Based on Ag + @Eu MOF / HOF resonance energy transfer electrochemiluminescence aptamer sensor and its application in acetamiprid detection

Abstract

The present invention belongs to electrochemiluminescent aptamer sensor and relates to an electrochemical luminescent aptamer sensor based on Ag. + @Eu MOF / HOF resonance energy transfer electrochemiluminescence aptamer sensor and its application in acetamiprid detection. Using rod-shaped hydrogen bonded organic framework as precursor, doped with lanthanide element Eu 3+ Using Ag + As a co-reactant promoter, the synthesis of Ag + @Eu MOF / HOF was used as the energy donor of the resonance energy transfer system, and leaf-shaped CdS@Au-cDNA was used as the energy acceptor to prepare the electrochemiluminescent aptamer sensor for resonance energy transfer. The sensor was used for the detection of ACE, and it had a wide linear range and a low detection limit. + @Eu MOF / HOF electrochemiluminescent material was first used in electrochemiluminescence detection to detect the residues of acetamiprid in water, opening up an effective and convenient strategy for the analysis and detection of pollutants in water environments.

Description

Technical Field

[0001] The present invention relates to the field of electrochemiluminescent aptamer sensors, and specifically involves a luminescent material Ag + @EuMOF / HOF is used as the luminescent body, and the resonance energy transfer method is used to realize signal conversion. The leaf-shaped CdS@Au-cDNA is used as the resonance energy transfer acceptor, and the quantitative analysis of acetamiprid is realized according to the regular ECL signal changes. Background Art

[0002] The monitoring and prevention of pesticide residues in water bodies have always been a research focus in the field of environmental chemistry. Acetamiprid (ACE) is a systemic contact insecticide that is widely used to control piercing-sucking pests as a substitute for traditional insecticides such as organophosphates. Its frequent and widespread use inevitably leads to the accumulation of ACE in soil and water environments, endangering people's health. Therefore, it is very necessary to find an efficient, sensitive, simple, convenient and low-cost ACE detection method. Existing methods for detecting acetamiprid residues include Raman spectroscopy, fluorescence and electrochemical methods. Although these methods meet the detection requirements, they involve expensive equipment, complex operating procedures and long detection times. Therefore, establishing a sensitive, simple and reliable acetamiprid rapid detection method is crucial to protecting human health.

[0003] Electrochemiluminescence combines the advantages of electrochemistry and chemiluminescence, and has the characteristics of low background interference, fast response speed and high sensitivity. Traditional electrochemiluminescent materials such as Ru(bpy)3 2+ , luminol, and quantum dots. However, these traditional luminophores require complex synthesis steps, have high water solubility resulting in unstable ECL efficiency, or exhibit relatively low ECL emission efficiency due to π-π stacking, which seriously restricts their practical application. Therefore, developing simple methods to prepare high-performance ECL luminophores without causing quenching due to molecular aggregation remains of great significance and challenge.

[0004] HOF materials are self-assembled through organic unit ligands due to hydrogen bonding interactions under the combined effects of van der Waals forces, π-π stacking interactions, and electrostatic interactions. In addition to the common advantages of tunable structures, high surface areas, and customized pore sizes similar to other ordered frameworks such as metal-organic frameworks (MOFs) and covalent organic frameworks (COFs), HOF materials can be constructed under milder conditions and have reversible and flexible hydrogen bonds. More importantly, the rich functional sites in HOF materials and the large number of active sites exposed from π-π conjugated linkers can be further assembled with metal ions to synthesize functional materials for various fields. However, currently only a small number of HOF materials are used as electrochemiluminescent materials for analytical detection.

[0005] Lanthanide metal-organic framework materials (Ln-MOFs) have the advantages of long luminescence lifetime, strong antenna effect, high specific surface area and active sites, and are an ideal type of ECL probe material. Among them, Eu-MOFs have attracted much attention due to their excellent luminescence properties. However, it is still difficult to obtain a high ECL signal solely by relying on the stepped electronic energy levels and 4f-4f transitions of lanthanide elements. In the electrochemiluminescence system, one of the strategies used to amplify ECL is to promote the electroreduction decomposition of co-reactants (such as K2S2O8) on the electrode surface. Co-reaction promoters can interact with co-reactants instead of luminophores to produce more reactive intermediate states, which will increase the oxygen reduction kinetics (ERR) of the luminophore and co-reactant, thereby generating a large number of excited state luminophores and emitting a strong ECL signal. Ag + It has excellent catalytic performance of K2S2O8 activation, Ag + It can promote K2S2O8 to generate more SO4·-, and ultimately amplify the ECL signal of the system.

[0006] The present invention uses lanthanide metal ions as the core to synthesize lanthanide-functionalized HOF materials. The lanthanide ion-induced partial conversion from HOF to MOF is achieved by a solvothermal method, and a Eu MOF / HOF composite material with satisfactory ECL performance is successfully prepared. Ag with catalytic properties is used. + , which not only increases the electrochemical active area of ​​HOF materials, but also acts as a co-reactant promoter to amplify the ECL signal of Eu MOF / HOF. The aptamer of ACE is modified on Ag by π-π conjugation. + @Eu MOF / HOF, used for specific recognition of ACE. CdS@Au-cDNA serves as the energy acceptor of the resonance energy transfer system. CdS@Au-cDNA is modified on the electrode surface through hybridization between cDNA and apt. CdS@Au-cDNA quenches Ag + The ECL signal of @EuMOF / HOF changes. When the sensor is used for ACE detection, due to the higher specificity of apt for ACE, the connection between apt and cDNA is disconnected, the CdS@Au-cDNA falls off the electrode, and the resonance energy transfer effect disappears, resulting in an increase in the ECL signal. The electrochemiluminescent aptamer sensor developed in this invention can sensitively detect ACE, demonstrating high selectivity and stability. Summary of the Invention

[0007] The first purpose of the present invention is to synthesize an electrochemiluminescent body of MOF / HOF material, with lanthanide metal ions as the center, and to realize the partial conversion of HOF to MOF induced by lanthanide ions by a solvothermal method, using Ag with catalytic properties.+ , which not only increases the electrochemical active area of ​​HOF materials, but also acts as a co-reactant promoter to amplify the ECL signal. + @Eu MOF / HOF exhibits satisfactory ECL performance.

