A covalent organic framework@quantum dot composite material and its preparation and application

Abstract

The present invention discloses a covalent organic framework@quantum dot composite material and its preparation and application. The covalent organic framework@quantum dot composite material comprises 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. By using the two-dimensional sheet-like covalent organic framework material MCA as a carrier of S-BNQDs, the present application not only reduces the trigger potential of S-BNQDs, but also greatly improves the ECL intensity and luminescence stability, thereby improving the sensor performance, realizing signal amplification, and improving the detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical technology, and in particular 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. 10 H 19 Malathion (O6S2P) is one of the most common commercial organophosphate insecticides, widely used as a protectant for grain storage and crop pest control. Despite its significant economic benefits in agricultural production, the development of highly sensitive biosensors for malathion detection is crucial to prevent further pesticide poisoning.

[0003] Quantum dot (QD)-based electrochemiluminescence (ECL) analysis offers a new platform for the analysis of pesticide residues 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. The use of nanomaterials as signal amplifiers is particularly important in biosensor design due to their advantages such as large surface area, strong electron transport capacity, easy labeling, and excellent biocompatibility. Porous materials such as metal-organic frameworks (MOFs), hydrogen-bonded organic frameworks (HOFs), and covalent organic frameworks (COFs) have been used as supports for immobilizing ECL luminophores due to their well-ordered porosity and high surface area, which significantly increase the number of immobilized luminophores and facilitate their electrochemical excitation. However, these materials have been found to be less effective for the detection of malathion, particularly in terms of stability and sensitivity.

[0004] Therefore, it is necessary to provide a novel COF-based electrochemiluminescence biosensor for 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, which are used to solve the problem of how to improve the detection sensitivity of COF-based electrochemiluminescence biosensors to malathion.

[0006] In order to achieve the above technical objectives, this application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a covalent organic framework@quantum dot composite material, comprising 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.

[0008] In a second aspect, the present application provides a method for preparing a covalent organic framework@quantum dot composite material, comprising the following steps:

[0009] Dissolve cyanuric acid and melamine in a solvent, add S-BNQDs quantum dot solution, and stir to react to obtain a covalent organic framework@quantum dot composite material.

[0010] Preferably, after the stirring reaction, the method further comprises the following steps: filtering the mixture after the stirring reaction, and then washing and vacuum drying.

[0011] Preferably, the solvent is dimethyl sulfoxide (DMSO); the volume ratio of DMSO to S-BNQDs solution is 1-2:1; and the molar ratio of cyanuric acid to melamine is 1:1-1.2. V(DMSO):V(S-BNQDs)=30-60mL:30mL. DMSO is an analytically pure organic solvent, and S-BNQDs are prepared as a liquid product.

[0012] Preferably, the stirring reaction temperature is 20-25° C., and the stirring reaction time is 60-72 h.

[0013] In a third aspect, the present application provides an ECL-RET aptamer biosensor comprising a covalent organic framework@quantum dot composite material.

[0014] In a fourth aspect, the present application provides a method for preparing an ECL-RET aptamer biosensor, comprising the following steps:

[0015] S1. Coating the covalent organic framework@quantum dot composite material on the pretreated glassy carbon electrode surface and drying it under infrared light to obtain the S-BNQD-MCA working electrode;

[0016] S2. The Mal-Apt aptamer solution was added dropwise to the surface of the S-BNQD-MCA working electrode, incubated and washed, and then BSA blocking solution was added dropwise, followed by incubation and washing again to obtain the S-BNQD-MCA / Mal-Apt / BSA working electrode;

[0017] S3. The quenching probe cDNA-Au@NH2-UiO-66 was dropped onto the surface of the S-BNQD-MCA / Mal-Apt / BSA working electrode. After incubation and washing, the ECL-RET aptamer biosensor was obtained.

[0018] Preferably, the pretreatment method of Mal-Apt aptamer is as follows:

[0019] Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to the Mal-Apt solution with modified 5'-terminal carboxyl group, and shake at room temperature to obtain the Mal-Apt aptamer.

[0020] The sequence of Mal-Apt with modified 5′-carboxyl group 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).

[0021] Preferably, the preparation method of the quenching probe cDNA-Au@NH2-UiO-66 is as follows:

[0022] After mixing tris(2-carboxyethyl)phosphine hydrochloride with thiol-modified cDNA, incubating and diluting to obtain a mixed solution, Au@NH2-UiO-66 was mixed with the mixed solution and incubated with shaking at room temperature to obtain the quenched probe cDNA-Au@NH2-UiO-66;

[0023] 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′ (SEQ ID NO. 2).

