Preparation method of electrochemical luminescence nano hybrid material, probe and sensor and application of electrochemical luminescence nano hybrid material, probe and sensor in determination of tumor-derived extracellular vesicles

Abstract

The invention belongs to the technical field of electrochemical sensors and extracellular vesicle detection, and relates to a preparation method of an electrochemical nano hybrid material, a probe and a sensor, and an application of the electrochemical nano hybrid material, the probe and the sensor in determination of tumor-derived extracellular vesicles. The electrochemical nano hybrid material is prepared from Ru (bpy) 3 < 2 + >, WO3-x QDs, H3BTC and Zn (NO3) 2.6 H2O, the electrochemical nano hybrid material is used for preparing an ECT probe, and tumor-derived extracellular vesicles are detected by combining a sensor with the ECT probe; the ECT probe reduces the electron transmission distance, improves the ECL efficiency, and improves the sensitivity of the sensor. The electrochemical sensor has good stability and excellent selectivity, can simply and rapidly detect the target TEVs, has excellent prospects in the field of cancer liquid biopsy, and provides a new auxiliary platform for detection of other tumor biomarkers.

Description

Technical field:

[0001] The present invention belongs to the technical field of electrochemiluminescent sensors and rapid detection of extracellular vesicles, and particularly relates to a preparation method of an electrochemiluminescent nanohybrid material, a probe, and a sensor, and their application in the detection of tumor-derived extracellular vesicles. Background technology:

[0002] Extracellular vesicles (EVs) are nanoscale vesicles secreted by cells that play a vital role in intercellular communication and are involved in various physiological and pathological processes. Due to the protection of the phospholipid bilayer membrane structure, EVs have greater stability than traditional liquid biopsy markers, providing more important and accurate biological information for capturing the state of the source cells; this includes tumor-derived extracellular vesicles (TEVs), which have received widespread attention in the field of tumor diagnosis in recent years. Diagnosing cancer by analyzing biomarkers on circulating EVs still has many shortcomings, such as complex operating steps, limited detection throughput, poor sensitivity, and a lack of clinically validated biomarkers. Therefore, the development of a rapid and reliable TEV detection method is necessary for monitoring cancer progression.

[0003] Electrochemiluminescence (ECL) materials have attracted widespread attention in many different fields due to their unique controllability, high sensitivity and low cost, such as food safety analysis, environmental health monitoring and clinical disease diagnosis. To date, a variety of ECL materials have been developed, including organic molecules, inorganic molecules and semiconductor nanomaterials. In particular, among the luminophores, Ru(bpy)3 2+ It has attracted widespread attention due to its unique properties, such as high ECL efficiency, reversible electrochemical reaction, good solubility in aqueous and non-aqueous solutions, and low biological toxicity. 2+ Efficient co-reactants through the "redox" mechanism. In recent years, a variety of co-reactants have been developed, including polyethyleneimine, biomolecules such as L-cysteine; nanomaterials such as carbon nanodots and boron nitride quantum dots. In the new co-reactants, nanomaterials not only act as co-reactants, but also as Ru(bpy)3 2+ Covalently immobilized coreactant-embedded ECL probes are constructed, and these probes are believed to expand the application of ECL in biomedicine because the addition of additional coreactants is eliminated.

[0004] Transition metal oxides (TMOs) have attracted increasing attention in many fields due to their diverse functions, non-toxicity, tunable electronic properties, excellent chemical and thermal stability, unique optical properties and strong oxidation ability. 3-x There is a report on using QDs quantum dots as precursors to synthesize co-reactants embedded ECL nanohybrid materials and their use in electrochemiluminescence sensors. Summary of the invention:

[0005] To address the deficiencies of the prior art, the present invention provides a method for preparing an electrochemiluminescent nanohybrid material, a probe, and a sensor, and their application in detecting tumor-derived extracellular vesicles. 2+ , WO 3-x An electrochemical nanohybrid material with co-reactant embedding was prepared by QDs, H3BTC and Zn(NO3)2·6H2O, which was used to prepare an ECT probe. The sensor was combined with an ECL probe to detect tumor-derived extracellular vesicles. The material had high sensitivity, good specificity, a wide linear range and a low detection limit, and could be applied to the detection of tumor-derived extracellular vesicles in samples.

[0006] In order to achieve the above object, the present invention provides a co-reactant embedded ECL nano-hybrid material WO 3- x QDs-Ru@ZnMOF was prepared as follows:

[0007] 0.5-40mg Ru(bpy)3 2+ , 0.5-40mg WO 3-x QDs powder, 100-400 mg H3BTC and 100-400 mg Zn(NO3)2·6H2O were added and dissolved in DMF / water (DMF:water=1:(0.1-10), v / v), with constant mechanical stirring, and reacted at room temperature for 5-60 minutes; then, the above reactants were transferred to a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 50-300°C for 1-30 hours; after cooling to room temperature, WO was obtained by centrifugation. 3-x The QDs-Ru@ZnMOF dispersion was then washed with ethanol and deionized water to eliminate any unreacted metal ions and H3BTC. The resulting precipitate was kept in a vacuum at 10-150 °C for 0.5-10 h. The resulting pale yellow powder was WO 3-x QDs-Ru@ZnMOF.

[0008] The Ru(bpy)3 of the present invention 2+ , WO 3-x The preferred amounts of QD powder, H3BTC, Zn(NO3)2·6H2O, and DMF / water are 5 mg, 5 mg, 216 mg, 147.5 mg, and 40 mL, respectively. The reaction time is preferably 30 minutes at room temperature. Preferably, the mixture is transferred to a stainless steel autoclave and reacted at 180°C for 10 hours. Preferably, the precipitate is maintained in a vacuum at 80°C for 2 hours.