[0008] The second purpose of this invention is to synthesize CdS@Au-cDNA as an energy acceptor for resonance energy transfer. + @EuMOF / HOF acts as an energy donor for resonance energy transfer. CdS@Au-cDNA can quench Ag + ECL signal of @Eu MOF / HOF. The unique leaf structure of CdS provides abundant loading sites for Au NPs, and the two are stably bonded through electrostatic adsorption.

[0009] The third objective of the present invention is to prepare an electrochemiluminescent aptamer sensor for detecting acetamiprid. The use of the aptamer improves the specificity of detection, can identify acetamiprid in complex environments, and achieve trace detection.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0011] 1. The present invention describes the synthesis of a HOF electrochemiluminescent material (Ag + @Eu MOF / HOF) is used as the energy donor in the resonance energy transfer system, and its preparation steps are as follows:

[0012] (1) Preparation of HOF

[0013] Weigh melamine into deionized water. Adjust the molar ratio of melamine to trimesic acid to between 1:1 and 3:1. Heat to 55-95°C with stirring until completely dissolved. Then, mix the melamine and trimesic acid solutions and stir at room temperature for 3 hours. Centrifuge at 12,000 rpm for 10 minutes to collect the crude product, which is then washed three times with deionized water. Finally, dry the white powder in a vacuum at 660°C for 10 hours.

[0014] (2) Ag + Preparation of @Eu MOF / HOF

[0015] Weigh HOF and Eu(NO3)3·6H2O in deionized water. The mass ratio of HOF and Eu(NO3)3·6H2O is 1:1~3:1. According to the molar ratio of Eu(NO3)3·6H2O and AgNO3 of 1:1~5:1, weigh the corresponding AgNO3 and add it to the above solution. After stirring evenly, add DMF and transfer to a high-pressure reactor at 60℃~100℃ for 2h. Centrifuge at 8000rpm for 10min and wash three times with deionized water. The obtained product is vacuum dried at 60℃ for 10h to obtain a white solid powder. Then the obtained Ag + @Eu MOF / HOF was dispersed in deionized water to obtain a dispersion of 1-5 mg / mL and stored at 4°C until use.

[0016] Furthermore, in step (1), by controlling the temperature of the HOF material synthesis, the dissolution rate of the raw material melamine can be controlled. When the temperature is too low, melamine cannot dissolve and participate in the coordination bond formation. When the temperature rises, the dissolution rate of melamine accelerates, and the rate of coordination with trimesic acid to form HOF accelerates, promoting the formation of HOF. When the temperature is too high, due to the weak thermal stability of the HOF material, the high temperature will destroy the stability of the HOF material. In addition, the high temperature will cause the HOF material to form too quickly, and the rod-shaped HOF material will transform into a fiber-shaped material, which is not conducive to the further formation of MOF material on its surface. Therefore, controlling the temperature between 55 and 95°C can produce an ideal rod-shaped HOF material. The surface of the rod-shaped HOF material has abundant active sites, and a large number of -NH2 and -COOH groups are distributed on its surface. These groups can coordinate with the lanthanide metal Eu, and ultimately generate Eu-MOF materials with excellent performance on the surface of the HOF material. Compared with traditional Eu MOF materials, traditional Eu MOF materials usually require high temperature and high pressure environment for synthesis, while HOF material precursor is first synthesized and then Eu is introduced. 3+ , Eu MOF materials can be generated under mild conditions. In addition, rod-shaped HOFs can provide a large number of active groups for Eu coordination, which greatly improves the coordination efficiency of Eu.

[0017] Furthermore, in step (2), Ag is added + When Ag + The amount of Ag + It has a positive effect on the ECL performance of Eu MOF / HOF materials. + When the concentration is too low, a small amount of Ag + It cannot significantly promote the Eu MOF / HOF. + When the concentration is too high, excess Ag +It will participate in the bonding of Eu MOF / HOF in large quantities, destroy the stable structure of Eu MOF / HOF, reduce the ECL efficiency of the central atom Eu, and reduce the ECL intensity. Therefore, the molar ratio of Eu(NO3)3·6H2O and AgNO3 is controlled to be 1:1~5:1.

[0018] 2. The synthetic leaf-shaped CdS@Au-cDNA described in the present invention is used as the energy acceptor in the resonance energy transfer system, and its preparation steps are as follows:

[0019] (1) Preparation of AuNPs

[0020] A 0.01 wt.% HAuCl₄ solution was added to a round-bottom flask and heated to boiling. A 1% (w / v) sodium citrate solution was then added dropwise to the HAuCl₄ solution, maintaining a volume ratio of HAuCl₄ to sodium citrate solution between 100:1 and 100:10. The solution was heated under reflux for 30 minutes, until the solution turned wine red. The resulting solution containing AuNPs was cooled to room temperature and stored at 4°C until further use. Furthermore, AuNPs were found to be stable in the reducing solution containing sodium citrate, allowing for further centrifugation and purification in subsequent experiments.

