD-MCA composite Figure 2 (c-f)EDS elemental surface scanning diagrams of S-BNQD-MCA composite (C; B; N; S) Figure 3 Comparison diagrams of MCA and S-BNQD-MCA composite in Example 1 Figure 3 (a)N2 adsorption-desorption isotherm Figure 3 (b)Pore size distribution diagram Figure 4 TEM images of AuNPs and Au@NH2-UIO-66 Figure 4 (a)TEM image of AuNPs Figure 4 (b, c)TEM images of Au@NH2-UIO-66 Figure 5 Analysis results of 3.5% agarose gel electrophoresis of different materials Figure 5 (a)Mal-Apt Figure 5 (b)cDNA Figure 5 (c)Mal-Apt + cDNA Figure 5 (d)cDNA-Au@NH2-UIO-66 Figure 5(e) is the result of Mal-Apt + EDC + NHS; Figure 6 are the EIS curves of different modified electrodes; Figure 7 are the ECL intensity-potential curves and ECL intensity-time curves of the S-BNQD-MCA modified electrode in different solutions; Figure 8 is the calibration curve of the ECL aptasensor for detecting malathion; Figure 9 is the result of the stability test of the ECL-RET aptasensor; Figure 10 is the result of the selectivity test of the ECL-RET aptasensor; Figure 11 is the result of the reproducibility test for detecting 10 pg / mL Mal by the ECL-RET aptasensors constructed in the same batch; Figure 12 is the schematic diagram of the construction process and principle of the ECL-RET aptasensor. Detailed implementation mode

[0020] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0021] This application provides a covalent organic framework@quantum dot composite material, including a two-dimensional sheet-like covalent organic framework material MCA (this COF is synthesized from melamine and cyanuric acid monomers, denoted as MCA), and S-BNQDs quantum dots (sulfur-doped boron nitride quantum dots) encapsulated inside the two-dimensional sheet-like covalent organic framework material MCA.

[0022] This application provides a preparation method of a covalent organic framework@quantum dot composite material, including the following steps: Dissolve cyanuric acid and melamine in a solvent, then add the S-BNQDs quantum dot solution, and stir and react to obtain the covalent organic framework@quantum dot composite material.

[0023] In some embodiments, after the stirring reaction, the following steps are further included: filtering the mixture after the stirring reaction, then washing and drying in vacuum; the solvent is dimethyl sulfoxide.

[0024] Specifically, the preparation method of the covalent organic framework @ quantum dot composite material (S-BNQD-MCA) includes the following steps: Dissolve cyanuric acid and melamine in dimethyl sulfoxide, then add the S-BNQDs solution, and stir vigorously at room temperature for 72 h; Use a 0.22 μm organic filter membrane to separate the product from the solvent by vacuum filtration to obtain a white solid product, and wash it with absolute ethanol; Dry it in a vacuum drying oven at 50-70 °C to obtain the luminescent material S-BNQD-MCA.

[0025] In some embodiments, the volume ratio of dimethyl sulfoxide to the S-BNQDs solution is 1-2:1; The molar ratio of cyanuric acid to melamine is 1:1-1.2.

[0026] In some embodiments, the temperature of the stirring reaction is 20-25 °C, which is the room temperature of this application, and the time of the stirring reaction is 60-72 h.

[0027] This application provides an ECL-RET aptasensor comprising a covalent organic framework @ quantum dot composite material.

[0028] The ECL-RET (Electrochemiluminescence Resonance Energy Transfer) aptasensor includes an electrode substrate material and a luminescent material, and the luminescent material is the covalent organic framework @ quantum dot composite material (S-BNQD-MCA); The ECL-RET aptasensor is used to detect the signal of Mal (malathion), and it is an "on-off" type ECL-RET aptasensor.

[0029] As Figure 12 shown, this application provides a preparation method of an ECL-RET aptasensor, including the following steps: S1. Coat the covalent organic framework @ quantum dot composite material on the surface of the pretreated glassy carbon electrode, and dry it under an infrared lamp to obtain the S-BNQD-MCA working electrode; S2. Drop the Mal-Apt aptamer solution onto the surface of the S-BNQD-MCA working electrode, incubate and wash it, then drop the BSA blocking solution, and incubate and wash it again to obtain the S-BNQD-MCA / Mal-Apt / BSA working electrode; S3. Drop the quenching probe cDNA-Au@NH2-UiO-66 onto the surface of the S-BNQD-MCA / Mal-Apt / BSA working electrode, incubate and wash it, and then obtain the ECL-RET aptasensor.