[0009] The WO of the present invention 3-xQDs powder is prepared according to the existing technology.

[0010] The present invention also provides a method for preparing an ECL probe, which comprises the following steps:

[0011] 0.01-1 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) aqueous solution (5-40 mg mL -1 ) and 0.01-1 mL of N-hydroxysuccinimide (NHS) aqueous solution (5-40 mg mL -1 ) added to 0.1-10 mL WO 3-x QDs-Ru@ZnMOF aqueous solution (10 mg mL -1 ), oscillated at 10-60°C for 0.5-10h, centrifuged and thoroughly washed to obtain an activated nanocomposite material, which was collected and resuspended in 0.1-10mL 0.1M PBS (pH 7.4), and then 0.1-10mL of a 0.1-10μM MUC1 aptamer solution was added to the above solution, and the reaction was oscillated at 10-60°C for 0.5-10h, followed by addition of 0.05-5mL of a 0.1-10wt% bovine serum albumin aqueous solution, and oscillated at 10-60°C for 0.5-10h to block nonspecific binding sites; finally, the ECL probe WO was obtained by centrifugation and washing with PBS solution. 3-x QDs-Ru@ZnMOF / Apt was suspended in 0.1M PBS solution to obtain an ECL probe (WO 3-x QDs-Ru@ZnMOF / Apt) solution (0.1-10 mg mL -1 ) and stored at 0-20 °C for further use.

[0012] The aptamer of the present invention is a single-stranded DNA with the sequence: 5'-NH2-GCAGTTGATCCTTTGGATACCCTGG-3' (SEQ ID NO. 1), which is used to specifically bind to the MUC1 protein on the TEVs membrane. The solvent of the aptamer solution is PBS solution.

[0013] The preferred concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution of the present invention is 20 mg·mL -1 , the dosage is 0.1mL; the preferred concentration of the N-hydroxysuccinimide (NHS) aqueous solution is 10mg·mL -1 , the amount is 0.1 mL; preferably, the oscillation temperature is 37 ° C, the time is 2 h; preferably, the concentration of the bovine serum albumin aqueous solution is 1 wt%, the amount is 0.5 mL; preferably, the concentration of the aptamer solution is 1 μM, the amount is 1 mL; preferably, the concentration of the ECT probe solution is 1 mg·mL-1 , stored at 4°C.

[0014] The present invention also provides an electrochemical sensor for measuring tumor-derived extracellular vesicles. The sensor comprises a substrate electrode surface coated with AuNPs, onto which an EpCAM antibody is immobilized. The EpCAM antibody specifically recognizes and reacts with the EpCAM protein on the surface membrane of the TEVs antigen. A TEVs antigen sample to be tested is added to the electrochemical sensor surface and incubated. The ECL probe is then added and incubated again to produce a sensor electrode to be tested. Electrochemiluminescence (ECL) detection is performed on the sensor electrode to determine the ECL intensity of the TEVs antigen sample to be tested. A standard curve of the relationship between ECL intensity and TEVs antigen sample concentration is used to determine the concentration of the TEVs antigen sample to be tested.

[0015] The present invention also provides a method for preparing an electrochemical sensor for measuring tumor-derived extracellular vesicles, comprising the following steps:

[0016] (1) Electrode pretreatment: grinding, polishing and ultrasonic cleaning of the base electrode;

[0017] (2) Place the electrode treated in step (1) in a solution with a concentration of 0.01-1 mg·mL -1 AuNPs were deposited in an aqueous solution of HAuCl4 at -3-0 V for 10-300 s. The electrode surface was thoroughly rinsed with ultrapure water, and the remaining water droplets were dried with nitrogen to obtain an AuNPs-modified electrode (AuNPs / electrode), which was then washed with distilled water and dried at room temperature.

[0018] (3) Covalently linking EpCAM antibodies: 1-10 μL of EpCAM antibody Ab solution was added to the electrode obtained in step (2) and placed at 0-20°C for 2-24 hours to allow Ab to fully link with AuNPs through gold-sulfur bonds; then washed with PBS solution and dried at room temperature;

[0019] (4) Blocking nonspecific sites: add 1-10 μL of BSA aqueous solution, leave at room temperature for 0.5-10 h to block unbound sites, wash with PBS solution, and dry at room temperature; thus, an electrochemical sensor for measuring tumor-derived extracellular vesicles is obtained.

[0020] The base electrode of the present invention is a glassy carbon electrode.

[0021] In step (1) of the present invention, the base electrode is polished with Al2O3 powders with particle sizes of 0.1-2 μm and 0.01-0.1 μm, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 1-10 minutes; preferably, it is polished with Al2O3 powders with particle sizes of 0.5 μm and 0.03 μm, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 5 minutes.

[0022] The preferred concentration of chloroauric acid in step (2) of the present invention is 0.05 mg·mL -1 ; Electrodeposition at -1.2 V for 100 s.

[0023] The concentration of Ab in step (3) of the present invention is 0.5-5 μg / mL; preferably, the concentration of Ab is 2 μg / mL; the amount of Ab solution added is 6 μL, and the mixture is placed at 4° C. for 12 hours.

[0024] The concentration of the BSA aqueous solution in step (4) of the present invention is 0.1-10 wt %; preferably, the concentration of the BSA aqueous solution is 1 wt %, the amount of the BSA aqueous solution added is 6 μL, and the mixture is placed at room temperature for 2 h.

[0025] The present invention also provides the use of the biosensor prepared by the preparation method in the quantitative detection of TEVs. The detection is for non-disease diagnosis purposes.