[0021] (2) Preparation of CdS

[0022] First, CH₄N₂S (thiourea) was dissolved in deionized water. CdCl₂·2.5H₂O was then added at a molar ratio of CH₄N₂S to CdCl₂·2.5H₂O of 1:1 to 7:1. The mixture was stirred at room temperature for 10 minutes to achieve uniform mixing. DMSO was then added dropwise to the solution, followed by stirring for 10 minutes. Finally, the solution was transferred to a stainless steel autoclave and reacted at 170°C for 8–16 hours. After the autoclave cooled to room temperature, the orange precipitate at the bottom was washed several times with anhydrous ethanol and deionized water and dried at 60°C to obtain an orange-yellow CdS powder.

[0023] (3) Preparation of CdS@Au-cDNA

[0024] CdS was added to ultrapure water, and the AuNPs solution was added dropwise to the mixture, stirring continuously for 6 hours. The mixture was then centrifuged, washed, and dried under vacuum at 60°C to obtain CdS@Au. The molar ratio of CdS to AuNPs was controlled to be between 10:1 and 20:1 during the preparation of CdS@Au-cDNA.

[0025] cDNA (3 μM) was added to the CdS@Au dispersion and stirred in an ice-water bath for 10 h. The product was centrifuged at 12,000 rpm for 15 min and washed three times with deionized water. The resulting precipitate was dispersed in deionized water to obtain a CdS@Au-cDNA dispersion with a concentration of 1 to 5 mg / mL.

[0026] Furthermore, the amount of added Au NPs was controlled to obtain CdS@Au-cDNA with the best RET effect. This is because too few Au NPs cannot ensure that the leaf-shaped CdS surface is loaded with enough AuNPs to bind to cDNA. If the number of nanoparticles is increased, too many Au NPs will aggregate on the CdS surface, hindering the adsorption of cDNA molecules on the nanoparticles, thereby limiting the ability of cDNA to base pair with apt. The ratio of nanoparticles to CdS is maintained in the above range to form a monolayer of nanoparticles on the surface and to have sufficient adsorption sites to coordinate with the amino groups (-NH2) on the cDNA.

[0027] 3. The preparation steps of the resonance energy transfer system electrochemiluminescence aptamer sensor described in the present invention are as follows:

[0028] (1) The glassy carbon electrode (GCE) was polished and then thoroughly cleaned using dilute nitric acid, anhydrous ethanol, and deionized water for 3 minutes each.

[0029] (2) Take 5 μL of 0.5-5 mg / mL Ag + @Eu MOF / HOF was added dropwise onto GCE and dried under infrared light to obtain Ag + @EuMOF / HOF / GCE;

[0030] (3) Add 5 μL of apt with a concentration of 1 to 6 μM to the surface of the modified electrode and incubate at 37°C for 6 h to obtain apt / Ag. + @Eu MOF / HOF / GCE;

[0031] (4) Add 5 μL of 0.5% bovine serum albumin (BSA) to block the nonspecific binding sites on the electrode surface, and dry naturally to obtain / BSA / apt / Ag + @Eu MOF / HOF / GCE;

[0032] (5) Add 1-5 μL of CdS@Au-cDNA dispersion to the electrode surface and allow it to fully bind to apt at 37°C for 2-8 h to obtain CdS@Au-cDNA / BSA / apt / Ag + @Eu MOF / HOF / GCE electrochemiluminescence sensor. Ag in steps (2) to (5) +The volume ratio of @Eu MOF / HOF, apt, bovine serum albumin, and CdS@Au-cDNA dispersion modification solution is 1~5:1~5:1~5:1~5.

[0033] 4. The electrochemiluminescent aptamer sensor prepared according to the present invention, wherein the aptamer sequence of acetamiprid is: 5'-(NH2 C6)-TGT AAT TTG TCT GCA GCG GTT CTT GAT CGC TGA CAC CATATTATG AAG A-3'. The cDNA sequence is: 5'-(NH2 C6)-TCT TCA TAA TAT GG-3'

[0034] 5. The present invention provides an electrochemiluminescent aptamer sensor for sensitive detection of acetamiprid, characterized in that the specific detection steps are as follows:

[0035] A1. Preparation of standard solutions containing different concentrations of acetamiprid:

[0036] The solid standard of acetamiprid was prepared with deionized water to a concentration of 1.0 × 10 -3 mol / L solution and diluted into a series of acetamiprid standard solutions with different concentrations, ranging from 1.0×10 -3 ~1.0×10 -17 mol / L;

[0037] A2. Drawing of standard curve:

[0038] A three-electrode system was used, with the prepared electrochemiluminescent aptamer sensor as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. + The @Eu MOF / HOF / GCE electrochemiluminescent aptamer sensor was immersed in a series of acetamiprid standard solutions of varying concentrations prepared in step A1 for 10–50 min (preferably 40 min). The electrode was removed, rinsed with PBS buffer, and allowed to air dry. ECL analysis was then performed in 0.1 M K₂S₂O₄-containing PBS buffer (0.1 M, pH 6.0–9.0). A linear relationship between acetamiprid concentration and the change in electrochemiluminescence intensity (ΔECL) was established, and the corresponding linear regression equation was obtained. The aptamer sequence for acetamiprid is: 5'-(NH₂ C₆)-TGTAATTTG TCT GCAGCG GTT CTT GAT CGC TGACAC CAT ATT ATG AAG A-3'. The cDNA sequence is: 5'-(NH₂ C₆)-TCT TCA TAA TAT GG-3'.

[0039] A3. Actual sample testing:

[0040] The standard addition method was used to perform ECL testing on acetamiprid in actual samples. The obtained ECL intensity was calculated using the linear regression equation obtained by A2 to obtain the acetamiprid concentration in the sample.