[0030] Specifically, the preparation method of the ECL-RET aptasensor includes the following steps: S1. Coat 5 - 7 μL of the luminescent material S - BNQD - MCA (1 mg / L) on the surface of the pretreated glassy carbon electrode and dry it under an infrared lamp to obtain the S - BNQD - MCA working electrode; S2. Drop 5 μL of the Mal - Apt aptamer solution (2.5 μmol / L) onto the surface of the S - BNQD - MCA - modified working electrode obtained in step S1, incubate at 25 °C for 3 - 12 h, gently rinse with PBS buffer to remove the excess unbound Mal - Apt solution, drop 5 - 10 μL of 0.5% BSA solution to block the excess binding sites on the electrode surface, and after incubating for 1 h, wash with PBS buffer to remove the excess unbound BSA solution to obtain the S - BNQD - MCA / Mal - Apt / BSA working electrode; S3. Drop the quenching probe cDNA - Au@NH2 - UiO - 66 onto the surface of the S - BNQD - MCA / Mal - Apt / BSA working electrode obtained in step S2, and after incubating at 25 °C for 3 h, gently rinse with PBS buffer to remove the excess unbound cDNA - Au@NH2 - UiO - 66, thus obtaining the ECL - RET aptasensor for Mal detection.

[0031] The present invention prepares the luminescent material S - BNQD - MCA by encapsulating quantum dots in a covalent organic framework material through a room - temperature stirring method, which improves the electrochemiluminescence intensity of S - BNQDs. Using S - BNQD - MCA as the luminophore, the nucleic acid aptamer of malathion (Mal - Apt) as the molecular recognition probe, and Au@NH2 - UIO - 66 as the double quencher, an electrochemiluminescent aptasensor GCE / S - BNQD - MCA / Mal - Apt / BSA / cDNA - Au@NH2 - UIO - 66 with a signal "on - off" detection mode is constructed based on the electrochemiluminescence resonance energy transfer (ECL - RET) mechanism, and the ECL detection of malathion is achieved through the change of the ECL signal.

[0032] In some embodiments, the pretreatment method of the Mal - Apt aptamer (malathion aptamer probe) is as follows: Add 1 - ethyl - 3 - (3 - dimethylaminopropyl) carbodiimide hydrochloride solution and N - hydroxysuccinimide solution to the Mal - Apt solution modified with a carboxyl group at the 5' end, and react with shaking at room temperature to obtain the Mal - Apt aptamer; The sequence of the Mal - Apt modified with a carboxyl group at the 5' end is: 5′ - COOH - ATC CGT CAC ACC TGC TCT TAT ACACAA TTG TTT TTC TCT TAA CTT CTT GAC TGC TGG TGT TGG CT - 3′.

[0033] Specifically, the pretreatment method of Mal-Apt is as follows: Take 100 μL of Mal-Apt solution modified with a carboxyl group at the 5'-end (10 μM), add 30 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution (10 mM) and 10 μL of N-hydroxysuccinimide (NHS) solution (10 mM), and react with shaking at 25 °C for 30 - 60 min.

[0034] Before Mal-Apt is immobilized on the surface of the S-BNQDs-MCA working electrode, take 100 μL of carboxyl-modified Mal-Apt solution (10 μM), add 30 μL of EDC solution (10 mM) and 10 μL of NHS solution (10 mM) thereto respectively, and react with shaking at 25 °C for 30 - 60 min to activate the carboxyl group at the 5'-end of the aptamer.

[0035] In some embodiments, the preparation method of the quenching probe cDNA-Au@NH2-UiO-66 is as follows: After mixing tris(2-carboxyethyl)phosphine hydrochloride with thiol-modified cDNA, incubate and dilute to obtain a mixture. Mix Au@NH2-UiO-66 with the mixture and incubate with shaking at room temperature to obtain the quenching probe cDNA-Au@NH2-UiO-66; The sequence of the thiol-modified cDNA (complementary strand) is: 5′-AGC CAA CAC CAG CAG TCA AGA AGT TAA GAG AAA AAC AAT TGT GTA TAA GAG CAG GTG TGA CGG AT-(CH2)6-HS-SH-3′.