[0026] The method for detecting tumor-derived extracellular vesicle antigens using the electrochemical sensor of the present invention is as follows:

[0027] (1) adding 1-10 μL of the TEVs antigen sample to be tested to the surface of the electrochemical sensor, incubating at 10-60°C for 10-60 minutes, and then washing the unbound antigen with a 0.01-1 M PBS solution; preferably, adding 5 μL of the TEVs antigen sample to be tested, incubating at 37°C for 30 minutes, and washing with a 0.1 M PBS solution;

[0028] (2) Add 6 μL of the above-mentioned ECL probe solution (WO 2) to the electrode surface obtained in step (1). 3-x QDs-Ru@ZnMOF / Apt), incubated at 10-60°C for 10-60 minutes, and washed with 0.01-1M PBS solution to obtain an electrochemical sensor electrode to be tested; preferably, incubated at 37°C for 30 minutes, and washed with 0.1M PBS solution;

[0029] (3) The electrochemical sensor electrode to be tested is tested in a 0.1 M PBS solution using an electrochemiluminescence detector to obtain the ECL intensity of the TEVs-specific antigen sample to be tested, and the concentration of the TEVs-specific antigen sample to be tested is determined using a standard relationship curve between the ECL intensity and the concentration of the TEVs-specific antigen sample.

[0030] The abbreviations of the technical terms in the present invention are as follows:

[0031] Glassy carbon electrode: GCE; tumor-derived extracellular vesicles: TEVs; gold nanoparticles: AuNPs; electrochemiluminescence: ECL; cyclic voltammetry: CV; differential pulse voltammetry: DPV; zinc metal organic framework: ZnMOF; tungsten oxide quantum dots: WO 3-x QDs; ruthenium terpyridine: Ru(bpy)3 2+ ; Chloroauric acid: HAuCl4; epithelial cell adhesion molecule: EpCAM; mucin 1: MUC1; aptamer: Apt; bovine serum albumin: BSA; 1,3,5-benzenetricarboxylic acid: H3BTC; X-ray photoelectron spectroscopy technology: XPS.

[0032] Compared with the prior art, the present invention uses a coreactant embedded ECL nano-hybrid material (WO 3-x QDs-Ru@ZnMOF) was used as a signal tag to construct a new type of ECL probe for detecting TEVs; ZnMOF was used as a carrier to detect Ru(bpy)3 2+ (as ECL emitter) and WO 3-x QDs (as core active agents) are captured. On the one hand, ZnMOF, as an ideal carrier candidate, can prevent quantum dots from being decomposed by the environment and further improve their stability. On the other hand, Ru(bpy)3 2+ and WO 3-x The short distance between QDs quantum dots ensures efficient co-reactant embedding into the ECL nanohybrid material, resulting in a relatively high reaction speed and low energy loss. The probe of the present invention reduces the electron transmission distance, greatly improves the ECL efficiency, and increases the sensitivity of the sensor. The electrochemical sensor of the present invention has good stability and excellent selectivity, can simply and quickly detect target TEVs, has excellent prospects in the field of cancer liquid biopsy, and provides a new auxiliary platform for the detection of other tumor biomarkers. The present invention is WO 3-x The application of QDs in ECL has opened up new avenues and demonstrated its wide applications in biosensing and clinical diagnosis. Description of the drawings:

[0033] Figure 1 Schematic diagram of the preparation and use of the electrochemical sensor for measuring TEVs according to the present invention.

[0034] Figure 2 This is the XPS characterization diagram of the co-reactant embedded ECL nanohybrid material synthesized in the present invention.

[0035] Figure 3 CV graphs of different modified electrodes of the present invention in 1.0 mM K3[Fe(CN)6] base solution.

[0036] Figure 4 Graphs showing the effects of (A) Ab concentration, (B) TEVs antigen-EpCAM antibody incubation time, and (C) TEVs antigen-ECL probe incubation time on electrochemical signals.

[0037] Figure 5 This is a linear relationship diagram between the ECL intensity of the electrochemical sensor for measuring TEVs and the TEVs concentration of the present invention. Specific implementation method:

[0038] The technical solution of the present invention is further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0039] Example 1:

[0040] This embodiment relates to a coreactant embedded ECL nanohybrid material WO 3-x The preparation method of QDs-Ru@ZnMOF includes the following steps:

[0041] 5mg Ru(bpy)3 2+ , 5mg WO 3-x QDs powder, 216 mg H3BTC and 147.5 mg Zn(NO3)2·6H2O were added to 40 mL DMF / water (DMF:water=1:6, v / v) and dissolved, with constant mechanical stirring, and reacted at room temperature for 30 minutes. Then, the reactants were transferred to a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 180°C for 10 hours. After cooling to room temperature, WO was obtained by centrifugation. 3-x The QDs-Ru@ZnMOF dispersion was then washed with ethanol and deionized water to eliminate any unreacted metal ions and H3BTC. The resulting precipitate was kept in a vacuum at 80 °C for 2 h. The resulting pale yellow powder was WO 3-x QDs-Ru@ZnMOF.

[0042] The WO in this embodiment 3-x QDs powder was prepared according to the existing technology, reference: Angew.Chem.Int.Ed.2020,59,16747–16754.

[0043] Example 2:

[0044] This embodiment relates to a coreactant embedded ECL nanohybrid material WO 3-x The preparation method of QDs-Ru@ZnMOF includes the following steps:

[0045] 0.5 mg Ru(bpy)3 2+ , 0.5mg WO 3-xQDs powder, 100 H3BTC and 100 mg Zn(NO3)2·6H2O were added and dissolved in 40 mL DMF / water (DMF:water=1:0.1, v / v) and stirred constantly for 5 minutes at room temperature. The reactants were then transferred to a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 50°C for 30 hours. After cooling to room temperature, WO was obtained by centrifugation. 3-x The QDs-Ru@ZnMOF dispersion was then washed with ethanol and deionized water to eliminate any unreacted metal ions and H3BTC. The resulting precipitate was kept in a vacuum at 10 °C for 10 h, and the resulting pale yellow powder was WO 3-x QDs-Ru@ZnMOF.

