Preparation, detection method and application of electrochemical luminescence detection probe

Abstract

The invention belongs to the technical field of electrochemiluminescence detection, and particularly relates to a preparation method, a detection method and application of an electrochemiluminescence detection probe, the electrochemiluminescence detection probe comprises a flower-shaped nanosphere hybrid material prepared from flower-shaped nanospheres and precious metal nanoparticles, the flower-shaped nanospheres are coated with the precious metal nanoparticles, and the precious metal nanoparticles are coated with the flower-shaped nanosphere hybrid material. Antibodies are enriched on the surfaces of the noble metal nanoparticles, so that the electrochemical luminescence detection probe, a glassy carbon electrode, a luminescent material, a detection antibody, a sealing agent and a detection sample can be prepared into an electrochemical luminescence detection sensor; the electrochemical luminescence detection probe or the electrochemical luminescence detection sensor can be applied to detection of protein and nucleic acid markers, the double-enhanced luminescence complex is utilized to improve an ECL signal, the ECL signal is stable, a signal opening state is formed, and the detection sensitivity is high. And the flower-like nanosphere hybrid material probe is used as a double quencher to close a signal, so that the detection range and the sensitivity are greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemiluminescence detection, and in particular relates to a preparation, detection method and application of an electrochemiluminescence detection probe. Background Art

[0002] Electrochemiluminescence (ECL) biosensing seamlessly combines the unique advantages of electrochemistry and chemiluminescence, offering advantages such as low background noise, simple optical setup, and superior spatiotemporal controllability. It has become one of the most accurate and established analytical methods. The principle of electrochemiluminescence resonance energy transfer (ECL-RET) lies in the overlap of the donor emission spectrum and the acceptor absorption spectrum. This overlap transfers energy from the donor excited state to the acceptor, thereby suppressing the ECL signal. Therefore, the development of an effective quenching tag is crucial to achieve a "signal-off" state and detect changes in trace analyte concentrations with high sensitivity. Summary of the Invention

[0003] In order to solve the problems in the prior art, the present invention provides a preparation, detection method and application of an electrochemiluminescence detection probe, which achieves a "signal off" state and detects changes in the concentration of trace analytes with high sensitivity.

[0004] The present invention solves the technical problem by adopting the following technical solutions:

[0005] The present invention aims to provide an electrochemiluminescence detection probe, comprising a flower-shaped nanosphere hybrid material made of flower-shaped nanospheres and noble metal nanoparticles, wherein the noble metal nanoparticles are coated on the flower-shaped nanospheres, and antibodies are enriched on the surface of the noble metal nanoparticles.

[0006] Furthermore, the flower-shaped nanospheres are CuS nanoflowers with a diameter of about 2.0 μm; and the noble metal nanoparticles are platinum noble metal nanoparticles with a diameter of 2 to 4 nm.

[0007] Flower-like nanospheres have a unique and stable spatial structure, which provides a larger specific surface area for the fixation of biological molecules, allowing them to load more precious metal nanoparticles to bind more antibody molecules and provide more binding sites.

[0008] A method for preparing an electrochemiluminescence detection probe comprises the following steps: adding flower-shaped nanospheres to 3-aminopropyltriethoxysilane, heating, stirring, and washing the mixture, and then mixing the product with noble metal nanoparticles to obtain a flower-shaped nanosphere hybrid material; redissolving the flower-shaped nanosphere hybrid material in deionized water; adding the solution to a detection antibody; and coupling the flower-shaped nanosphere hybrid material and the detection antibody via a Pt-N bond to obtain a flower-shaped nanosphere hybrid material probe, i.e., an electrochemiluminescence detection probe.

[0009] Furthermore, the preparation method of flower-shaped nanospheres includes: dissolving copper chloride dihydrate in ethylene glycol, with a molar ratio of copper chloride dihydrate to ethylene glycol of 1:9-10; mixing it with an ethylene glycol solution of thiourea under strong magnetic stirring conditions, with a molar ratio of thiourea to ethylene glycol of 3.8-4:9-10; transferring the obtained mixture to a polytetrafluoroethylene-lined stainless steel autoclave for reaction, controlling the temperature at 120°C, and reacting for 12 hours to obtain CuS flower-shaped nanosphere material.

[0010] An electrochemiluminescence detection sensor comprises the electrochemiluminescence detection probe or the electrochemiluminescence detection probe prepared by the preparation method of the electrochemiluminescence detection probe, as well as a glassy carbon electrode, a luminescent material, a detection antibody, a blocking agent, and a detection sample.