[0036] Specifically, the preparation method of the quenching probe cDNA-Au@NH2-UiO-66 is as follows: Treat the thiol-modified complementary strand (cDNA) with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (10 mM) for more than 1 h. Mix 10 μM cDNA with TCEP to reduce the disulfide bond (S-S bond) at the 3'-end of cDNA, and then dilute it to a concentration of 2 μM with 10 mM PBS buffer. Take 500 μL of 1 mg / mL Au@NH2-UIO-66 and mix it with 500 μL of cDNA, and incubate with shaking at room temperature for 4 - 12 h through thiol-gold linkage. Then, centrifuge the mixed solution at 8000 rpm for 10 min to remove the unbound cDNA, and wash it 1 - 3 times with PBS buffer (pH = 7.4). Finally, disperse the precipitate in 500 μL of PBS buffer containing BSA (0.5% w / v) and store it at 0 - 8 °C.

[0037] The preparation method of the double quencher Au@NH2-UIO-66 is as follows: Disperse UiO-66-NH2 (amino-functionalized metal-organic framework, 40 mg) in 40 mL of ultrapure water and sonicate for 3 - 5 min. Add 20 mL of AuNPs solution and stir at room temperature for 12 - 24 h. Subsequently, use a 0.22 μm aqueous filter membrane to filter and collect the product Au@UiO-66-NH2, and wash it with ultrapure water. Finally, dry the collected product under vacuum at 55 °C for 12 - 24 h, grind it, weigh it, and disperse it in ultrapure water to prepare a 1 mg / mL dispersion.

[0038] Among them, the preparation method of the AuNPs solution is: Add 3 mL of 1% HAuCl4·3H2O to 100 - 200 mL of ultrapure water, stir evenly, and cool and place it in a refrigerator at 4 °C for 12 h. Then disperse 1 mL of K2CO3 solution (0.2 mol / L) into the above solution, quickly add 9 mL of NaBH4 (0.5 mg / mL), and stir vigorously for 5 min to obtain a wine-red AuNPs solution.

[0039] The present application provides a method for detecting Mal based on an ECL-RET aptamer biosensor, including the following steps: K1. Drop different concentrations of the target Mal onto the ECL-RET aptamer biosensor, and after incubation, obtain the sensor after incubating the target. K2. Place the sensor after incubating the target in an ECL detection solution, use the modified glassy carbon electrode as the working electrode, the platinum sheet electrode as the counter electrode, and the saturated Ag / AgCl electrode as the reference electrode to detect the ECL intensity of different concentrations of the target Mal, and draw a standard curve. K3. Detect the ECL intensity of the Mal to be measured, and calculate the concentration of the Mal to be measured according to the standard curve.

[0040] In some embodiments, the ECL detection solution is a 0.01 M phosphate buffer solution containing 0.1 M K2S2O8.

[0041] In some embodiments, the volume of the dropped target Mal in step S1 is 10 μl, and the incubation time is 2 - 3 h.

[0042] In some embodiments, a three - electrode system is adopted. The modified glassy carbon electrode (3 mm) serves as the working electrode, the platinum sheet electrode (10*10*0.1) serves as the counter electrode, and the saturated Ag / AgCl electrode serves as the reference electrode. The photomultiplier tube PMT is set to 1200 V, the scanning voltage range is set from - 2.3 V to 0 V, and the scanning rate is 100 mV / s. The photomultiplier tube PMT is set to 1200 V, the voltage range is set from - 2.3 V to 0 V, and the scanning rate is 100 mV / s.

[0043] The following further illustrates this solution through specific embodiments.

[0044] Raw material preparation: Example 1 A covalent organic framework @ quantum dot composite material includes two - dimensional sheet - like covalent organic framework material MCA and S - BNQDs quantum dots encapsulated inside the two - dimensional sheet - like covalent organic framework material MCA.

[0045] The preparation method of the covalent organic framework @ quantum dot composite material includes the following steps: Dissolve boric acid (0.495 g), melamine (0.17 g) and thiourea (0.36 g) in 30 - 60 mL of ultrapure water. Transfer the mixture to a sealed stainless - steel autoclave and carry out a hydrothermal reaction at 200 °C for 24 h. Subsequently, filter the dispersion liquid using a 0.22 - μm organic filter head to obtain the S - BNQDs filtrate; collect the S - BNQDs filtrate and store it in a refrigerator at 0 - 8 °C. Characterize the prepared S - BNQDs by transmission electron microscopy (TEM). It can be observed from Figure 2 a that the prepared QDs have a small particle size (≤20 nm) and relatively uniform dispersion, indicating the successful preparation of S - BNQDs.