[0024] In a fifth aspect, the present application provides a method for detecting Mal based on an ECL-RET aptamer biosensor, comprising the following steps:

[0025] K1. Add different concentrations of target Mal to the ECL-RET aptamer biosensor and incubate to obtain a sensor after incubation of the target;

[0026] K2. Place the sensor after target incubation in an ECL detection solution. Using a modified glassy carbon electrode as the working electrode, a platinum electrode as the counter electrode, and a saturated Ag / AgCl electrode as the reference electrode, measure the ECL intensity of the target Mal at different concentrations and plot a standard curve.

[0027] K3. Detect the ECL intensity of the Mal to be tested and calculate the concentration of the Mal to be tested based on the standard curve.

[0028] The beneficial effects of this application are as follows:

[0029] The present invention utilizes ECL-RET technology to encapsulate sulfur-doped boron nitride quantum dots within a covalent organic framework to create the luminescent material S-BNQD-MCA. Using Au@NH2-UIO-66 as a dual quencher, an electrochemiluminescent aptasensor is fabricated. This sensor achieves ultrasensitive detection of malathion using an on-off detection mode. Compared to traditional malathion detection methods, the present method avoids the costly equipment and offers high sensitivity, strong specificity, rapid response, and simple operation.

[0030] Compared to single S-BNQDs, using the two-dimensional sheet-like covalent organic framework material MCA as a carrier for S-BNQDs not only reduces the trigger potential of S-BNQDs but also greatly improves the ECL intensity and luminescence stability, thereby enhancing sensor performance and achieving signal amplification while increasing detection sensitivity. Furthermore, the process is simple to operate, uses inexpensive raw materials, has low toxicity, and is environmentally friendly.

[0031] By mixing the prepared AuNPs and UIO-66-NH2 at room temperature and stirring vigorously, the AuNPs are evenly distributed on the surface of the octahedral UIO-66-NH2 to obtain the double quencher Au@NH2-UIO-66. The preparation method is simple and can be prepared in large quantities. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 SEM images of MCA prepared at 80℃ and 25℃. Figure 1 (a) is the SEM image of MCA prepared under the conditions of Comparative Example 4, Figure 1 (b) SEM image of MCA prepared at room temperature (25°C);

[0033] Figure 2 This is the identification diagram 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 material; Figure 2 (cf) EDS element surface scanning images of S-BNQD-MCA composite material (C; B; N; S);

[0034] Figure 3 A comparison diagram of the MCA and S-BNQD-MCA composite materials in Example 1; Figure 3 (a) is the N2 adsorption-desorption isotherm, Figure 3 (b) is the pore size distribution diagram;

[0035] 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;

[0036] Figure 5 The results of 3.5% agarose gel electrophoresis analysis of different materials are shown; Figure 5 (a) is Mal-Apt, Figure 5 (b) is cDNA, Figure 5 (c) is Mal-Apt+cDNA, Figure 5 (d) cDNA-Au@NH2-UIO-66, Figure 5 (e) is the result of Mal-Apt+EDC+NHS;

[0037] Figure 6 are the EIS curves of different modified electrodes;

[0038] Figure 7 The ECL intensity-potential curves and ECL intensity-time curves of the S-BNQD-MCA modified electrode in different solutions;

[0039] Figure 8 is the calibration curve of the ECL aptasensor for the detection of malathion;

[0040] Figure 9 This is the stability test result of ECL-RET aptamer biosensor;

[0041] Figure 10 This is the selectivity test result of ECL-RET aptamer biosensor;

[0042] Figure 11 Reproducibility test results of the ECL-RET aptamer biosensor constructed in the same batch for detecting 10 pg / mL Mal;

[0043] Figure 12 Schematic diagram of the construction process and principle of the ECL-RET aptamer biosensor. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] The present application provides a covalent organic framework@quantum dot composite material, including a two-dimensional sheet covalent organic framework material MCA (the COF is synthesized from melamine and cyanuric acid monomers, represented by MCA), and S-BNQDs quantum dots (sulfur-doped boron nitride quantum dots) encapsulated inside the two-dimensional sheet covalent organic framework material MCA.

[0046] The present application provides a method for preparing a covalent organic framework@quantum dot composite material, comprising the following steps:

[0047] Dissolve cyanuric acid and melamine in a solvent, add S-BNQDs quantum dot solution, and stir to react to obtain a covalent organic framework@quantum dot composite material.

[0048] In some embodiments, after the stirring reaction, the following steps are further included: filtering the mixture after the stirring reaction, and then washing and vacuum drying; the solvent is dimethyl sulfoxide.