[0011] In the construction of the electrochemiluminescence detection sensor, after the luminescent material is incubated on the glassy carbon electrode, the detection antibody is added, and the IgG detection antibody is connected to the luminescent material through an Au-N bond to couple the luminescent material and the detection antibody. After blocking with a blocking agent, the test sample is added and captured using the electrochemiluminescence detection probe containing the present invention. The incubation concentration of the luminescent material is 0.1667-0.2μg / mL, preferably 0.1667μg / mL.

[0012] Furthermore, the sensor also includes a luminescent material, which includes a metal-organic framework material, a metal-organic framework hybrid material made of noble metal nanoparticles and graphite carbon nitride, wherein the noble metal nanoparticles are coated on the surface of the graphite carbon nitride and then covered on the metal-organic framework material.

[0013] Preferably, the noble metal nanoparticles are gold noble metal nanoparticles with a diameter of 14 to 16 nm.

[0014] First, graphite carbon nitride is mixed with noble metal nanoparticles, connected through Au-N bonds, stirred for reaction and then washed, and then the product is mixed with metal-organic framework materials and connected through Au and carboxyl groups to obtain the final product; the obtained metal-organic framework hybrid material is redissolved in deionized water.

[0015] Furthermore, the metal-organic framework material is MIL-100(Fe), and its preparation method includes: dissolving trimesic acid in a 1 mol / L sodium hydroxide aqueous solution to obtain a first solution, and then dissolving ferrous chloride tetrahydrate in deionized water to obtain a second solution. After both solutions are completely clarified, the first solution is added dropwise to the second solution under stirring, and stirring is continued at room temperature for 24 hours. The mixture is centrifuged and washed with deionized water three times, and then washed once with anhydrous ethanol, and then vacuum dried at 60°C for 12 hours. The molar ratio of ferrous chloride tetrahydrate, trimesic acid, sodium hydroxide and water is: 1.4-1.5:0.9-1.0:2.9-3.0:877-880.

[0016] Furthermore, the sensor also includes a detection antibody, and the mass ratio of the electrochemiluminescent detection probe to the detection antibody is 0.02-0.028:1, preferably 0.02:1; the incubation time of the electrochemiluminescent detection probe and the detection antibody is 40-80 minutes, preferably 40 minutes.

[0017] Application of an electrochemiluminescence detection probe or an electrochemiluminescence detection sensor in detecting protein and nucleic acid markers.

[0018] Furthermore, the protein marker is soluble growth-stimulating gene 2 protein (sST2). The flower-shaped nanosphere hybrid material probe is combined with a detection antibody, which is an antibody against soluble growth-stimulating gene 2 protein. The flower-shaped nanosphere hybrid material probe can specifically bind to the target to be detected through the detection antibody.

[0019] Furthermore, the buffer solution includes a potassium persulfate solution with a concentration of 0.06 M and a pH value of 7.4, a phosphate buffer solution with a concentration of 0.01 M and a pH value of 7.4, and a bovine serum albumin solution with mass concentrations of 0.6 wt % and 5.0 wt %.

[0020] Compared with the prior art, the beneficial technical effects of the present invention are:

[0021] 1. The electrochemiluminescence detection probe of the present invention adopts a flower-shaped nanosphere hybrid material probe. The flower-shaped nanosphere hybrid material probe includes flower-shaped nanospheres and noble metal nanoparticles. The noble metal nanoparticles are adsorbed on the surface of the flower-shaped nanospheres. The surface of the noble metal nanoparticles is enriched with detection antibodies. The noble metal nanoparticles coated with the electrochemiluminescence detection probe of the present invention become a qualified electrochemiluminescence receptor due to its wide absorption range, and CuS has size-dependent light absorption characteristics. Its absorption spectrum is highly matched with the ECL emission spectrum of the luminescent material we selected. At the same time, the synthesized flower-shaped nanosphere hybrid material broadens the absorption range of the flower-shaped nanospheres, improves the absorption intensity of the flower-shaped nanospheres and the noble metal material in the ultraviolet-visible region, makes it an ideal energy receptor material in the ECL resonance energy transfer (ECL-RET) system, and reduces the electrochemiluminescence intensity. Therefore, the use of this detection probe can achieve ultrasensitive detection, providing a very promising analytical tool for the early clinical diagnosis of diseases.