[0046] Dissolve equimolar amounts of cyanuric acid (0.51 g) and melamine (0.50 g) in 30 mL of dimethyl sulfoxide and stir vigorously. Add 30 mL of the S - BNQDs solution prepared in step 1 and stir vigorously at room temperature (25 °C) for 72 h. Use a 0.22 - μm organic filter membrane for vacuum filtration to obtain a white solid product, and wash it with absolute ethanol. Finally, dry it in a vacuum drying oven at 55 °C for 24 h, grind it for standby, and obtain the S - BNQD - MCA composite material, which is the covalent organic framework @ quantum dot composite material.

[0047] Example 2 A preparation method of an ECL - RET aptasensor includes the following steps: S1. Coat the luminescent material S - BNQD - MCA (5 μL, 1 mg / L) prepared in Example 1 on the surface of the pretreated glassy carbon electrode and dry it under an infrared lamp to obtain the S - BNQD - MCA working electrode; S2. Drop the Mal-Apt aptamer solution (5 μL, 2.5 μmol / L) onto the surface of the S-BNQD-MCA modified electrode, incubate for 12 h, gently rinse with PBS buffer, add 5 μL of 0.5% BSA, and after incubating for 1 h, rinse with PBS buffer to obtain the S-BNQD-MCA / Mal-Apt / BSA working electrode; S3. Drop the quenching probe cDNA-Au@NH2-UiO-66 onto the surface of the S-BNQD-MCA / Mal-Apt / BSA working electrode, and after incubating for 3 h, rinse with PBS buffer to obtain the ECL-RET aptamer biosensor for Mal detection.

[0048] Among them, the preparation of the double quencher Au@NH2-UIO-66 includes the following steps: (1) AuNPs: Add 3 mL of 1% HAuCl4·3H2O to 100 mL of ultrapure water, stir evenly, and cool and place in a refrigerator at 4 °C for 12 h. Then disperse 1 mL of K2CO3 solution (0.2 mol / L) into the above solution, quickly add 9 mL of NaBH4 (0.5 mg / mL), and stir vigorously for 5 min to obtain a wine-red AuNPs solution. Figure 4 a is the transmission electron microscope (TEM) image of the prepared AuNPs. The prepared AuNPs are spherical-like and have good monodispersity and crystallinity.

[0049] (2) UiO-66-NH2: Dissolve ZrCl4 (0.32 g) and 2-aminoterephthalic acid (0.248 g) in 70 mL of DMF solution, and stir vigorously for 10 min. Then slowly add 6 mL of acetic acid and stir for 15 min. Put the mixture into a stainless steel autoclave and carry out a hydrothermal reaction at 120 °C for 24 h. Use a 0.22 μm organic filter membrane to filter and collect the solid product, wash it with DMF and methanol solutions respectively, and finally dry it under vacuum at 55 °C for 12 h.

[0050] (3) Au@NH2-UIO-66: Disperse UiO-66-NH2 (40 mg) in 40 mL of ultrapure water and sonicate for 3 min. Add 20 mL of the AuNPs solution prepared in step 1 and stir at room temperature for 24 h. Use a 0.22 μm aqueous filter membrane to filter and collect the product Au@UiO-66-NH2, and wash it with ultrapure water. Finally, dry the collected product under vacuum at 55 °C for 24 h, grind it, weigh it, and disperse it in ultrapure water to prepare a 1 mg / mL dispersion. Figure 4(b, c) is the TEM image of the Au@NH2-UIO-66 composite material. UIO-66-NH2 presents an octahedral topological structure. AuNPs are evenly distributed on the surface of UIO-66-NH2, and the structural integrity of the UIO-66-NH2 main body is completely retained. AuNPs are successfully loaded on the surface of UIO-66-NH2 to prepare the Au@NH2-UIO-66 composite material.