[0049] Specifically, the preparation method of the covalent organic framework@quantum dot composite material (S-BNQD-MCA) includes the following steps: dissolving cyanuric acid and melamine in dimethyl sulfoxide, then adding the S-BNQDs solution, and vigorously stirring at room temperature for 72 hours; using a 0.22μm organic filter membrane, separating the product from the solvent by vacuum filtration to obtain a white solid product, and washing with anhydrous ethanol; drying in a vacuum drying oven at 50~70℃ to obtain the luminescent material S-BNQD-MCA.

[0050] In some embodiments, the volume ratio of dimethyl sulfoxide to S-BNQDs solution is 1-2:1; and the molar ratio of cyanuric acid to melamine is 1:1-1.2.

[0051] In some embodiments, the stirring reaction temperature is 20-25° C., which is the room temperature of the present application, and the stirring reaction time is 60-72 h.

[0052] The present application provides an ECL-RET aptamer biosensor comprising a covalent organic framework@quantum dot composite material.

[0053] The ECL-RET (Electrochemiluminescence Resonance Energy Transfer) aptamer biosensor includes an electrode substrate material and a luminescent material. The luminescent material is a covalent organic framework@quantum dot composite material (S-BNQD-MCA). The ECL-RET aptamer biosensor is used to detect the signal of Mal (malathion) and is an "on-off" type ECL-RET aptamer biosensor.

[0054] like Figure 12 As shown, the present application provides a method for preparing an ECL-RET aptamer biosensor, comprising the following steps:

[0055] S1. Coating the covalent organic framework@quantum dot composite material on the pretreated glassy carbon electrode surface and drying it under infrared light to obtain the S-BNQD-MCA working electrode;

[0056] S2. The Mal-Apt aptamer solution was added dropwise to the surface of the S-BNQD-MCA working electrode, incubated and washed, and then BSA blocking solution was added dropwise, followed by incubation and washing again to obtain the S-BNQD-MCA / Mal-Apt / BSA working electrode;

[0057] S3. The quenching probe cDNA-Au@NH2-UiO-66 was dropped onto the surface of the S-BNQD-MCA / Mal-Apt / BSA working electrode. After incubation and washing, the ECL-RET aptamer biosensor was obtained.

[0058] Specifically, the preparation method of the ECL-RET aptamer biosensor includes the following steps:

[0059] 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 an S-BNQD-MCA working electrode.

[0060] S2. Add 5 μL of Mal-Apt aptamer solution (2.5 μmol / L) dropwise to the surface of the S-BNQD-MCA-modified working electrode obtained in step S1 and incubate at 25°C for 3–12 h. Gently rinse with PBS buffer to remove excess unbound Mal-Apt solution, then add 5–10 μL of 0.5% BSA solution dropwise to block excess binding sites on the electrode surface. After incubation for 1 h, rinse with PBS buffer to remove excess unbound BSA solution, obtaining an S-BNQD-MCA / Mal-Apt / BSA working electrode.

[0061] S3. The quenching probe cDNA-Au@NH2-UiO-66 was added dropwise to the surface of the S-BNQD-MCA / Mal-Apt / BSA working electrode obtained in step S2. After incubation at 25°C for 3 h, the excess unbound cDNA-Au@NH2-UiO-66 was gently rinsed with PBS buffer to obtain the ECL-RET aptamer biosensor for Mal detection.

[0062] This study encapsulates quantum dots in a covalent organic framework (COF) using a room-temperature stirring method to produce the luminescent material S-BNQD-MCA, enhancing the electrochemiluminescence intensity of the S-BNQDs. Furthermore, using S-BNQD-MCA as the luminophore, a malathion nucleic acid aptamer (Mal-Apt) as the molecular recognition probe, and Au@NH2-UIO-66 as the dual quencher, an electrochemiluminescence aptamer biosensor (GCE / S-BNQD-MCA / Mal-Apt / BSA / cDNA-Au@NH2-UIO-66) with an "on-off" detection mode was constructed based on the electrochemiluminescence resonance energy transfer (ECL-RET) mechanism. Malathion can be detected by ECL signal changes.

[0063] In some embodiments, the pretreatment method of the Mal-Apt aptamer (malathion aptamer probe) is as follows:

[0064] Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to the Mal-Apt solution with modified 5'-terminal carboxyl group, and shake at room temperature to obtain the Mal-Apt aptamer.

[0065] The sequence of Mal-Apt with modified 5'-terminal carboxyl group 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'.

[0066] Specifically, the pretreatment method of Mal-Apt aptamer is as follows: take 100 μL of 5'-terminal carboxyl-modified Mal-Apt solution (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.