[0041] Beneficial results of the present invention:

[0042] (1) The present invention synthesizes a rod-shaped HOF material by temperature control. The HOF material of this morphology has a large number of active sites, which is conducive to further assembly with metal ions on its surface to synthesize functional materials.

[0043] (2) An electrochemiluminescent material (Ag + @Eu MOF / HOF), a solvothermal method was used to achieve the partial conversion of HOF to MOF induced by lanthanide ions, and Ag + As a co-reactant promoter, it amplifies the ECL signal and broadens the application scope of HOF materials in the field of electrochemiluminescence.

[0044] (3) The present invention adopts a resonance energy transfer strategy to obtain an "off-on-off" ECL signal. This strategy has the significant advantages of high sensitivity and low background noise, overcoming the weakness of false positives caused by single signal changes.

[0045] (4) Leaf-shaped CdS@Au-cDNA was synthesized as a resonance energy transfer acceptor, where the UV-vis spectrum of CdS@Au-cDNA was similar to that of the donor (Ag + The ECL spectra of CdS@Au and cDNA are well overlapped. CdS@Au is linked to cDNA through Au-N bonds. cDNA can specifically recognize specific DNA sequence fragments on the acetamiprid aptamer, improving the specificity of the sensor and the detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0047] Figure 1 Based on Ag + Schematic diagram of the electrochemiluminescence aptamer resonance energy transfer sensor based on @Eu MOF / HOF for the detection of acetamiprid (A: preparation diagram of AuNPs; B: preparation diagram of CdS@Au; C: Ag + @Eu MOF / HOF preparation diagram; D: Simple schematic diagram of sensor preparation).

[0048] Figure 2 : HOF (A), Eu MOF / HOF (B), Ag obtained at 80℃ + @Eu MOF / HOF (C) is the SEM image; (D) is the SEM image of HOF obtained under 110℃ conditions.

[0049] Figure 3 It is HOF(A), Eu MOF / HOF(B), Ag + @Eu MOF / HOF(C) ECL-time curve.

[0050] Figure 4 Ag was tested in PBS at different pH values. + @Eu ECL intensity of MOF / HOF.

[0051] Figure 5 are SEM images of CdS (A) and CdS@Au (B).

[0052] Figure 6 ECL-time curves of different working electrodes (a: GCE, b: Ag + @Eu MOF / HOF / GCE, c:apt / Ag + @Eu MOF / HOF / GCE,d:BSA / apt / Ag + @Eu MOF / HOF / GCE, e: CdS@Au-cDNA / BSA / apt / Ag + @Eu MOF / HOF / GCE, f: ACE / CdS @Au-cDNA / BSA / apt / Ag + @Eu MOF / HOF / GCE).

[0053] Figure 7 The linear curves of different concentrations of acetamiprid detected by the prepared electrochemiluminescent aptamer sensor are shown;

[0054] in Figure 7 A is the ECL-time curve of different concentrations of acetamiprid; Figure 7 B is the standard curve of ΔECL and the logarithm of acetamiprid concentration. DETAILED DESCRIPTION

[0055] The present invention will be further described below with reference to specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0056] Example 1

[0057] 1. A method for preparing an electrochemiluminescent body of HOF material, comprising the following steps:

[0058] (1) Preparation of HOF

[0059] Dissolve 0.5 mmol of melamine and 0.5 mmol of trimesic acid in 50 mL of deionized water, stirring and heating to 80°C until completely dissolved. Then, combine the melamine and trimesic acid solutions and stir at room temperature for 1 hour. Centrifuge at 12,000 rpm for 1 minute to collect the crude product, which is then washed three times with deionized water. Finally, dry the white powder under vacuum at 60°C for 12 hours.

[0060] (2) Ag + Preparation of @Eu MOF / HOF

[0061] 25 mg of HOF, 0.04 mmol of Eu(NO3)3·6H2O, and 0.01 mmol of AgNO3 were weighed and dissolved in 10 mL of deionized water. After stirring evenly, 2 mL of DMF was added and the mixture was transferred to a 25 mL autoclave and reacted at 80°C for 2 h. Centrifuged at 8000 rpm for 10 min, the mixture was washed three times with deionized water, and the obtained product was vacuum dried at 60°C for 10 h to obtain a white solid powder. + @Eu MOF / HOF was dispersed in deionized water to obtain a 2 mg / mL dispersion and stored at 4°C until use.

[0062] 2. Prepared Ag + @Eu MOF / HOF for ECL detection

[0063] (1) Preparation of electrochemiluminescence sensor

[0064] 1) A 3 mm diameter glassy carbon electrode was polished using 0.3 μm and 0.05 μm Al2O3 powders, respectively. The surface was then ultrasonically cleaned for 3 min using 1% dilute nitric acid and deionized water, respectively. Finally, the surface was dried using N2.

[0065] 2) Add 5 μL of 2 mg / mL Ag + @Eu MOF / HOF was drop-coated on the GCE surface and dried naturally to obtain Ag + @EuMOF / HOF / GCE;

[0066] (2) The electrochemiluminescence sensor of the present invention is used for ECL detection

[0067] (1) The prepared Ag + @Eu MOF / HOF / GCE was used as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode for ECL testing;

[0068] (2) The parameters of the electrochemiluminescence detection instrument were set as follows: the scan rate was set to 100 mV / s, and the photomultiplier tube high voltage was set to 800 V;

[0069] (3) The electrochemical workstation parameters were set as follows: cyclic voltammetry scan voltage range was 0 to -1.8 V; scan rate was set to 100 mV / s;

[0070] (4) Using a PBS buffer solution containing K2S2O8, the intensity of the generated electrochemiluminescence signal was recorded; the PBS buffer solution was a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution with a pH of 7.5 containing 0.1 mol / L K2S2O8.