[0051] cDNA-Au@NH2-UiO-66: Mix 10 μM cDNA with 10 mM TCEP and incubate on a shaker at room temperature for 1 h, then dilute to a concentration of 2 μM with 10 mM PBS buffer. Take 500 μL of 1 mg / mL Au@NH2-UIO-66 and mix it with 500 μL of cDNA, and incubate with shaking at room temperature for 12 h. Centrifuge at 8000 rpm for 10 min to remove unbound cDNA, and wash 3 times with PBS buffer (pH = 7.4). Finally, disperse the precipitate in 500 μL of PBS buffer containing BSA (0.5% w / v).

[0052] Mal-Apt: Take 100 μL of the Mal-Apt solution (10 μM) with a carboxyl modification at the 5' end, add 30 μL of the EDC solution (10 mM) and 10 μL of the NHS solution (10 mM), and react with shaking at 25 °C for 30 min.

[0053] Example 3 A method for detecting Mal based on an ECL-RET aptamer biosensor, comprising the following steps: K1. Drop 10 μl of different concentrations of the target Mal onto the surface of the ECL-RET aptamer biosensor electrode constructed in Example 2, and incubate at 25 °C for 2 h to obtain the sensor after incubating the target. K2. Place the sensor after incubating the target in a 0.01 M phosphate buffer solution containing 0.1 M K2S2O8 to measure its ECL intensity. Use a three-electrode system, with the modified glassy carbon electrode (3 mm) as the working electrode, the platinum plate electrode (10*10*0.1) as the counter electrode, and the saturated Ag / AgCl electrode as the reference electrode; set the photomultiplier PMT to 1200 V, set the scanning voltage range to -2.3 ~ 0 V, and the scanning rate to 100 mV / s to detect the ECL intensity of different concentrations of the target Mal. According to the relationship between the obtained ECL intensity (y ECL ) and the logarithm of the Mal concentration (lgc), plot a calibration curve. As Figure 8 shown, in the range of 0.1 fg / mL to 1 ng / mL, there is a good linear relationship between the ECL intensity and the logarithm of the Mal concentration, with a correlation coefficient of 0.999 and a detection limit of 0.048 fg / mL; K3. Detect the ECL intensity of the Mal to be measured, and calculate the concentration of the Mal to be measured according to the standard curve.

[0054] Comparative Example 1 A covalent organic framework @ quantum dot composite material, with other contents the same as in Example 1, except that the room temperature condition is changed to 80 °C.

[0055] Comparative Example 2 A covalent organic framework @ quantum dot composite material, with other contents the same as in Example 1, except that the vacuum drying is changed to forced-air drying.

[0056] Comparative Example 3 A covalent organic framework material, and its preparation method is as follows: Dissolve equimolar amounts of cyanuric acid (0.51 g) and melamine (0.50 g) in 20 mL and 10 mL of dimethyl sulfoxide respectively, and stir vigorously at 25 °C room temperature for 72 h to form a uniform white solution. Use a 0.22 μm organic filter membrane to vacuum filter to obtain a white solid product, and wash it with absolute ethanol and ultrapure water. Finally, dry it in a forced-air drying oven at 55 °C for 12 h to obtain the material MCA, and grind it for standby.

[0057] Comparative Example 4 A covalent organic framework material, with other contents the same as in Comparative Example 3, except that it is stirred vigorously at 80 °C for 72 h.

[0058] Comparative Example 5 A covalent organic framework material, with other contents the same as in Comparative Example 3, except that the forced-air drying is changed to vacuum drying.

[0059] Comparative Example 6 A covalent organic framework material, with other contents the same as in Comparative Example 3, except that it is stirred vigorously at 80 °C for 72 h and the forced-air drying is changed to vacuum drying.

[0060] Testing and Evaluation Test the material morphology of Comparative Example 3 and Comparative Example 4 and the specific surface area of Comparative Example 5 and Comparative Example 6, as Figure 1 shown, the MCA prepared under different temperature conditions shows significant morphological differences. As Figure 3 can be seen from a, the BET specific surface area of the MCA prepared at 25 °C room temperature is as high as 204.14 m² / g, while the specific surface area of the preparation system at 80 °C drops sharply to 104.26 m² / g. In addition, compared with Figure 2 the sample treated by vacuum drying in b, the surface structure of the MCA sample treated by non-vacuum drying has changed significantly ( Figure 1), specifically manifested as the edge curling phenomenon of the two-dimensional sheet-like MCA samples, which confirmed that the vacuum degree of the drying environment has an important regulatory effect on the final morphology of the material.