[0067] Before Mal-Apt was immobilized on the surface of the S-BNQDs-MCA working electrode, 100 μL of carboxyl-modified Mal-Apt solution (10 μM) was taken, and 30 μL of EDC solution (10 mM) and 10 μL of NHS solution (10 mM) were added to it, respectively, and the reaction was shaken at 25°C for 30-60 min to activate the carboxyl group at the 5' end of the aptamer.

[0068] In some embodiments, the preparation method of the quenching probe cDNA-Au@NH2-UiO-66 is as follows:

[0069] After mixing tris(2-carboxyethyl)phosphine hydrochloride with thiol-modified cDNA, incubating and diluting to obtain a mixed solution, Au@NH2-UiO-66 was mixed with the mixed solution and incubated with shaking at room temperature to obtain the quenched probe cDNA-Au@NH2-UiO-66;

[0070] The sequence of the thiol-modified cDNA (complementary strand) is: 5′-AGC CAA CAC CAG CAG TCA AGA AGT TAAGAG AAA AAC AAT TGT GTA TAA GAG CAG GTG TGA CGG AT-(CH2)6-HS-SH-3′.

[0071] Specifically, the preparation method of the quenching probe cDNA-Au@NH2-UiO-66 is as follows:

[0072] The thiol-modified complementary strand (cDNA) was treated with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (10 mM) for at least 1 hour. 10 μM cDNA was mixed with TCEP to reduce the 3'-terminal disulfide bonds (SS bonds) of the cDNA, and then diluted to a concentration of 2 μM in 10 mM PBS buffer. 500 μL of 1 mg / mL Au@NH2-UIO-66 was mixed with 500 μL of cDNA, and the reaction was incubated with shaking at room temperature for 4–12 hours to allow for thiol-gold linkage. The mixture was then centrifuged at 8000 rpm for 10 minutes to remove unbound cDNA and washed one to three times with PBS buffer (pH 7.4). Finally, the precipitate was dispersed in 500 μL of PBS buffer containing 0.5% w / v BSA and stored at 0–8°C.

[0073] The dual quencher Au@NH2-UIO-66 was prepared as follows: 40 mg of UiO-66-NH2 (an amino-functionalized metal-organic framework) was dispersed in 40 mL of ultrapure water and sonicated for 3–5 minutes. 20 mL of the AuNPs solution was added and stirred at room temperature for 12–24 hours. The product, Au@UiO-66-NH2, was then collected by filtration using a 0.22 μm aqueous filter and washed with ultrapure water. Finally, the collected product was dried under vacuum at 55°C for 12–24 hours, ground, weighed, and dispersed in ultrapure water to prepare a 1 mg / mL dispersion.

[0074] The AuNPs solution was prepared by adding 3 mL of 1% HAuCl₄·3H₂O to 100–200 mL of ultrapure water, stirring thoroughly, and cooling in a 4°C refrigerator for 12 hours. 1 mL of 0.2 mol / L K₂CO₃ solution was then dispersed into the solution, and 9 mL of 0.5 mg / mL NaBH₄ was quickly added. The mixture was stirred vigorously for 5 minutes to obtain a wine-red AuNPs solution.

[0075] The present application provides a method for detecting Mal based on an ECL-RET aptamer biosensor, comprising the following steps:

[0076] K1. Add different concentrations of target Mal to the ECL-RET aptamer biosensor and incubate to obtain a sensor after incubation of the target;

[0077] K2. Place the sensor after target incubation in an ECL detection solution. Using a modified glassy carbon electrode as the working electrode, a platinum electrode as the counter electrode, and a saturated Ag / AgCl electrode as the reference electrode, measure the ECL intensity of the target Mal at different concentrations and plot a standard curve.

[0078] K3. Detect the ECL intensity of the Mal to be tested and calculate the concentration of the Mal to be tested based on the standard curve.

[0079] In some embodiments, the ECL detection solution is a 0.01 M phosphate buffer solution containing 0.1 M K2S2O8.

[0080] In some embodiments, the volume of target Mal added dropwise in step S1 is 10 μl, and the incubation time is 2-3 h.

[0081] In some embodiments, a three-electrode system is used, with a modified glassy carbon electrode (3 mm) as the working electrode, a platinum electrode (10*10*0.1) as the counter electrode, and a saturated Ag / AgCl electrode as the reference electrode; the photomultiplier tube PMT is set to 1200 V, the scanning voltage setting range is -2.3 to 0 V, and the scanning rate is 100 mV / s; the photomultiplier tube PMT is set to 1200 V, the voltage setting range is -2.3 to 0 V, and the scanning rate is 100 mV / s.

[0082] The present invention is further described below through specific examples.