[0046] Example 3:

[0047] This embodiment relates to a coreactant embedded ECL nanohybrid material WO 3-x The preparation method of QDs-Ru@ZnMOF includes the following steps:

[0048] 40mg Ru(bpy)3 2+ 、40mg WO 3-x QDs powder, 400 mg H3BTC and 400 mg Zn(NO3)2·6H2O were added and dissolved in 40 mL DMF / water (DMF:water=1:10, v / v) and stirred constantly at room temperature for 60 min. Then, the reactants were transferred to a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 300 °C for 1 h. After cooling to room temperature, WO was obtained by centrifugation. 3-x The QDs-Ru@ZnMOF dispersion was then washed with ethanol and deionized water to eliminate any unreacted metal ions and H3BTC. The resulting precipitate was kept in a vacuum at 150 °C for 0.5 h, and the resulting pale yellow powder was WO 3-x QDs-Ru@ZnMOF.

[0049] Example 4:

[0050] This embodiment relates to a coreactant embedded ECL nanohybrid material WO 3-x The preparation method of QDs-Ru@ZnMOF includes the following steps:

[0051] 20mg Ru(bpy)3 2+ , 25mg WO 3-xQDs powder, 300 mg H3BTC and 200 mg Zn(NO3)2·6H2O were added and dissolved in 40 mL DMF / water (DMF:water=1:4, v / v) and stirred constantly at room temperature for 35 min. Then, the reactants were transferred to a stainless steel autoclave lined with polytetrafluoroethylene and reacted at 220°C for 18 h. After cooling to room temperature, WO was obtained by centrifugation. 3-x The QDs-Ru@ZnMOF dispersion was then washed with ethanol and deionized water to eliminate any unreacted metal ions and H3BTC. The resulting precipitate was kept in a vacuum at 60 °C for 3 h, and the resulting pale yellow powder was WO 3-x QDs-Ru@ZnMOF.

[0052] Example 5:

[0053] This example involves the XPS characterization of ECL nanohybrid materials with embedded co-reactants, and the specific steps include:

[0054] The co-reactant embedded ECL nanohybrid material WO prepared in Example 1 was studied by X-ray photoelectron spectroscopy (XPS) 3-x Elemental composition information of QDs-Ru@ZnMOF. 3-x The full XPS spectrum of the QDs-Ru@ZnMOF nanohybrid material shows the peaks of Zn2p, W4f, Ru3p, O1s and C1s, which proves that the nanocomposite material contains elements such as Zn, W, Ru, O and C ( Figure 2 ), thus demonstrating the successful synthesis of co-reactant embedded ECL nanohybrid materials.

[0055] Example 6:

[0056] This embodiment relates to a method for preparing an ECL probe, and the specific steps are as follows:

[0057] 0.1 mL of EDC aqueous solution (20 mg mL -1 ) and 0.1 mL of NHS aqueous solution (10 mg mL -1 ) was added to the WO prepared in Example 1 3-x Aqueous solution of QDs-Ru@ZnMOF (1 mL, 10 mg mL -1) and shaken at 37°C for 2h. After centrifugation and thorough washing, the activated nanocomposite was obtained, collected and resuspended in 1mL 0.1M PBS (pH 7.4). Then, 1mL of 1μM MUC1 aptamer solution was added to the above solution and shaken at 37°C for 2h. Then, 0.5mL of 1wt% BSA aqueous solution was added and shaken at 37°C for 2h to block nonspecific binding sites. Finally, the ECL probe WO was obtained by centrifugation and washing. 3- x QDs-Ru@ZnMOF / Apt was resuspended in PBS solution to obtain an ECL probe (WO 3-x QDs-Ru@ZnMOF / Apt) solution (1 mg mL -1 ) and stored at 4 °C for further use.

[0058] The aptamer DNA sequence described in this example is: 5'-NH2-GCAGTTGATCCTTTGGATACCCTGG-3'.

[0059] Example 7:

[0060] This embodiment relates to a method for preparing an ECL probe, and the specific steps are as follows:

[0061] 0.01 mL of EDC aqueous solution (5 mg mL -1 ) and 0.01 mL of NHS aqueous solution (5 mg mL -1 ) was added to the WO prepared in Example 2 3-x Aqueous solution of QDs-Ru@ZnMOF (0.1 mL, 10 mg mL -1 ), shaken at 10°C for 10 h, centrifuged and thoroughly washed to obtain the activated nanocomposite material, which was collected and resuspended in 0.1 mL 0.1 M PBS (pH 7.4). Then, 0.1 mL of 0.1 μM MUC1 aptamer solution was added to the above solution and shaken at 10°C for 10 h. Then, 0.05 mL of 0.1 wt% BSA aqueous solution was added and shaken at 10°C for 10 h to block nonspecific binding sites. Finally, the ECL probe WO was obtained by centrifugation and washing with PBS solution. 3-x QDs-Ru@ZnMOF / Apt was suspended in PBS solution to obtain an ECL probe (WO 3-x QDs-Ru@ZnMOF / Apt) solution (0.1 mg mL -1 ) and stored at 0-20 °C for further use.

[0062] The aptamer DNA sequence described in this example is: 5'-NH2-GCAGTTGATCCTTTGGATACCCTGG-3'.