[0061] By observing the S-BNQD-MCA composite material of Example 1 through SEM-EDS elemental surface scanning ( Figure 2 c-f), it was found that S-BNQDs were successfully loaded in the framework structure of the MCA substrate. In addition to the inherent elements C and N of the matrix material MCA, B element and S element could be clearly detected in the composite material. As Figure 3 shown in a, in Example 1, after loading S-BNQDs, the BET specific surface area of MCA (Comparative Example 5) decreased to 121.93 m² / g, with a decrease of 40.3%. By calculating the pore size distribution through the BJH model ( Figure 3 b), its average pore size increased from 5.99 nm to 7.14 nm, which formed a synergistic evidence with the decreasing trend of the specific surface area. At the same time, comparing Example 1 and Comparative Example 1, except that the preparation temperature was changed to 80 °C and the others remained unchanged, S-BNQD-MCA was prepared. The specific surface area of the 80 °C preparation system decreased sharply to 72.32 m² / g, and the average pore size decreased from 5.65 nm to 5.39 nm. The above comparison shows that the room temperature synthesis conditions are more conducive to constructing a mesoporous structure with a high specific surface area, while the high-temperature reflux process may cause excessive shrinkage of the framework and pore collapse. The room temperature synthesis strategy achieved high-density uniform encapsulation of S-BNQDs in the covalent organic framework MCA by optimizing the quantum dot-framework interface interaction. As Figure 7 shown in c, in Example 1, as the carrier of S-BNQDs, the ECL intensity of the modified electrode increased greatly, indicating that MCA can significantly enhance the ECL intensity of S-BNQDs. This may be because MCA makes S-BNQDs more dispersed, thus weakening the self-quenching effect and effectively increasing the ECL intensity. Using MCA as the carrier of S-BNQDs has a positive effect.

[0062] To evaluate whether Mal-Apt and cDNA in Example 2 can effectively bind, and the successful coupling of cDNA and Au@NH2-UIO-66, agarose gel electrophoresis was used for verification. The results are as Figure 5, compared with Mal-Apt (lane a) and cDNA (lane b), a new band (lane c) appeared, indicating the stable formation of Mal-Apt@cDNA. In lane e, the band was observed to maintain the same position as lane a, indicating that the activators EDC and NHS had no effect on the structure of the aptamer Mal-Apt. Lane d was after the coupling of cDNA and Au@NH2-UIO-66. Comparing with lane b, a band with a larger molecular weight could be observed, which was caused by the successful binding of a large amount of cDNA to Au@NH2-UIO-66 to form cDNA-Au@NH2-UIO-66, indicating that cDNA-Au@NH2-UIO-66 was successfully prepared and could be used for the assembly of the sensor.

[0063] In Example 2, by using 5.0 mM Fe[(CN)6] 3- / 4- as the electroactive probe, the EIS curves of different modified electrodes were successively tested to prove the successful construction of the ECL aptasensor. The results are as Figure 6 , where the EIS curves of bare GCE (I), GCE / S-BNQD-MCA (II), GCE / S-BNQD-MCA / Mal-Apt (III), GCE / S-BNQD-MCA / Mal-Apt / BSA (IV), GCE / S-BNQD-MCA / Mal-Apt / BSA / cDNA-Au@NH2-UIO-66 (V) in 0.01 M PBS buffer containing 5 mM [Fe(CN)6] 3− / 4− (as the redox probe) and 0.1 M KCl were tested, and the EIS was recorded in the frequency range of 0.1 Hz - 100 kHz with an amplitude of 5 mV. After modifying the GCE (glassy carbon electrode) with the S-BNQD-MCA nanocomposite, the Rct value decreased to 422 Ω. Due to the negative charge on the surface of the DNA strand preventing the free movement of electrons, the addition of Mal-Apt led to an increase in the semicircle diameter (Rct = 1482 Ω). To block the excess binding sites on the surface of the modified electrode, BSA was dropped on the surface of GCE / S-BNQD-MCA / Mal-Apt. Non-conductive BSA is a biological macromolecule that hinders the electron transfer process on the electrode surface. Therefore, the modification of BSA led to a further increase in the electrode Rct (Rct = 2293 Ω). After assembling cDNA-Au@NH2-UIO-66 on the electrode surface, due to the relatively good conductivity of cDNA-Au@NH2-UIO-66, the Rct decreased to 1917 Ω, indicating the successful construction of the ECL aptasensor.