[0022] 2. The present invention realizes the function of ultrasensitive quantitative analysis through the flower-shaped nanosphere hybrid material probe, and uses the flower-shaped nanosphere hybrid material probe as a double quencher to effectively quench the ECL signal, thereby improving the detection sensitivity.

[0023] 3. The CuS nanoflowers of the present invention have a high specific surface area and can adsorb a large number of precious metal nanoparticles, thereby binding a large number of antibodies to capture target antigens.

[0024] 4. The present invention utilizes a dual-enhanced luminescence complex to enhance the ECL signal, stabilize the ECL signal, and form a "signal-on" state. The flower-shaped nanosphere hybrid material probe is then used as a dual quencher to enable "signal-off" and greatly improve the detection range and sensitivity.

[0025] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 TEM images of the flower-shaped nanospheres, noble metal nanoparticles and flower-shaped nanosphere hybrid materials of the present invention.

[0027] Figure 2 These are the ECL signal diagrams of the quenching effect of noble metal nanoparticles, flower-shaped nanospheres, and equal amounts of noble metal nanoparticles and flower-shaped nanospheres on the luminescent material.

[0028] Figure 3 This is a standard curve diagram for detecting sST2 in serum samples in Example 6 of the present invention. DETAILED DESCRIPTION

[0029] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0030] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0031] Example 1

[0032] Preparation method of flower-shaped nanosphere material:

[0033] 1. Dissolve 0.171 g of CuCl2·2H2O in 10 mL of ethylene glycol (EG). The solution immediately turns clear and light green, yielding Solution 1.

[0034] 2. 0.304 g of thiourea (CH4N2S) was dissolved in EG to obtain solution 2;

[0035] 3. Under strong magnetic stirring, slowly add solution 1 to solution 2;

[0036] 4. After stirring at room temperature for 3 h, the mixture was transferred to a polytetrafluoroethylene-lined stainless steel autoclave and reacted at 120 °C for 12 h;

[0037] 5. When the reactor is cooled to room temperature, take it out and wash the product 5 times with anhydrous ethanol to remove any residual substances;

[0038] 6. Then vacuum drying was performed at 80℃ for 6h to obtain CuS flower-like nanospheres. The transmission electron microscopy image is shown in Figure 1 (a).

[0039] Example 2

[0040] Preparation method of precious metal nanoparticles:

[0041] 1. Dissolve 25.7 mg of polyvinylpyrrolidone (PVP) and 2.5 mL of 20 mmol / L H2PtCl6 aqueous solution in 22.5 mL of deionized water.

[0042] 2. Slowly add 15 mL of 15.5 mmol / L sodium borohydride (NaBH4) to the mixed solution;

[0043] 3. Under magnetic stirring, the color of the solution gradually turned dark brown. The obtained platinum nanoparticle (Pt NPs) solution was stirred continuously at room temperature for 2 hours and then stored at 4°C for later use. Transmission electron microscopy showed Figure 1 (b).

[0044] Example 3

[0045] Preparation method of flower-shaped nanosphere hybrid material:

[0046] 1. Take 20 mg of the flower-shaped nanospheres (CuS NFs) prepared in Example 1 and redissolve them in 20 mL of anhydrous ethanol and vortex;

[0047] 2. Add 5 mL of 3-aminopropyltriethoxysilane (APTES) and heat to 70°C with stirring for 4 h to modify the CuSNFs with amino groups;

[0048] 3. After centrifugation and multiple washes with anhydrous ethanol, the collected precipitate was redispersed in 20 mL of anhydrous ethanol;

[0049] 4. Next, 5 mL of the noble metal nanoparticle (Pt NPs) solution prepared in Example 2 was added to 5 mL of the APTES-modified CuS NFs supernatant and stirred for 12 h to obtain a flower-like nanosphere hybrid material (Pt@CuS);

[0050] 5. Finally, the Pt@CuS was centrifuged (5000 rpm, 5 min) and thoroughly rinsed with anhydrous ethanol and deionized water;

[0051] 6. The precipitate obtained above was redispersed in 5 mL of deionized water to obtain a Pt@CuS solution. The transmission electron microscopy image is shown in Figure 1 (c) and (d).