[0071] Comparative Example 1

[0072] 1. A method for preparing an electrochemiluminescent body of HOF material, comprising the following steps:

[0073] (1) Preparation of HOF

[0074] Dissolve 0.5 mmol of melamine and 0.5 mmol of trimesic acid in 50 mL of deionized water, stirring and heating to 80°C until completely dissolved. Then, combine the melamine and trimesic acid solutions and stir at room temperature for 1 hour. Centrifuge at 12,000 rpm for 1 minute to collect the crude product, which is then washed three times with deionized water. Finally, dry the white powder under vacuum at 60°C for 12 hours.

[0075] (2) Preparation of Eu MOF / HOF

[0076] 25 mg of HOF and 0.04 mmol of Eu(NO3)3·6H2O were weighed and dissolved in 10 mL of deionized water. After stirring evenly, 2 mL of DMF was added and the mixture was transferred to a 25 mL autoclave and reacted at 80°C for 2 h. Centrifuged at 8000 rpm for 10 min and washed three times with deionized water. The obtained product was vacuum dried at 60°C for 10 h to obtain a white solid powder. Then the obtained Ag + @Eu MOF / HOF was dispersed in deionized water to obtain a 2 mg / mL dispersion and stored at 4°C until use.

[0077] 2. Use the prepared HOF or Eu MOF / HOF for ECL detection

[0078] (1) Preparation of electrochemiluminescence sensor

[0079] (I) A 3 mm diameter glassy carbon electrode (GCE) was polished and thoroughly cleaned using dilute nitric acid, anhydrous ethanol, and deionized water for 3 min each by ultrasonication.

[0080] (II) 5 μL of 2 mg / mL HOF or Eu MOF / HOF was drop-coated on the GCE surface and allowed to dry naturally to obtain HOF / GCE;

[0081] (2) The electrochemiluminescence sensor of the present invention is used for ECL detection

[0082] (I) ECL test was performed using the prepared HOF / GCE and Eu MOF / HOF / GCE as working electrodes, platinum wire as counter electrode, and Ag / AgCl as reference electrode;

[0083] (II) The parameters of the electrochemiluminescence detection instrument were set as follows: the scan rate was set to 100 mV / s, and the photomultiplier tube high voltage was set to 800 V;

[0084] (III) The electrochemical workstation parameters were set as follows: cyclic voltammetry scan voltage range of 0 to -1.8 V; scan rate of 100 mV / s;

[0085] (IV) Using a PBS buffer solution containing K2S2O8, the intensity of the generated electrochemiluminescence signal was recorded; the PBS buffer solution was a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution with a pH of 7.5 containing 0.1 mol / L K2S2O8.

[0086] Figure 2 A is the prepared HOF, Eu MOF / HOF, Ag + @Eu MOF / HOF SEM image, Figure 1 A is the SEM image of HOF, and its surface appears as long and thin nanowires. Figure 2 B is the morphology of Eu MOF / HOF, and Eu 3+ After coordination, HOF changes from a linear shape with a diameter of about 80nm to a rough rod shape with a diameter of about 600nm. This transformation maintains the original rod-like characteristics of HOF, and many newly generated fine particles are attached to its surface. These fine irregular particles are generated on the surface of HOF material by Eu 3+ Induced generation of MOF materials. Figure 2 C is Ag + @Eu MOF / HOF SEM image, we can see that Ag + After coordination, the morphology of Eu MOF / HOF is not changed, and Ag + The ionic form is fixed on the HOF material through Ag-amino coordination bonds.

[0087] Figure 3 The prepared HOF / GCE, Eu MOF / HOF / GCE, Ag +@Eu MOF / HOF / GCE electrode ECL test obtained by ECL-time curve. As can be seen from the figure, the ECL value of HOF / GCE is about 12000a.u. (curve a), the ECL value of EuMOF / HOF / GCE is about 25000a.u. (curve b), and the ECL value of Ag is about 12000a.u. (curve a). + @Eu MOF / HOF / GCE's ECL value is about 46000a.u. (curve c). By comparison, Ag + @Eu MOF / HOF / GCE has the best performance. Lanthanide elements can improve the ECL performance of HOF materials. This is because the lanthanide element Eu 3+ As the main ECL center, it can emit a strong signal in the co-reaction medium of K2S2O8 due to the ligand antenna effect between the HOF ligand. + Ag is fixed on the HOF material through Ag-amino coordination bonds. + It will further increase the ECL value of Eu MOF / HOF, which is due to the fact that Ag + As a metal catalyst, it can catalyze the decomposition of K2S2O8 into SO4· - , thereby amplifying Ag + @Eu ECL signal of MOF / HOF.

[0088] Comparative Example 2

[0089] 1. The preparation steps of HOF precursor are as follows:

[0090] Dissolve 0.5 mmol of melamine and 0.5 mmol of trimesic acid in 50 mL of deionized water, stirring and heating to 110°C until completely dissolved. Then, combine the melamine and trimesic acid solutions and stir at room temperature for 1 hour. The crude product is collected by centrifugation at 12,000 rpm and washed three times with deionized water. Finally, dry the white powder under vacuum at 60°C for 12 hours.

[0091] The stirring temperature was set to 110 °C, and the SEM image of the obtained HOF material was as follows: Figure 2 As shown in D. Figure 2 D shows that when the temperature is too high, the HOF material is a thin and long nanofiber, which is not conducive to the formation of MOF material induced by lanthanide central atom Eu on its surface. + @Eu MOF / HOF also has a low ECL intensity (ECL≈9000 a.u.).

[0092] Comparative Example 3

[0093] 1. A method for preparing an electrochemiluminescent body of HOF material, comprising the following steps:

[0094] Prepare Ag according to the method of Example 1 + @Eu MOF / HOF / GCE.