[0083] Raw material preparation:

[0084] Example 1

[0085] A covalent organic framework@quantum dot composite material comprises 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.

[0086] The preparation method of the covalent organic framework@quantum dot composite material comprises the following steps:

[0087] Boric acid (0.495 g), melamine (0.17 g) and thiourea (0.36 g) were dissolved in 30-60 mL of ultrapure water, and the mixture was transferred to a sealed stainless steel autoclave for hydrothermal reaction at 200 ° C for 24 h. Subsequently, the dispersion was filtered using a 0.22 μm organic filter to obtain the S-BNQDs filtrate; the S-BNQDs filtrate was collected and stored in a refrigerator at 0-8 ° C. The prepared S-BNQDs were characterized by transmission electron microscopy (TEM). Figure 2 In a, it can be observed that the prepared QDs have a small particle size (≤20 nm) and are relatively evenly dispersed, indicating that S-BNQDs were successfully prepared.

[0088] Dissolve equimolar amounts of cyanuric acid (0.51 g) and melamine (0.50 g) in 30 mL of dimethyl sulfoxide (DMSO) and stir vigorously. Add 30 mL of the S-BNQD solution prepared in Step 1 and stir vigorously at 25°C for 72 hours. Vacuum filter the mixture using a 0.22 μm organic filter membrane to obtain a white solid product, which is then washed with anhydrous ethanol. Finally, dry the solid in a vacuum oven at 55°C for 24 hours and grind it for later use. This yields the S-BNQD-MCA composite, a covalent organic framework-quantum dot composite.

[0089] Example 2

[0090] A method for preparing an ECL-RET aptamer biosensor comprises the following steps:

[0091] S1. The luminescent material S-BNQD-MCA (5 μL, 1 mg / L) prepared in Example 1 was coated on the surface of the pretreated glassy carbon electrode and dried under an infrared lamp to obtain an S-BNQD-MCA working electrode;

[0092] S2. A Mal-Apt aptamer solution (5 μL, 2.5 μmol / L) was added dropwise to the surface of the S-BNQD-MCA modified electrode and incubated for 12 h. The electrode was gently rinsed with PBS buffer and then dropwise added with 5 μL of 0.5% BSA. After incubation for 1 h, the electrode was rinsed with PBS buffer to obtain the S-BNQD-MCA / Mal-Apt / BSA working electrode.

[0093] S3. The quenching probe cDNA-Au@NH2-UiO-66 was added dropwise onto the surface of the S-BNQD-MCA / Mal-Apt / BSA working electrode. After incubation for 3 h, the electrode was rinsed with PBS buffer to obtain the ECL-RET aptamer biosensor for Mal detection.

[0094] The preparation of the double quencher Au@NH2-UIO-66 includes the following steps:

[0095] (1) AuNPs: Add 3 mL of 1% HAuCl4·3H2O to 100 mL of ultrapure water, stir well, and cool 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 microscopy (TEM) image of the prepared AuNPs. The prepared AuNPs are spherical and have good monodispersity and crystallinity.

[0096] (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. Place the mixture in a stainless steel autoclave and hydrothermally react at 120°C for 24 h. Collect the solid product by filtration using a 0.22 μm organic filter membrane, wash with DMF and methanol, and finally dry it in a vacuum at 55°C for 12 h.

[0097] (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 to collect the product Au@UiO-66-NH2 by suction filtration and wash with ultrapure water. Finally, the collected product was dried at 55 °C under vacuum for 24 h, ground, weighed, and dispersed in ultrapure water to prepare a 1 mg / mL dispersion. Figure 4 (b, c) are TEM images of the Au@NH2-UIO-66 composite material. UIO-66-NH2 presents an octahedral topological structure, and AuNPs are evenly distributed on the surface of UIO-66-NH2, while 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.

[0098] cDNA-Au@NH2-UiO-66: Mix 10 μM cDNA with 10 mM TCEP and incubate on a shaker at room temperature for 1 hour. Then dilute to a 2 μM concentration with 10 mM PBS buffer. Mix 500 μL of 1 mg / mL Au@NH2-UIO-66 with 500 μL of cDNA and incubate at room temperature with shaking for 12 hours. Centrifuge at 8000 rpm for 10 minutes to remove unbound cDNA. Wash three times with PBS buffer (pH 7.4). Finally, disperse the pellet in 500 μL of PBS buffer containing 0.5% w / v BSA.

[0099] Mal-Apt: Take 100 μL of Mal-Apt solution (10 μM) with modified carboxyl group at the 5' end, add 30 μL of EDC solution (10 mM) and 10 μL of NHS solution (10 mM), and shake at 25°C for 30 min.