[0052] Example 4

[0053] Preparation method of flower-shaped nanosphere hybrid material probe:

[0054] 1. Take 10 μL of the Pt@CuS solution of Example 3 above and mix with 1 mL of sST2 antibody (10 μg / mL) and incubate at 4°C for 12 h;

[0055] 2. Centrifuge (5000 rpm, 5 min) and rinse twice with PBS (0.01 M, pH 7.4);

[0056] 3. The collected flower-like nanosphere hybrid material probes were redissolved in 1 mL of PBS (0.01 M, pH 7.4) and reacted with 10 μL of BSA blocking solution (5.0 wt %) at 4 °C for 2 h to prevent nonspecific binding;

[0057] 4. The prepared flower-like nanosphere hybrid material probe was collected by centrifugation (5000 rpm, 5 min) and rinsed twice with PBS (0.01 M, pH 7.4);

[0058] 5. Disperse into 1 mL PBS (0.01 M, pH 7.4) at 4°C for further use.

[0059] Example 5

[0060] Immunosensor electrochemiluminescence detection mechanism:

[0061] 1. A glassy carbon electrode (GCE, Φ = 5 mm) was polished with alumina for 5 min to obtain a mirror-like surface, and then sonicated in deionized water and dried with nitrogen.

[0062] 2. Then, the cleaned GCE was incubated with 10 μL of luminescent material (Au-g-C3N4@MIL-100) and dried in a 37°C oven;

[0063] 3. Incubate the modified electrode with noble metal nanoparticles (Pt NPs), flower-like nanospheres (CuS NFs), and equal amounts of noble metal nanoparticles (Pt NPs) and flower-like nanospheres (CuS NFs), and dry them in a 37°C oven;

[0064] 4. Finally, the MPI-E electrochemiluminescence analysis system was used to detect that the superposition of precious metal nanoparticles (PtNPs) and flower-like nanospheres (CuS NFs) effectively weakened the electrochemiluminescence signal of Au-g-C3N4@MIL-100 by nearly three times, and the ECL quenching signal was observed. Figure 2 .

[0065] Example 6

[0066] Detection method of sST2 in serum:

[0067] 1. A glassy carbon electrode (GCE, Φ = 5 mm) was polished with alumina for 5 min to obtain a mirror-like surface, and then sonicated in deionized water and dried with nitrogen.

[0068] 2. Then, the cleaned GCE was incubated with 10 μL of luminescent material (Au-g-C3N4@MIL-100) and dried in a 37°C oven;

[0069] 3. Incubate the modified electrode with 10 μL of 10 μg / mL sST2 antibody and non-luminescent material (Au-g-C3N4@MIL-100) at 4°C for at least 12 h.

[0070] 4. Rinse the modified electrode surface three times with PBS (0.01 M, pH 7.4);

[0071] 5. Then, 0.6 wt% BSA was incubated on the electrode to block non-specific sites and rinsed three times with PBS (0.01 M, pH 7.4).

[0072] 6. Serial dilutions of sST2 stock solution of known concentrations were performed with PBS (0.01 M, pH 7.4) to the following concentrations: 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 500 pg / mL, 100 pg / mL, 50 pg / mL, 10 pg / mL, 5 pg / mL, and 1 pg / mL.

[0073] 7. Take 10 μL of sST2 dilution solution of different concentrations and add it to the modified electrode and incubate at 37°C for 1.5 hours;

[0074] 8. After the detection antibody and sST2 are bound by a specific immune reaction, the unbound free antigen is removed by washing three times with PBS (0.01 M, pH 7.4);

[0075] 9. The modified electrode was reacted with 10 μL of the flower-shaped nanosphere hybrid material probe (Pt@CuS) prepared in Example 4 at 37°C for 40 min. The solution was then rinsed three times with PBS (0.01 M, pH 7.4) and detected using an MPI-E electrochemiluminescence analysis system. The ECL signal decreased with increasing sST2 concentration. The standard curve of sST2 is shown in Figure 2. Figure 3 .

[0076] The present invention provides an electrochemiluminescence detection probe and sensor based on a sandwich immunoassay model. The electrochemiluminescence detection probe is a complex of CuS nanoflowers (Cu NFs) and Pt NPs. Pt NPs have a wide absorption range, and copper sulfide (CuS) has excellent visible light absorption capacity, which has a wide range of applications in photoelectric conversion and biosensing. It also has size-dependent light absorption characteristics, and its absorption spectrum is highly matched with the ECL emission spectrum of the luminescent material we selected. In addition, Cu NFs have a unique and stable spatial structure, providing a larger specific surface area for the fixation of biomolecules. At the same time, the synthesized flower-shaped nanosphere hybrid material broadens the absorption range of the flower-shaped nanospheres, improves the absorption intensity of the flower-shaped nanospheres and precious metal nanoparticles in the ultraviolet-visible region, making it an ideal energy acceptor material in the ECL-RET system and reducing the electrochemiluminescence intensity. Therefore, the electrochemiluminescence detection probe of the present invention can achieve ultrasensitive detection, providing a very promising analytical tool for the early clinical diagnosis of diseases.