[0063] Example 8:

[0064] This embodiment relates to a method for preparing an ECL probe, and the specific steps are as follows:

[0065] 1 mL of EDC aqueous solution (40 mg mL -1 ) and 1 mL of NHS aqueous solution (30 mg mL -1 ) was added to the WO prepared in Example 3 3-x QDs-Ru@ZnMOF aqueous solution (8 mL, 10 mg mL -1 ), oscillated at 60°C for 0.5h, centrifuged and thoroughly washed to obtain the activated nanocomposite material, which was collected and resuspended in 10mL 0.1M PBS (pH7.4). Then, 10mL of 10μM MUC1 aptamer solution was added to the above solution and oscillated at 60°C for 0.5h. Then, 5mL of 10wt% BSA aqueous solution was added and oscillated at 60°C for 0.5h to block nonspecific binding sites. Finally, the ECL probe WO was obtained by centrifugation and washing with PBS solution. 3-x QDs-Ru@ZnMOF / Apt was suspended in PBS solution to obtain an ECL probe (WO 3-x QDs-Ru@ZnMOF / Apt) solution (5 mg mL -1 ) and stored at 20 °C for further use.

[0066] The aptamer DNA sequence described in this example is: 5'-NH2-GCAGTTGATCCTTTGGATACCCTGG-3'.

[0067] Example 9:

[0068] This embodiment relates to a method for preparing an ECL probe, and the specific steps are as follows:

[0069] 0.5 mL of EDC aqueous solution (30 mg mL -1 ) and 0.5 mL of NHS aqueous solution (20 mg mL -1 ) was added to the WO prepared in Example 1 3-x QDs-Ru@ZnMOF aqueous solution (10 mL, 10 mg mL -1), oscillated at 25°C for 6 h, centrifuged and thoroughly washed to obtain the activated nanocomposite material, which was collected and resuspended in 5 mL of 0.1 M PBS (pH 7.4). 5 mL of 5 μM MUC1 aptamer solution was then added to the above solution and oscillated at 25°C for 6 h. 25 mL of 5 wt% BSA aqueous solution was then added and oscillated at 25°C for 6 h to block nonspecific binding sites. Finally, the ECL probe WO was obtained by centrifugation and washing with PBS solution. 3-x QDs-Ru@ZnMOF / Apt was suspended in PBS solution to obtain an ECL probe (WO 3-x QDs-Ru@ZnMOF / Apt) solution (10 mg mL -1 ) and stored at 0 °C for further use.

[0070] The aptamer DNA sequence described in this example is: 5'-NH2-GCAGTTGATCCTTTGGATACCCTGG-3'.

[0071] Example 10:

[0072] This embodiment relates to a method for preparing an electrochemical sensor for measuring TEVs, and the specific steps are as follows:

[0073] (1) Electrode pretreatment: The glassy carbon electrode was polished and then polished with Al2O3 powder with a particle size of 0.5 μm and 0.03 μm, respectively. It was then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 5 min.

[0074] (2) Place the electrode treated in step (1) in a solution with a concentration of 0.05 mg mL -1 The AuNPs were electroplated in a HAuCl4 aqueous solution at -1.2 V for 100 s. After the electrode surface was thoroughly rinsed with ultrapure water, the remaining water droplets were dried with nitrogen to obtain AuNPs-modified GCE (GCE / AuNPs), which was then washed with distilled water and dried at room temperature.

[0075] (3) Covalently linking EpCAM antibodies: 6 μL of EpCAM antibody (2 μg / mL, Ab) solution was added dropwise to the electrode obtained in step (2), and the solution was placed at 4°C for 12 h to allow Ab to fully link with AuNPs. The solution was then washed with PBS solution and dried at room temperature to obtain GCE / AuNPs / Ab.

[0076] (4) Blocking nonspecific sites: 5 μL of 1 wt% BSA aqueous solution was added dropwise and allowed to stand at room temperature for 2 h to block the unbound sites. The solution was washed with PBS solution and dried at room temperature to obtain GCE / AuNPs / Ab / BSA, which is the electrochemical sensor for measuring TEVs.

[0077] The method for detecting tumor-derived extracellular vesicle antigens using the electrochemical sensor of this embodiment is as follows:

[0078] (1) adding 5 μL of the TEVs antigen sample to be tested to the surface of the electrochemical sensor, incubating at 37°C for 30 minutes, and then washing the unbound antigen with 0.1 M PBS solution to obtain GCE / AuNPs / Ab1 / BSA / TEVs;

[0079] (2) Add 6 μL of the ECL probe solution (WO 200) prepared in Example 6 to the surface of the electrode obtained in step (1). 3-x QDs-Ru@ZnMOF / Apt), incubated at 37 °C for 30 min, and the unbound ECL probe was washed with 0.1 M PBS solution to obtain GCE / AuNPs / Ab1 / BSA / TEVs / Apt / WO 3-x QDs-Ru@ZnMOF, which is the electrochemical sensor electrode to be tested;

[0080] (3) The electrochemical sensor electrode to be tested is tested in a 0.1 M PBS solution using an electrochemiluminescence detector to obtain the ECL intensity of the TEVs-specific antigen sample to be tested, and the concentration of the TEVs-specific antigen sample to be tested is determined using a standard relationship curve between current intensity and TEVs-specific antigen sample concentration.

[0081] The glassy carbon electrode obtained in each step of this example was placed in a solution containing 1.0 mM K3[Fe(CN)6] and subjected to cyclic voltammetry scanning at a rate of 0.1 V / s. The results are shown in FIG. Figure 3 As shown. Compared with GCE, a pair of significantly enhanced redox peaks can be observed after AuNPs modification of the electrode, indicating that AuNPs can improve the conductivity of the electrode and the electron transfer rate in the solution. Due to the poor conductivity of the protein, the peak value of the redox current decreases. As BSA is gradually immobilized on the electrode, the impedance of the modified electrode surface increases and the peak current gradually decreases. Next, since TEVs are non-conductive, the peak current further decreases after TEVs are immobilized on the modified electrode. Finally, the ECL signal probe (WO 3-x QDs-Ru@ZnMOF / Apt), the peak current decreased, which can be attributed to the 3-x The electronic conductivity of QDs-Ru@ZnMOF is poor. According to these results in the electrode modification process, the proposed electrochemical sensor was successfully prepared.