[0064] Meanwhile, for the ECL reaction mechanism of the S-BNQD-MCA / K2S2O8 system in Example 2, the bare GCE and GCE / S-BNQD-MCA were measured in different solutions with a potential scanning range from -2.3 V to 0 V. Figure 7 (a) CV and (b) ECL intensity-potential curves of the S-BNQD-MCA modified electrode in 0.01 M PBS solution and 0.01 M PBS solution containing 0.1 M K2S2O8, and the bare GCE in 0.01 M PBS solution containing 0.1 M K2S2O8. ECL intensity-time curves of different modified electrodes: (c) bare GCE, GCE / MCA, GCE / S-BNQD, and GCE / S-BNQD-MCA, and (b) GCE / S-BNQD-MCA and GCE / S-BNQD-MCA / Au@NH2-UIO-66 in 0.01 M PBS solution containing 0.1 M K2S2O8. As Figure 7 b shows, when measuring GCE / S-BNQD-MCA in PBS, the obtained ECL signal is extremely low. After adding K2S2O8 to PBS, the ECL intensity of the bare GCE has no obvious difference from that of GCE / S-BNQD-MCA in the PBS solution. When measuring GCE / S-BNQD-MCA in PBS-S2O8 2- the ECL intensity increases to 95975 a.u., because the coreactant K2S2O8 participates in the reaction and promotes the ECL emission of S-BNQD-MCA. Meanwhile, the CV curves of the bare GCE and GCE / S-BNQD-MCA in the range of -2.3~0 V were obtained ( Figure 7 a). When measuring GCE / S-BNQD-MCA in PBS, there is no obvious reduction peak. In addition, the CV curve of GCE / S-BNQD-MCA in PBS-S2O8 2- shows an obviously stronger reduction peak current (1.08 mA, -1.44 V), and compared with the bare GCE in PBS-S2O8 2- (0.7985 mA, -1.52 V), the peak potential shifts negatively, indicating that K2S2O8 is reduced in this process. Figure 7 c shows the bare GCE, GCE / MCA, GCE / S-BNQDs, and GCE / S-BNQD-MCA in PBS-S2O8 2-ECL intensity test in [the relevant context]. Compared with the bare GCE, GCE / MCA and GCE / S-BNQD have higher ECL intensity. When using S-BNQD-MCA to modify the electrode, a significant enhancement in ECL intensity occurs, indicating that MCA can significantly enhance the ECL intensity of S-BNQDs. This is because MCA makes S-BNQDs more dispersed, thereby weakening the self-quenching effect and effectively increasing the ECL intensity. Using MCA as the carrier of S-BN QDs has positive significance. At the same time, according to conventional methods, GCE / S-BNQD-MCA and GCE / S-BNQD-MCA / Au@NH2-UIO-66 were prepared, and in PBS-S2O8 2- the ECL curves in [the relevant context] show that GCE / S-BNQD-MCA / Au@NH2-UIO-66 obtained an extremely low ECL signal ( Figure 7 d). Therefore, effective ECL-RET can occur between Au@NH2-UIO-66 and the luminophore.

[0065] Investigation of the stability, specificity and reproducibility of the sensor constructed in Example 2: Sensor stability test: The prepared sensor was used to measure the ECL emission of 11 consecutive CV scans. The results are as Figure 9 shown, Figure 9 which is the ECL emission stability of the constructed ECL aptasensor after 11 consecutive CV scans in a 0.01 M PBS solution containing 0.1 M K2S2O8. Through 11 ECL test cycles, it was found that the measured signals had good stability, and the relative standard deviation (RSD) was 0.99%.

[0066] Sensor specificity test: Some pesticides (carbendazim, acetamiprid, diazinon) were selected as interfering factors, Figure 10 which is the selectivity of the prepared ECL aptasensor for Mal and its interferents: blank control, 10 pg / mL carbendazim, 10 pg / mL acetamiprid, 10 pg / mL diazinon, and a mixture containing 100 fg / mL malathion and 10 pg / mL interferent. It can be seen from Figure 10 that the interfering ECL signals are basically the same as those of the blank sample, the ECL intensity is lower than that of Mal, and at the same time, the ECL signal of Mal is close to that of the mixture (100 fg / mL malathion + 10 pg / mL interferent). The above results prove that the constructed sensor can perform specific Mal detection.