[0095] 2. Prepared Ag + @Eu MOF / HOF for ECL detection

[0096] (1) Preparation of electrochemiluminescence sensor

[0097] 1) A 3 mm diameter glassy carbon electrode (GCE) was polished and then thoroughly cleaned using dilute nitric acid, anhydrous ethanol, and deionized water for 3 minutes each.

[0098] 2) Add 5 μL of 2 mg / mL Ag + @Eu MOF / HOF was drop-coated on the GCE surface and dried naturally to obtain Ag + @EuMOF / HOF / GCE;

[0099] (2) The electrochemiluminescence sensor of the present invention is used for ECL detection

[0100] (1) The prepared Ag + @Eu MOF / HOF / GCE was used as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode for ECL testing;

[0101] (2) The parameters of the electrochemiluminescence detection instrument were set as follows: the scan rate was set to 100 mV / s, and the photomultiplier tube high voltage was set to 800 V;

[0102] (3) The electrochemical workstation parameters were set as follows: cyclic voltammetry scan voltage range was 0 to -1.8 V; scan rate was set to 100 mV / s;

[0103] (4) Using a PBS buffer solution containing K2S2O8, the intensity of the generated electrochemiluminescence signal was recorded; the PBS buffer solution was a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution containing 0.1 mol / L K2S2O8, with pH values ​​selected from 6.0, 6.5, 7.0, 8.0, 8.5, and 9.0, respectively.

[0104] Ag + @Eu MOF / HOF ECL-time curves under different pH conditions are shown in Figure 4As can be seen from the figure, the ECL value gradually increases in the range of pH = 6.0 to 7.5, reaching a maximum of about 46000 a.u. at pH = 7.5, and then ECL decreases with increasing pH. This is mainly because when the pH is less than 7.5, the protons around the electrode are easily reduced to H2, which inhibits the formation of S2O8 2- reduction, resulting in SO4 ·- The generation of SO4 is reduced, which ultimately reduces the ECL intensity. Under alkaline conditions, excess anions are dispersed around the electrode, preventing SO4 ·- Close to the electrode surface, the ECL signal decreases. Therefore, the pH value was selected as 7.5 throughout the experiment.

[0105] Example 2

[0106] The preparation method of the resonance energy transfer receptor CdS@Au-cDNA has the following specific steps:

[0107] (1) Preparation of AuNPs

[0108] 0.01% HAuCl4 (100 mL) was heated to boiling at 120°C, and then 3.5 mL of 1% (w / v) sodium citrate was added to the solution. The solution was heated under reflux for 30 minutes until the solution turned wine red. The resulting AuNPs were cooled to room temperature and stored at 4°C until ready for use.

[0109] (2) Preparation of CdS

[0110] First, 0.475 g of CdCl₂·2.5H₂O and 0.152 g of CH₄N₂S (thiourea) were dissolved in 70 mL of deionized water and stirred at room temperature for 10 minutes to achieve uniform mixing. Then, 0.3 mL of DMSO was added dropwise to the solution and stirred for 10 minutes. Finally, the solution was transferred to a 100 mL Teflon-lined stainless steel autoclave and reacted in a 170°C drying oven for 8 hours. After the autoclave cooled to room temperature, the orange precipitate at the bottom was washed several times with anhydrous ethanol and deionized water and dried at 60°C to obtain an orange-yellow CdS powder.

[0111] (3) Preparation of CdS@Au-cDNA

[0112] 3 mg of CdS was added to 3 mL of ultrapure water, and 2 mL of a 0.22 mM Au NP solution was gradually added to the mixture, stirring continuously for 6 hours. The mixture was then centrifuged, washed, and dried under vacuum at 60°C to obtain CdS@Au. 70 μL of cDNA (3 μM) was added to the CdS@Au dispersion, stirred in an ice-water bath for 10 hours, and the product was centrifuged at 12,000 rpm for 15 minutes and washed three times with deionized water. The resulting precipitate was dispersed in deionized water to obtain a 1 mg / mL CdS@Au-cDNA dispersion.

[0113] Figure 5 The SEM images of CdS and CdS@Au are shown in the figure. By comparing the morphology of the two, it can be seen that CdS presents a unique leaf-like structure ( Figure 5 A), which can provide abundant binding sites for AuNPs. Many small particles ( Figure 5 B), these newly emerged particles are a large number of AuNPs loaded on the CdS surface.

[0114] Example 3

[0115] 1. An Ag-based + The preparation process of the @Eu MOF / HOF resonance energy transfer electrochemiluminescence aptamer sensor for acetamiprid is as follows:

[0116] Prepare Ag according to the method in Example 1 + @Eu MOF / HOF, CdS@Au-cDNA was prepared according to the method in Example 2. 5 μL of 2 mg / mL Ag + @Eu MOF / HOF drop coating on GCE surface to obtain Ag + @Eu MOF / HOF / GCE. Then 5μL 3μM apt was added to the electrode surface and incubated at 37℃ for 6h to obtain apt / Ag + @Eu MOF / HOF / GCE. 5.0 μL of 0.5% bovine serum albumin (BSA) was then added to block the nonspecific binding sites on the electrode surface. 5 μL of 2 mg / mL CdS@Au-cDNA was then added to the electrode surface and allowed to fully bind to apt at 37°C for 2 h to obtain CdS@Au-cDNA / BSA / apt / Ag + @Eu MOF / HOF / GCE. Finally, the obtained sensor was immersed in 10 -10 The electrode was placed in ACE solution of 0.1 M for 40 min, then taken out, rinsed with PBS solution, and tested in PBS (0.1 M, pH = 7.5) containing 0.1 M K2S2O8.