[0082] Example 11:

[0083] This embodiment relates to a method for preparing an electrochemical sensor for measuring TEVs, and the specific steps are as follows:

[0084] (1) Electrode pretreatment: The glassy carbon electrode was ground, polished, and ultrasonically cleaned. Specifically, the electrode was polished with 2 μm and 0.1 μm Al2O3 powders, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 10 min.

[0085] (2) Place the electrode treated in step (1) in a solution with a concentration of 0.01 mg mL -1 AuNPs were deposited in an aqueous solution of HAuCl4 at -3 V for 10 s. The electrode surface was thoroughly rinsed with ultrapure water, and the remaining water droplets were dried with nitrogen to obtain an AuNPs-modified electrode (AuNPs / electrode), which was then washed with distilled water and dried at room temperature.

[0086] (3) Covalently linking EpCAM antibodies: 1 μL of EpCAM antibody Ab solution was added to the electrode obtained in step (2) and placed at 0°C for 24 h to allow Ab to fully link with AuNPs through gold-sulfur bonds; then washed with PBS solution and dried at room temperature;

[0087] (4) Blocking nonspecific sites: add 1 μL of 0.1 wt% BSA aqueous solution, leave at room temperature for 10 h to block unbound sites, wash with PBS solution, and dry at room temperature; thus, an electrochemical sensor for measuring tumor-derived extracellular vesicles is obtained.

[0088] The concentration of Ab in step (3) of this example is 0.5 μg / mL.

[0089] The method for detecting tumor-derived extracellular vesicle antigens using the electrochemical sensor of this embodiment is as follows:

[0090] (1) Add 1 μL of the TEVs antigen sample to be tested to the surface of the electrochemical sensor, incubate at 10°C for 60 minutes, and then wash the unbound antigen with 0.01 M PBS solution;

[0091] (2) Add 6 μL of the ECL probe solution (WO 9) obtained in step (1) to the surface of the electrode. 3-x QDs-Ru@ZnMOF / Apt), incubated at 10 °C for 60 min, and the unbound ECL probe was washed with 0.01 M PBS solution to obtain the electrochemical sensor electrode to be tested;

[0092] (3) The electrochemical sensor electrode to be tested is tested in a 0.1 M PBS solution using an electrochemiluminescence detector to obtain the ECL intensity of the TEVs-specific antigen sample to be tested, and the concentration of the TEVs-specific antigen sample to be tested is determined using a standard relationship curve between the ECL intensity and the concentration of the TEVs-specific antigen sample.

[0093] Example 12:

[0094] This embodiment relates to a method for preparing an electrochemical sensor for measuring TEVs, and the specific steps are as follows:

[0095] (1) Electrode pretreatment: The glassy carbon electrode was ground, polished, and ultrasonically cleaned. Specifically, it was polished with Al2O3 powder with a particle size of 0.1 μm and 0.01 μm, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 1-10 min.

[0096] (2) Place the electrode treated in step (1) in a solution with a concentration of 1 mg mL -1 AuNPs were deposited in an aqueous solution of HAuCl4 at 0 V for 300 s. The electrode surface was thoroughly rinsed with ultrapure water, and the remaining water droplets were dried with nitrogen to obtain an AuNPs-modified electrode (AuNPs / electrode), which was then washed with distilled water and dried at room temperature.

[0097] (3) Covalently linking EpCAM antibodies: 10 μL of EpCAM antibody Ab solution was added to the electrode obtained in step (2) and placed at 20°C for 2 h to allow Ab to fully link with AuNPs through gold-sulfur bonds; then washed with PBS solution and dried at room temperature;

[0098] (4) Blocking nonspecific sites: add 10 μL of 10 wt% BSA aqueous solution, let it stand at room temperature for 0.5 h to block the unbound sites, wash with PBS solution, and dry at room temperature; thus, an electrochemical sensor for measuring tumor-derived extracellular vesicles is obtained.

[0099] The concentration of Ab in step (3) of this example is 5 μg / mL.

[0100] The method for detecting tumor-derived extracellular vesicle antigens using the electrochemical sensor of this embodiment is as follows:

[0101] (1) Add 10 μL of the TEVs antigen sample to be tested to the surface of the electrochemical sensor, incubate at 60°C for 10 minutes, and then wash the unbound antigen with 1 M PBS solution;

[0102] (2) Add 6 μL of the ECL probe solution (WO 200) prepared in Example 7 to the surface of the electrode obtained in step (1). 3-x QDs-Ru@ZnMOF / Apt), incubated at 60 °C for 10 min, and the unbound ECL probe was washed with 1 M PBS solution to obtain the electrochemical sensor electrode to be tested;

[0103] (3) The electrochemical sensor electrode to be tested is tested in a 0.1 M PBS solution using an electrochemiluminescence detector to obtain the ECL intensity of the TEVs-specific antigen sample to be tested, and the concentration of the TEVs-specific antigen sample to be tested is determined using a standard relationship curve between the ECL intensity and the concentration of the TEVs-specific antigen sample.

[0104] Example 13:

[0105] This embodiment relates to a method for preparing an electrochemical sensor for measuring TEVs, and the specific steps are as follows:

[0106] (1) Electrode pretreatment: The glassy carbon electrode was ground, polished, and ultrasonically cleaned. Specifically, the electrode was polished with Al2O3 powders with particle sizes of 0.5 μm and 0.06 μm, respectively, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 5 min.

[0107] (2) Place the electrode treated in step (1) in a solution with a concentration of 0.1 mg mL -1 AuNPs were deposited in an aqueous solution of HAuCl4 at -2 V for 150 s. The electrode surface was thoroughly rinsed with ultrapure water, and the remaining water droplets were dried with nitrogen to obtain an AuNPs-modified electrode (AuNPs / electrode), which was then washed with distilled water and dried at room temperature.