[0100] Example 3

[0101] A method for detecting Mal based on an ECL-RET aptamer biosensor comprises the following steps:

[0102] K1. 10 μl of target Mal at different concentrations were added dropwise to the surface of the ECL-RET aptamer biosensor electrode constructed in Example 2 and incubated at 25°C for 2 h to obtain a sensor after target incubation;

[0103] K2. After incubation with the target, the sensor was placed in a 0.01M phosphate buffer solution containing 0.1M K2S2O8 to measure its ECL intensity. A three-electrode system was used, with a modified glassy carbon electrode (3mm) as the working electrode, a platinum electrode (10*10*0.1) as the counter electrode, and a saturated Ag / AgCl electrode as the reference electrode. The photomultiplier tube (PMT) was set to 1200V, the scan voltage range was -2.3~0V, and the scan rate was 100mV / s. The ECL intensity of the target Mal at different concentrations was detected. The obtained ECL intensity (y ECL ) and the logarithmic value of Mal concentration (lgc), and draw a calibration curve. Figure 8 As 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;

[0104] K3. Detect the ECL intensity of the Mal to be tested and calculate the concentration of the Mal to be tested based on the standard curve.

[0105] Comparative Example 1

[0106] A covalent organic framework@quantum dot composite material, the other contents are the same as those in Example 1, except that the room temperature condition is changed to 80°C.

[0107] Comparative Example 2

[0108] A covalent organic framework@quantum dot composite material, the other contents are the same as those in Example 1, except that vacuum drying is replaced by forced air drying.

[0109] Comparative Example 3

[0110] A covalent organic framework material, the preparation method of which is:

[0111] Equimolar amounts of cyanuric acid (0.51 g) and melamine (0.50 g) were dissolved in 20 mL and 10 mL of dimethyl sulfoxide, respectively. The mixture was stirred vigorously at 25°C for 72 hours to form a homogeneous white solution. Vacuum filtration using a 0.22 μm organic filter membrane yielded a white solid, which was then washed with anhydrous ethanol and ultrapure water. Finally, the solid was dried in a forced air drying oven at 55°C for 12 hours to obtain MCA, which was then ground and used for further processing.

[0112] Comparative Example 4

[0113] A covalent organic framework material, the other contents are the same as those of Comparative Example 3, except that it is vigorously stirred at 80° C. for 72 hours.

[0114] Comparative Example 5

[0115] A covalent organic framework material, the other contents are the same as those of comparative example 3, except that the forced air drying is replaced by vacuum drying.

[0116] Comparative Example 6

[0117] A covalent organic framework material, the other contents are the same as those of comparative example 3, except that it is vigorously stirred at 80° C. for 72 hours and is dried in vacuo instead of being dried with forced air.

[0118] Testing and Evaluation

[0119] The material morphology of Comparative Example 3 and Comparative Example 4 and the specific surface area of ​​Comparative Example 5 and Comparative Example 6 were tested. Figure 1 As shown in Figure 2, MCA prepared under different temperature conditions showed significant morphological differences. Figure 3 As can be seen in a, the BET specific surface area of ​​MCA prepared at room temperature at 25℃ is as high as 204.14m² / g, while the specific surface area of ​​the system prepared at 80℃ is sharply reduced to 104.26m² / g. Figure 2 Compared with the vacuum-dried sample in b, the surface structure of the MCA sample without vacuum drying treatment changed significantly ( Figure 1), specifically manifested as edge curling of the two-dimensional sheet-like MCA sample, which confirms that the vacuum degree of the drying environment has an important regulatory effect on the final morphology of the material.

[0120] The S-BNQD-MCA composite material of Example 1 was observed by SEM-EDS element surface scanning ( Figure 2 cf) 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 and S elements can be clearly detected in the composite material. Figure 3 As shown in a, in Example 1, the BET specific surface area of ​​MCA (Comparative Example 5) after loading S-BNQDs decreased to 121.93m² / g, a decrease of 40.3%. The pore size distribution was calculated by the BJH model and found that ( Figure 3 b), its average pore diameter increased from 5.99nm to 7.14nm, which is a synergistic evidence of the decreasing trend of specific surface area. At the same time, by comparing Example 1 and Comparative Example 1, except that the preparation temperature was changed to 80°C, the others remained unchanged, and S-BNQD-MCA was prepared. The specific surface area of ​​the system prepared at 80°C dropped sharply to 72.32m² / g, and the average pore diameter was reduced from 5.65nm to 5.39nm. The above comparison shows that room temperature synthesis conditions are more conducive to the construction of high specific surface area mesoporous structures, while the high temperature reflux process may cause excessive shrinkage of the framework and collapse of the pores. The room temperature synthesis strategy achieves high-density and uniform encapsulation of S-BNQDs in the covalent organic framework MCA by optimizing the quantum dot-framework interface. As Figure 7 As shown in Figure c, in Example 1, MCA was used as a carrier of S-BNQDs, and the ECL intensity of the modified electrode was greatly enhanced, indicating that MCA can significantly enhance the ECL intensity of S-BNQDs. This may be because MCA makes S-BNQDs more dispersed, thereby weakening the self-quenching effect and effectively increasing the ECL intensity. Using MCA as a carrier of S-BNQDs has a positive effect.