[0077] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0078] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. An electrochemiluminescence detection probe, characterized in that The method comprises flower-shaped nanospheres and noble metal nanoparticles. The noble metal nanoparticles are coated on the flower-shaped nanospheres, and antibodies are enriched on the surface of the noble metal nanoparticles.

2. The electrochemiluminescence detection probe according to claim 1, wherein: The flower-shaped nanospheres are CuS nanoflowers with a diameter of about 2.0 μm; the noble metal nanoparticles are platinum noble metal nanoparticles with a diameter of 2 to 4 nm.

3. The method for preparing an electrochemiluminescence detection probe according to claim 1 or 2, wherein: Flower-shaped nanospheres are added to 3-aminopropyltriethoxysilane, and after heating, stirring and washing, the product is mixed with noble metal nanoparticles to obtain a flower-shaped nanosphere hybrid material. The flower-shaped nanosphere hybrid material is redissolved in deionized water, and the solution is added to the detection antibody for coupling to obtain an electrochemiluminescence detection probe.

4. The method for preparing an electrochemiluminescence detection probe according to claim 3, wherein: The preparation method of flower-shaped nanospheres includes: dissolving copper chloride dihydrate in ethylene glycol, with a molar ratio of copper chloride dihydrate to ethylene glycol of 1:9-10; mixing the solution with an ethylene glycol solution of thiourea under strong magnetic stirring, with a molar ratio of thiourea to ethylene glycol of 3.8-4:9-10; transferring the obtained mixture to a polytetrafluoroethylene-lined stainless steel autoclave for reaction, controlling the temperature at 120°C, and reacting for 12 hours to obtain a CuS flower-shaped nanosphere material.

5. An electrochemiluminescence detection sensor, characterized in that: The electrochemiluminescent detection probe comprises an electrochemiluminescent detection probe according to claim 1 or 2 or an electrochemiluminescent detection probe prepared by the preparation method of the electrochemiluminescent detection probe according to claim 3 or 4.

6. The electrochemiluminescence detection sensor according to claim 5, characterized in that: The sensor also includes a luminescent material, which includes a metal organic framework material, noble metal nanoparticles and graphite carbon nitride. The noble metal nanoparticles are coated on the surface of the graphite carbon nitride and then covered on the metal organic framework material.

7. The electrochemiluminescence detection sensor according to claim 6, characterized in that: The preparation method of the metal-organic framework material includes: dissolving trimesic acid in a 1 mol / L sodium hydroxide aqueous solution to obtain a first solution, then dissolving ferrous chloride tetrahydrate in deionized water to obtain a second solution, after both solutions are completely clarified, adding the first solution dropwise to the second solution under stirring, continuing stirring at room temperature for 24 hours, centrifuging and washing with deionized water three times, then washing once with anhydrous ethanol, and then vacuum drying at 60°C for 12 hours, wherein the molar ratio of ferrous chloride tetrahydrate, trimesic acid, sodium hydroxide and water is: 1.4-1.5:0.9-1.0:2.9-3.0:877-880.

8. The electrochemiluminescence detection sensor according to claim 5, characterized in that: The buffer solution includes a potassium persulfate solution with a concentration of 0.06M and a pH value of 7.4, a phosphate buffer solution with a concentration of 0.01M and a pH value of 7.4, and a bovine serum albumin solution with mass concentrations of 0.6wt% and 5.0wt%.

9. The electrochemiluminescence detection sensor according to claim 5, characterized in that: The sensor also includes a detection antibody. The mass ratio of the electrochemiluminescent detection probe to the detection antibody is 0.02-0.028:1, preferably 0.02:

1. The incubation time of the electrochemiluminescent detection probe and the detection antibody is 40-80 minutes, preferably 40 minutes.

10. Use of the electrochemiluminescent detection probe according to claim 1 or 2, or the method for preparing the electrochemiluminescent detection probe according to claim 3 or 4, or the electrochemiluminescent detection sensor according to any one of claims 5 to 9 in detecting protein and nucleic acid markers, characterized in that: The protein marker is soluble growth-stimulated gene-expressed 2 protein.

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