[0117] 2.ECL test

[0118] (1) ECL tests were performed using a bare GCE with a diameter of 3 mm and the modified electrodes prepared in each step as working electrodes, a platinum wire as a counter electrode, and Ag / AgCl as a reference electrode;

[0119] (2) The parameters of the electrochemiluminescence detection instrument were set as follows: the scan rate was set to 100 mV / s, and the photomultiplier tube high voltage was set to 800 V;

[0120] (3) The electrochemical workstation parameters were set as follows: cyclic voltammetry scan voltage range was 0 to -1.8 V; scan rate was set to 100 mV / s;

[0121] (4) Using a PBS buffer solution containing K2S2O8, the intensity of the generated electrochemiluminescence signal was recorded; the PBS buffer solution was a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution with a pH of 7.5 containing 0.1 mol / L K2S2O8.

[0122] Figure 6 It is the ECL intensity curve of each modified electrode. As can be seen from the figure, the ECL intensity of bare GCE is ≈1600 a.u. (curve a). + @Eu MOF / HOF / GCE ECL intensity increased significantly to ≈46000 a.u. (curve b), which indicates that Ag + @Eu MOF / HOF was successfully modified on the electrode and had a strong ECL signal. After modification with apt and BSA, the ECL signal dropped to ≈44650 a.u. (curve c) and ≈40000 a.u. (curve d), respectively. Subsequently, when the acceptor CdS@Au-cDNA was added to the electrode surface, the ECL intensity dropped significantly to 10200 a.u. (curve e). This is because the donor Ag + The resonance energy transfer between Ag@Eu MOF / HOF and the acceptor CdS@Au makes Ag@Eu + The ECL intensity of @Eu MOF / HOF was weakened. Finally, the prepared sensor was immersed in a solution with a concentration of 1×10 -10 After removing the electrode from the ACE sample and rinsing with PBS to remove any incompletely bound ACE, the ECL value measured was approximately 320,000 a.u. (curve f). This is because the specific binding of apt to the ACE target replaces the weaker interaction between apt and cDNA, causing the CdS@Au-cDNA to fall off the electrode, resulting in the disappearance of the resonance energy transfer effect and a rebound in the ECL intensity. These results demonstrate that the electrochemiluminescence sensor was successfully fabricated and can be used for ACE detection.

[0123] Example 4

[0124] 1. An Ag-based + The method for detecting acetamiprid using a resonance energy transfer electrochemiluminescence aptamer sensor based on @Eu MOF / HOF is as follows:

[0125] Prepare Ag according to the method in Example 1 + @Eu MOF / HOF, CdS@Au-cDNA was prepared according to the method in Example 2. 5 μL of 2 mg / mL Ag + @Eu MOF / HOF drop coating on GCE surface to obtain Ag + @Eu MOF / HOF / GCE. Then 5μL 3μM apt was added to the electrode surface and incubated at 37℃ for 6h to obtain apt / Ag + @Eu MOF / HOF / GCE. 5.0 μL of 0.5% bovine serum albumin (BSA) was then added to block the nonspecific binding sites on the electrode surface. 5 μL of 2 mg / mL CdS@Au-cDNA was then added to the electrode surface and allowed to fully bind to apt at 37°C for 2 h to obtain CdS@Au-cDNA / BSA / apt / Ag + @Eu MOF / HOF / GCE electrochemiluminescence aptamer sensor.

[0126] A1. Preparation of standard solutions containing different concentrations of acetamiprid:

[0127] The solid standard of acetamiprid was prepared with deionized water to a concentration of 1.0 × 10 -3 mol / L solution and diluted into a series of acetamiprid standard solutions with different concentrations, ranging from 1.0×10 -3 ~1.0×10 -17 mol / L;

[0128] A2. Drawing of standard curve:

[0129] A three-electrode system was used, with the prepared electrochemiluminescence aptamer sensor as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The prepared electrochemiluminescence aptamer sensor was placed in a series of standard solutions of different concentrations of acetamiprid prepared in step A1 and soaked for 40 minutes. The electrode was taken out and rinsed with PBS buffer solution. After natural drying, the electrochemiluminescence test was performed in a PBS buffer solution containing 0.1M K2S2O8 (0.1M, pH=7.5). A linear relationship between acetamiprid and the change in electrochemiluminescence intensity (ΔECL) was established, and the corresponding linear regression equation was obtained. The parameters of the electrochemiluminescence detection instrument were set as follows: the scan rate was set to 100mV / s, the photomultiplier tube high voltage was set to 800V; the electrochemical workstation parameters were set as follows: the cyclic voltammetry scan voltage range was 0 to -1.8V; the scan rate was set to 100mV / s;

[0130] Figure 7 A is based on the luminescent material Ag + @Eu MOF / HOF prepared electrochemiluminescent aptamer sensor (CdS@Au-cDNA / BSA / apt / Ag + @Eu MOF / HOF / GCE) was used to detect acetamiprid. As can be seen from the figure, when the acetamiprid concentration was 10 -8 ~10 -15 Within the range of M, the ECL value first increases with the increase of acetamiprid concentration. Figure 7 B is the linear regression curve of the ECL change value (ΔECL) of the sensor and the logarithm of the acetamiprid concentration. The two show a good linear relationship. The linear regression equation is ΔECL = 41351.19048 + 2020.19048 × lgC, R 2 =0.9983, the detection limit is 7.33×10 -16 M(3σ / slope).