[0121] To evaluate whether Mal-Apt and cDNA can effectively bind, and whether cDNA and Au@NH2-UIO-66 can be successfully coupled, agarose gel electrophoresis was used to verify the results. Figure 5Compared to Mal-Apt (lane a) and cDNA (lane b), a new band appeared (lane c), indicating the stable formation of Mal-Apt@cDNA. In lane e, the band was observed to maintain the same position as in lane a, indicating that the activators EDC and NHS had no effect on the structure of the aptamer Mal-Apt. Lane d shows the coupling of cDNA and Au@NH2-UIO-66. Compared to lane b, a band with a larger molecular weight can be observed. This is 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 can be used for sensor assembly.

[0122] In Example 2, 5.0 mM Fe[(CN)6] 3- / 4- As an electroactive probe, the EIS curves of different modified electrodes were tested to prove the successful construction of ECL aptamer sensor. Figure 6 Among them, 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) were prepared in the presence of 5 mM [Fe(CN)6] 3− / 4− Electrochemical impedance spectroscopy (EIS) curves in 0.01 M PBS buffer (used as a redox probe) and 0.1 M KCl were recorded over a frequency range of 0.1 Hz to 100 kHz with an amplitude of 5 mV. Modification of the GCE (glassy carbon electrode) with the S-BNQD-MCA nanocomposite reduced the Rct value to 422 Ω. Because the negative surface charge of the DNA strands prevents free electron movement, the addition of Mal-Apt resulted in an increase in the semicircular diameter (Rct = 1482 Ω). To block excess binding sites on the modified electrode surface, BSA was added dropwise to the GCE / S-BNQD-MCA / Mal-Apt surface. BSA, a non-conductive biomacromolecule, hinders electron transfer at the electrode surface, resulting in a further increase in the electrode Rct (Rct = 2293 Ω). After assembling cDNA-Au@NH2-UIO-66 on the electrode surface, Rct decreased to 1917Ω due to the relatively good conductivity of cDNA-Au@NH2-UIO-66, indicating the successful construction of the ECL aptamer biosensor.

[0123] Meanwhile, the ECL reaction mechanism of the S-BNQD-MCA / K2S2O8 system in Example 2 was investigated. 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 S-BNQD-MCA modified electrode in 0.01 M PBS solution and 0.1 M K2S2O8 in 0.01 M PBS solution, and 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. Figure 7 b, When measuring GCE / S-BNQD-MCA in PBS, the ECL signal obtained is extremely low. After adding K2S2O8 to PBS, there is no significant difference in the ECL intensity of bare GCE and GCE / S-BNQD-MCA in PBS solution. 2- When measuring GCE / S-BNQD-MCA, the ECL intensity increased to 95975a.u. This is because the co-reactant K2S2O8 participated in the reaction and promoted the ECL emission of S-BNQD-MCA. At the same time, the CV curves of bare GCE and GCE / S-BNQD-MCA in the range of -2.3~0V were obtained ( Figure 7 a). When measuring GCE / S-BNQD-MCA in PBS, there is no obvious reduction peak. In addition, GCE / S-BNQD-MCA in PBS-S2O8 2- The CV curves in PBS-S2O8 showed a significantly stronger reduction peak current (1.08 mA, -1.44 V) and were significantly higher than those in the bare GCE. 2- Compared with the medium phase (0.7985 mA, -1.52 V), the peak potential shifted negatively, indicating that K2S2O8 was 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. Compared with bare GCE, GCE / MCA and GCE / S-BNQD have higher ECL intensity. When S-BNQD-MCA is used to modify the electrode, the ECL intensity is greatly enhanced, 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 a carrier of S-BN QDs has positive significance. At the same time, GCE / S-BNQD-MCA and GCE / S-BNQD-MCA / Au@NH2-UIO-66 were prepared by conventional means, and the ECL intensity of the electrode was greatly enhanced in PBS-S2O8. 2- The ECL curves in Figure 2 show that GCE / S-BNQD-MCA / Au@NH2-UIO-66 obtained an extremely low ECL signal ( Figure 7 d). Therefore, efficient ECL-RET can occur between Au@NH2-UIO-66 and the luminophore.