[0108] (3) Covalently linking EpCAM antibodies: 5 μL of EpCAM antibody Ab solution was added to the electrode obtained in step (2) and placed at 5°C for 15 h to allow Ab to fully link with AuNPs through gold-sulfur bonds; then washed with PBS solution and dried at room temperature;

[0109] (4) Blocking nonspecific sites: add 6 μL of 5 wt% BSA aqueous solution, let it stand at room temperature for 5 h to block the unbound sites, wash with PBS solution, and dry at room temperature; thus, an electrochemical sensor for measuring tumor-derived extracellular vesicles is obtained.

[0110] The concentration of Ab in step (3) of this example is 2 μg / mL.

[0111] The method for detecting tumor-derived extracellular vesicle antigens using the electrochemical sensor of this embodiment is as follows:

[0112] (1) Add 5 μL of the TEVs antigen sample to be tested to the surface of the electrochemical sensor, incubate at 37°C for 30 minutes, and then wash the unbound antigen with 0.1 M PBS solution;

[0113] (2) Add 6 μL of the ECL probe solution (WO 200) prepared in Example 8 to the surface of the electrode obtained in step (1). 3-x QDs-Ru@ZnMOF / Apt), incubated at 37 °C for 30 min, and the unbound ECL probe was washed with 0.1 M PBS solution to obtain the electrochemical sensor electrode to be tested;

[0114] (3) The electrochemical sensor electrode to be tested is tested in a 0.1 M PBS solution using an electrochemiluminescence detector to obtain the ECL intensity of the TEVs-specific antigen sample to be tested, and the concentration of the TEVs-specific antigen sample to be tested is determined using a standard relationship curve between the ECL intensity and the concentration of the TEVs-specific antigen sample.

[0115] Example 14:

[0116] This example studies the concentration of anti-EpCAM Ab, the reaction time of TEVs and anti-EpCAM, and the effect of WO 3-x Effect of incubation time of QDs-Ru@ZnMOF / Apt with TEVs on the sensitivity of the sensor. Electrochemical sensors with different Ab concentrations (0.1-10 μg / mL) were prepared according to the method of Example 10, and electrochemical detection was performed using DPV (differential pulse voltammetry) in 1.0 mM K3[Fe(CN)6]. The results are shown in Figure 1. Figure 4 As shown in A, as the concentration of antibody Ab1 increased from 0.1 μg·mL -1 Increase to 5 μg mL -1 , the peak current of the biosensor decreased, and further increasing the Ab1 concentration did not lead to a significant change in the current intensity. -1 The specific binding of TEVs surface membrane protein and antibody is the key step in constructing this biosensor. TEVs were detected according to the detection method and prepared electrochemiluminescence sensor of Example 10. TEVs antigen (concentration of 10000 μL) was added dropwise. -1 ) and then changed the incubation time to 10-60 min, and detected with an electrochemiluminescence detector in 0.1 M PBS solution. The results are as follows Figure 4 As shown in B, the ECL intensity response increases with the increase of immune response time. When the reaction time exceeds 30 min, the ECL intensity response tends to be constant, indicating that the fixed amount of TEVs tends to be saturated at this time. Therefore, 30 min was selected as the optimal incubation time. 3-x The effect of capture time between QDs-Ru@ZnMOF / Apt and TEVs was investigated. TEVs were detected using the electrochemical sensor prepared according to the detection method of Example 10. After adding the ECL label, the incubation time was changed to 10-60 min. Electrochemical detection was performed using the DPV (differential pulse voltammetry) technique in 0.1 M PBS solution. The results are shown in FIG. Figure 4 As shown in C. Figure 4 C shows that the ECL intensity of the biosensor increased as the incubation time increased from 10 min to 30 min and then remained constant, indicating that the optimized incubation time was 30 min.

[0117] Example 15: Determination of standard curve

[0118] Prepare different concentrations (100 μL -1 , 500 μL -1 , 1000 μL -1 , 5000 μL -1 , 10000 μL -1 , 100,000 μL -1 and 1,000,000 μL -1 ) of TEVs antigen solution were added dropwise to the surface of the biosensor prepared in Example 10, incubated at 37°C for 30 minutes, and then unbound antigens were washed with 0.1M PBS solution; 6 μL of the ECL probe solution (WO 200) prepared in Example 5 was added dropwise to the electrode surface. 3-x QDs-Ru@ZnMOF / Apt) was incubated at 37°C for 30 minutes, and the unbound ECL probe was washed with 0.1M PBS solution to obtain the electrochemical sensor electrode to be tested; the electrochemical sensor electrode to be tested was placed in a 0.1M PBS solution and tested with an electrochemiluminescence detector, and the ECL intensity corresponding to different concentrations of TEVs was obtained and plotted into a standard relationship curve. The results are shown in FIG. Figure 5 shown. Figure 5 The ECL intensity of the prepared biosensor was (100~1.0)×10 6 Particles·μL -1 The range is linearly dependent on the logarithm of the number of TEVs. As the concentration of TEVs increases, the ECL signal intensity (Y) of the biosensor increases. The standard relationship curve is Y = 2286.3logC TEVs -3903.7(R 2 =0.989), and the detection limit was 31 μL -1 , indicating that the sensor has high sensitivity.