[0067] Sensor reproducibility test: Six different sensors were prepared to measure the target Mal at 10 pg / mL. The ECL signal intensity is as Figure 11 shown, and the RSD is 1.53%, indicating that the sensor has good reproducibility.

[0068] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A covalent organic framework @ quantum dot composite material, characterized in that, It includes two-dimensional sheet-like covalent organic framework material MCA and S-BNQDs quantum dots encapsulated inside the two-dimensional sheet-like covalent organic framework material MCA.

2. A preparation method of the covalent organic framework @ quantum dot composite material as described in claim 1, characterized in that, It includes the following steps: Dissolve cyanuric acid and melamine in a solvent, then add the S-BNQDs quantum dot solution, and stir and react to obtain the covalent organic framework @ quantum dot composite material.

3. The preparation method of the covalent organic framework @ quantum dot composite material according to claim 2, wherein, After the stirring reaction, it also includes the following steps: filter the mixture after the stirring reaction, then wash and vacuum dry.

4. The preparation method of the covalent organic framework @ quantum dot composite material according to claim 2, wherein, The solvent is dimethyl sulfoxide; the volume ratio of the dimethyl sulfoxide to the S-BNQDs solution is 1-2:1; the molar ratio of cyanuric acid to melamine is 1:1-1.

2.

5. The preparation method of the covalent organic framework @ quantum dot composite material according to claim 2, characterized in that, The temperature of the stirring reaction is 20-25 °C, and the time of the stirring reaction is 60-72 h.

6. An ECL-RET aptamer biosensor comprising the covalent organic framework @ quantum dot composite material as described in claim 1.

7. A method for preparing the ECL-RET aptamer biosensor according to claim 6, characterized in that, It includes the following steps: S1. Coat the covalent organic framework @ quantum dot composite material on the surface of a pretreated glassy carbon electrode and dry it under an infrared lamp to obtain the S-BNQD-MCA working electrode. S2. Drop the Mal-Apt aptamer solution onto the surface of the S-BNQD-MCA working electrode, incubate and wash it, then drop the BSA blocking solution and incubate and wash it again to obtain the S-BNQD-MCA / Mal-Apt / BSA working electrode. S3. Drop the quenching probe cDNA-Au@NH2-UiO-66 onto the surface of the S-BNQD-MCA / Mal-Apt / BSA working electrode, incubate and wash it to obtain the ECL-RET aptamer biosensor.

8. The preparation method of the ECL-RET aptamer biosensor according to claim 7, wherein The pretreatment method of the Mal-Apt aptamer is as follows: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to the Mal-Apt solution modified with a carboxyl group at the 5' end, and react with oscillation at room temperature to obtain the Mal-Apt aptamer. The sequence of the Mal-Apt modified with a carboxyl group at the 5' end is: 5′-COOH-ATC CGT CAC ACC TGC TCT TAT ACACAA TTG TTT TTC TCT TAA CTT CTT GAC TGC TGG TGT TGG CT-3′.

9. The preparation method of the ECL-RET aptamer biosensor according to claim 7, characterized in that, The preparation method of the quenching probe cDNA-Au@NH2-UiO-66 is as follows: Mix tris(2-carboxyethyl)phosphine hydrochloride with the cDNA modified with a thiol group, incubate and dilute to obtain a mixture, mix Au@NH2-UiO-66 with the mixture, and incubate with oscillation at room temperature to obtain the quenching probe cDNA-Au@NH2-UiO-66. The sequence of the thiol-modified cDNA is: 5′-AGC CAA CAC CAG CAG TCA AGA AGT TAA GAG AAAAAC AAT TGT GTA TAA GAG CAG GTG TGA CGG AT-(CH2)6-HS-SH-3′.

10. A method for detecting Mal based on the ECL-RET aptamer biosensor as described in claim 6, characterized in that, It includes the following steps: K1. Drop different concentrations of the target Mal onto the ECL-RET aptamer biosensor, and after incubation, obtain the sensor after incubating the target; K2. Place the sensor after incubating the target in the ECL detection solution, use the modified glassy carbon electrode as the working electrode, the platinum sheet electrode as the counter electrode, and the saturated Ag / AgCl electrode as the reference electrode to detect the ECL intensity of different concentrations of the target Mal and plot the standard curve; K3. Detect the ECL intensity of the Mal to be measured, and calculate the concentration of the Mal to be measured according to the standard curve.

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