[0124] The stability, specificity and reproducibility of the sensor constructed in Example 2 were investigated:

[0125] Sensor stability test: The prepared sensor was used to measure the ECL emission of 11 consecutive CV scans. The results are shown in Figure 2. Figure 9 As shown, Figure 9 Figure 3. ECL emission stability of the constructed ECL aptasensor after 11 consecutive CV scans in 0.01 M PBS solution containing 0.1 M K₂S₂O₃. The measured signal showed good stability with a relative standard deviation (RSD) of 0.99% over 11 ECL test cycles.

[0126] Sensor specificity test: some pesticides (carbendazim, acetamiprid, diazinon) were selected as interference factors. Figure 10 The selectivity of the prepared ECL aptamer sensor to Mal and its interferents: blank control, 10pg / mL carbendazim, 10pg / mL acetamiprid, 10pg / mL diazinon, and a mixture containing 100fg / mL malathion and 10pg / mL interferents can be obtained from Figure 10 As can be seen in the figure, the ECL signal of the interfering sample is basically the same as that of the blank sample, and the ECL intensity is lower than that of Mal. At the same time, the ECL signal of Mal is close to that of the mixture (100 fg / mL malathion + 10 pg / mL interfering compound). These results demonstrate that the constructed sensor can perform specific Mal detection.

[0127] Sensor reproducibility test: Six different sensors were prepared and the target Mal was measured at 10 pg / mL. The ECL signal intensity was as follows: Figure 11As shown, the RSD is 1.53%, indicating that the sensor has good reproducibility.

[0128] The above are only preferred specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A covalent organic framework@quantum dot composite material, characterized in that: The invention comprises 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; the preparation method of the covalent organic framework@quantum dot composite material comprises the following steps: dissolving cyanuric acid and melamine in a solvent, adding an S-BNQDs quantum dot solution, stirring and reacting, filtering the mixture after the stirring reaction, and then washing and vacuum drying; the stirring reaction temperature is 20-25°C, the stirring reaction time is 60-72h, and the molar ratio of cyanuric acid to melamine is 1:1-1.

2.

2. The covalent organic framework@quantum dot composite material according to claim 1, characterized in that The solvent is dimethyl sulfoxide; the volume ratio of the dimethyl sulfoxide to the S-BNQDs solution is 1-2:

1.

3. An ECL-RET aptamer biosensor comprising the covalent organic framework@quantum dot composite material according to any one of claims 1 to 2.

4. A method for preparing the ECL-RET aptamer biosensor according to claim 3, characterized in that: The following steps are involved: S1. Coating the covalent organic framework@quantum dot composite material on the pretreated glassy carbon electrode surface and drying under infrared light to obtain an S-BNQD-MCA working electrode; S2. The Mal-Apt aptamer solution was added dropwise to the surface of the S-BNQD-MCA working electrode, incubated and washed, and then BSA blocking solution was added dropwise, followed by incubation and washing again to obtain the S-BNQD-MCA / Mal-Apt / BSA working electrode; S3. The quenching probe cDNA-Au@NH2-UiO-66 was added dropwise to the surface of the S-BNQD-MCA / Mal-Apt / BSA working electrode, and after incubation and washing, the ECL-RET aptamer biosensor was obtained; The pretreatment method of the Mal-Apt aptamer is as follows: Adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to the Mal-Apt solution modified with the 5'-terminal carboxyl group, and shaking the mixture at room temperature to obtain the Mal-Apt aptamer; The sequence of the 5′-terminal 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′.

5. The method for preparing the ECL-RET aptamer biosensor according to claim 4, wherein: 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 to obtain a mixed solution, mixing Au@NH2-UiO-66 with the mixed solution, and incubating with shaking at room temperature to obtain the quenched 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′.

6. A method for detecting Mal based on the ECL-RET aptamer biosensor according to claim 3, characterized in that: The following steps are involved: K1. Adding different concentrations of target Mal to the ECL-RET aptamer biosensor, incubating, and obtaining a sensor after incubation of the target; K2. The sensor after incubation of the target was placed in an ECL detection solution, with a modified glassy carbon electrode as the working electrode, a platinum electrode as the counter electrode, and a saturated Ag / AgCl electrode as the reference electrode. The ECL intensity of the target Mal at different concentrations was detected, and a standard curve was plotted; K3. Detect the ECL intensity of the Mal to be tested and calculate the concentration of the Mal to be tested based on the standard curve.

Images