Claims

1. An ECL nanohybrid material WO 3-x QDs-Ru@ZnMOF, characterized by Prepare as follows: Ru(bpy)3 2+ , WO 3-x QDs powder, 1,3,5-benzenetricarboxylic acid and Zn(NO3)2·6H2O were added and dissolved in DMF / water, stirred mechanically and reacted at room temperature; then, the reactants were placed at 50-300°C for 1-30 hours; after cooling to room temperature, WO was obtained by centrifugation. 3-x The QDs-Ru@ZnMOF dispersion was washed and the resulting precipitate was kept in a vacuum at 10-150 °C for 0.5-10 hours. The resulting light yellow powder was WO 3-x QDs-Ru@ZnMOF.

2. ECL nano hybrid material WO according to claim 1 3-x QDs-Ru@ZnMOF, characterized by Ru(bpy)3 2+ , WO 3-x The amounts of QDs powder, 1,3,5-benzenetricarboxylic acid and Zn(NO3)2·6H2O are 0.5-40 mg, 0.5-40 mg, 100-400 mg and 100-400 mg respectively, and the reaction time at room temperature is 5-60 minutes.

3. A method for preparing an ECL probe, characterized in that: The specific steps are: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution and N-hydroxysuccinimide aqueous solution to the WO according to claim 1 3-x The activated nanocomposite was obtained by shaking, centrifuging and washing in an aqueous solution of QDs-Ru@ZnMOF, collecting and resuspending in a PBS solution. Then, the aptamer solution of MUC1 was added to the above solution and shaken, and then a bovine serum albumin aqueous solution was added and shaken to block nonspecific binding sites. Finally, the ECL probe WO was obtained by centrifugation and washing. 3-x QDs-Ru@ZnMOF / Apt.

4. The method for preparing the ECL probe according to claim 3, wherein The aptamer DNA sequence of the present invention is: 5'-NH2-GCAGTTGATCCTTTGGATACCCTGG-3', the concentration of the MUC1 aptamer solution is 0.1-10 μM, and the dosage is 0.1-10 mL; the concentration of the bovine serum albumin aqueous solution is 0.1-10 wt%, and the dosage is 0.05-5 mL; the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution is 5-40 mg·mL -1 The dosage is 0.01-1 mL; the concentration of N-hydroxysuccinimide aqueous solution is 5-40 mg·mL -1 , the dosage is 0.01-1mL; WO 3-x The concentration of QDs-Ru@ZnMOF aqueous solution was 10 mg mL -1 .

5. The method for preparing the ECL probe according to claim 3, wherein The shaking temperature is 10-60° C., and the shaking time is 0.5-10 h.

6. An electrochemical sensor for measuring tumor-derived extracellular vesicles, characterized in that The structure is as follows: AuNPs are covered on the surface of a base electrode, EpCAM antibodies are fixed on the surface of the AuNPs, and the EpCAM antibodies can react specifically with the EPCAM protein on the surface membrane of the TEVs antigen; a TEVs antigen sample to be tested is added dropwise to the surface of the electrochemical sensor and incubated, and then the ECL probe according to any one of claims 3 to 6 is added dropwise and incubated again to obtain a sensor electrode to be tested; electrochemiluminescence detection is performed on the sensor electrode to obtain the ECL intensity of the TEVs antigen sample to be tested, and the concentration of the TEVs antigen sample to be tested is determined using a standard relationship curve between the ECL intensity and the concentration of the TEVs antigen sample.

7. A method for preparing an electrochemical sensor for measuring tumor-derived extracellular vesicles, characterized in that: The following steps are involved: (1) Electrode pretreatment: grinding, polishing and ultrasonic cleaning of the base electrode; (2) placing the electrode treated in step (1) in an aqueous solution of chloroauric acid, electroplating, rinsing, and drying to obtain an AuNPs-modified electrode; (3) Covalently linking EpCAM antibodies: adding EpCAM antibody Ab solution to the electrode obtained in step (2) to fully connect Ab and AuNPs through gold-sulfur bonds; then washing and drying; (4) Blocking nonspecific sites: Add BSA aqueous solution to block the unbound sites, thereby obtaining an electrochemical sensor for measuring tumor-derived extracellular vesicles.

8. The method for preparing an electrochemical sensor for measuring tumor-derived extracellular vesicles according to claim 7, characterized in that: The base electrode is a glassy carbon electrode; in step (1), the base electrode is polished with Al2O3 powders with particle sizes of 0.1-2 μm and 0.01-0.1 μm, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 1-10 minutes; in step (2), the concentration of the chloroauric acid aqueous solution is 0.01-1 mg·mL -1 ; Electrodeposition is performed at -3-0 V for 10-300 s; in the step (3), the concentration of Ab is 0.5-5 μg / mL, the amount of Ab solution added is 1-10 μL, and the mixture is placed at 0-20°C for 2-24 h; in the step (4), the concentration of the BSA aqueous solution is 0.1-10 wt%, and the mixture is placed at room temperature for 0.5-10 h.

9. Use of the electrochemical sensor according to claim 6 or the electrochemical sensor prepared by the method according to any one of claims 7 to 8 in the quantitative detection of tumor-derived extracellular vesicles.

10. Use of the electrochemical sensor according to claim 9 for quantitative detection of tumor-derived extracellular vesicles, characterized in that: The application method is: (1) adding a TEVs antigen sample to be tested to the surface of the electrochemical sensor, incubating at 10-60° C. for 10-60 minutes, and then washing the unbound antigen with PBS solution; (2) adding the ECL probe solution of claim 3 to the surface of the electrode obtained in step (1), incubating at 10-60° C. for 10-60 minutes, and washing the unbound ECL probe with PBS solution to obtain an electrochemical sensor electrode to be tested; (3) The electrochemical sensor electrode to be tested is tested in a 0.1 M PBS solution using an electrochemiluminescence detector to obtain the ECL intensity of the TEVs-specific antigen sample to be tested, and the concentration of the TEVs-specific antigen sample to be tested is determined using a standard relationship curve between the ECL intensity and the concentration of the TEVs-specific antigen sample.

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