[0131] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An Ag-based + @Eu MOF / HOF resonance energy transfer electrochemiluminescence aptamer sensor, characterized by: Including as a donor to modify the electrode surface Ag + @Eu MOF / HOF luminescent material, aptamer apt modified on the surface of Ag+@Eu MOF / HOF luminescent material and bovine serum albumin, as a receptor modified CdS@Au-cDNA on the electrode surface, to obtain CdS@Au-cDNA / BSA / apt / Ag + @Eu MOF / HOF / GCE aptamer sensor; CdS@Au-cDNA and Ag + @Eu A resonance energy transfer mechanism is formed between MOF / HOF.

2. According to claim 1, based on Ag + @Eu MOF / HOF resonance energy transfer electrochemiluminescence aptamer sensor, characterized by: Take Ag + @Eu MOF / HOF dispersion was added dropwise to GCE and dried under infrared light to obtain Ag + @EuMOF / HOF / GCE modified electrode; apt was added to the surface of the modified electrode and incubated to obtain apt / Ag + @Eu MOF / HOF / GCE; add bovine serum albumin and dry naturally to obtain BSA / apt / Ag + @Eu MOF / HOF / GCE; finally, CdS@Au-cDNA was added dropwise to the electrode surface to obtain the resonance energy transfer electrochemiluminescence aptamer sensor.

3. According to claim 2, based on Ag + @Eu MOF / HOF resonance energy transfer electrochemiluminescence aptamer sensor, characterized by: Ag + The preparation steps of @Eu MOF / HOF are as follows: (1) Preparation of HOF Weigh melamine in deionized water, weigh trimesic acid and add it to the above liquid, stir and heat until completely dissolved, collect the crude product by centrifugation, wash and dry; wherein the molar ratio of melamine to trimesic acid is 1:1 to 3:1; (2) Ag + Preparation of @EuMOF / HOF Weigh HOF and Eu(NO3)3·6H2O in deionized water, add AgNO3, After stirring evenly, add DMF, transfer to a high-pressure reactor and react at 60℃~100℃ for 2h, centrifuge, wash and dry to obtain a white solid powder, which is Ag. + @EuMOF / HOF is dispersed in deionized water to obtain a dispersion, which is stored at a low temperature for future use; wherein the molar ratio of Eu(NO3)3·6H2O and AgNO3 is 1:1 to 5:1; and the mass ratio of HOF and Eu(NO3)3·6H2O is 1:1 to 3:

1.

4. According to claim 2, based on Ag + @Eu MOF / HOF resonance energy transfer electrochemiluminescence aptamer sensor, characterized by: The preparation method of CdS@Au-cDNA is as follows: CdS is weighed and added to ultrapure water, and the AuNPs solution is added dropwise thereto and continuously stirred and mixed. Then, the mixture is centrifuged and washed, and vacuum dried to obtain CdS@Au; cDNA is added to the CdS@Au dispersion, stirred in an ice-water bath, the product is centrifuged and washed, and the resulting precipitate is dispersed with deionized water to obtain a CdS@Au-cDNA dispersion.

5. According to claim 1, based on Ag + @Eu MOF / HOF resonance energy transfer electrochemiluminescence aptamer sensor, characterized by: Ag at a concentration of 2 mg / mL + @Eu MOF / HOF drop coating on GCE surface to obtain Ag + @Eu MOF / HOF / GCE; then 3 μM apt was added to the electrode surface and incubated at 37 °C for 6 h to obtain apt / Ag + @Eu MOF / HOF / GCE; then, 0.5% bovine serum albumin was added dropwise, and then CdS@Au-cDNA dispersion was added dropwise to the electrode surface, and fully combined with apt at 37°C for 2 hours.

6. The Ag-based + A method for detecting acetamiprid using a resonance energy transfer electrochemiluminescence aptamer sensor based on @Eu MOF / HOF is characterized by: The electrochemiluminescent aptamer sensor according to claim 1 is used as a working electrode, a platinum wire is used as a counter electrode, and Ag / AgCl is used as a reference electrode. In a three-electrode system, the concentration of acetamiprid is detected by electrochemiluminescence detection; the sequence of the apt DNA chain is: 5'-(NH2 C6)-TGT AAT TTGTCT GCA GCG GTT CTT GAT CGC TGA CAC CAT ATTATG AAG A-3'; the sequence of the cDNA is 5'-(NH2C6)-TCT TCATAATATGG-3.

7. The Ag-based method according to claim 6. + A method for detecting acetamiprid using a resonance energy transfer electrochemiluminescence aptamer sensor based on @Eu MOF / HOF is characterized by: (1) Preparation of standard solutions containing different concentrations of acetamiprid: The solid standard of acetamiprid was prepared with deionized water to a concentration of 1.0 × 10 -3 mol / L solution and diluted into a series of acetamiprid standard solutions with different concentrations, ranging from 1.0×10 -3 ~1.0×10 -17 mol / L; (2) A three-electrode system was used, with the prepared electrochemiluminescence aptamer sensor as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The prepared electrochemiluminescence aptamer sensor was placed in a series of standard solutions of different concentrations of acetamiprid prepared in step (1) and soaked for 10 to 50 minutes. The electrode was taken out and rinsed with PBS buffer solution. After natural drying, an ECL test was performed in a PBS buffer solution containing 0.1M K2S2O8. A linear relationship between acetamiprid and the change in electrochemiluminescence intensity was established, and the corresponding linear regression equation was obtained. (3) The ECL test of acetamiprid in the actual sample was performed using the standard addition method. The obtained ECL intensity was calculated using the linear regression equation obtained in step (2) to obtain the acetamiprid concentration in the sample.

8. The Ag-based method according to claim 7 + A method for detecting acetamiprid using a resonance energy transfer electrochemiluminescence aptamer sensor based on @Eu MOF / HOF is characterized by: The pH of the PBS buffer solution is 6.0-9.0, and the concentration is 0.1M.

Images