Preparation method of novel electrochemical luminescence biosensor for detecting MicroRNA-126 by using nano confinement reactor WS2atCOF-LuCDs

Abstract

The invention relates to a preparation method and application of a novel electrochemical luminescence biosensor for detecting MicroRNA-126 by using a nano confinement reactor WS2 (at) COF-LuCDs, and belongs to the technical field of novel functional materials and biosensing detection. According to the invention, luminol-derived carbon dots (LuCDs) of a luminophor are assembled on a heterojunction WS2 (at) COF, and strong and stable ECL performance is shown. The enhanced performance is attributed to a limited pore environment of a COF structural domain, so that H2O2 is effectively enriched at a WS2 catalytic edge site. A self-designed DNA nano machine is applied to a sensing platform constructed by a nano constraint reactor, so that the detection accuracy is effectively improved, high sensitivity, wide detection range and extremely low detection limit can be shown for a target object, rapid and sensitive determination of miR-126 in human serum can be realized, and the method has a relatively high application value for clinical judgment of infection types of patients.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of an electrochemiluminescent biosensor based on WS2@COF-LuCDs nanoparticles, and specifically to an electrochemiluminescent biosensor for sensitively detecting MicroRNA-126 using WS2@COF-LuCDs nanoparticles as a substrate material, belonging to the technical field of novel functional materials and biosensor detection. Background Art

[0002] Electrochemiluminescence (ECL) has become a prominent focus in the field of sensing and analysis due to its advantages of low background noise and fast response time. New nanoluminescent materials such as carbon dots, non-metal-based quantum dots, and nanoclusters have attracted increasing attention due to their good biocompatibility, low toxicity, and unique optical properties. However, the low luminescence efficiency and energy loss between the sensing interfaces restrict their ECL emission and limit their usability. Therefore, exploring new strategies to enhance the ECL emission of nanofluorophores is of great significance for ECL-based bioassays; The co-reaction acceleration strategy is widely used in bioassays, in which co-reactant accelerators such as catalytic enzymes or noble metal particles analyze co-reactants to generate more active free radicals, injecting more electrons or holes into the energy level of the nanoluminophore to enhance ECL emission, which is the most common and easiest strategy to enhance ECL emission. However, with the limitation of the ECL reaction layer, traditional detection methods face challenges related to limited co-reactant contact and short lifetime of co-reactant radicals, which makes it difficult to meet the growing demand for high-sensitivity trace detection technology. Potential problems such as energy transfer between noble metals and ECL emitters or inefficient electron utilization may also hinder the use of amplifiers and the correlation between ECL intensity. Solving the underutilization of co-reactants is the key to improving ECL intensity. In this regard, local amplification of co-reactants at the electrode interface may become a potential cost-effective signal enhancement method; Tungsten disulfide (WS2) is known for its large spin-orbit coupling (≈420 meV) and unique excitonic properties, leading to an efficient peroxidase-like effect to catalyze the generation of free radicals. Unfortunately, the catalytic activity of these materials often relies on a certain cutoff boundary, showing low catalytic activity and limited adsorption activity on the basal surface, which hinders their application in ECL catalysis. A promising approach to improve their adsorption performance is nanoconfinement, where nanoconfinement serves as a reaction vessel and provides a general approach with broad applications in catalysis, hydrogen storage, and adsorption. This approach exploits the unique dimensional constraints and nanoconfined space to improve adsorption. Previous studies have demonstrated various nanoconfinement behaviors aimed at enhancing adsorption, thereby achieving enhanced ECL in nanoconfinement reactors. Covalent organic frameworks (COFs) have high porosity and unique topological environments, which promote the adsorption of small molecules in their limited pore structures. Therefore, in-depth investigation of the structure-property relationship of species transformation by integrating COFs and WS2 into heterostructures can lay the foundation for understanding the ECL enhancement mechanism and its potential significance. Summary of the invention

[0003] One of the purposes of the present invention is to obtain a WS2@COF-LuCDs composite material with superior performance by ultrasound and polymerization, and to further optimize the electrochemiluminescence activity by modification; The second purpose of the present invention is to construct a DNA nano-working machine combined with WS2@COF-LuCDs. The DNA nano-working machine obtains Target DNA by inputting MicroRNA, and prepares an electrochemiluminescent biosensor with high sensitivity, strong specificity and fast detection speed, thereby realizing ultra-sensitive detection of MicroRNA-126 (miR-126).

[0004] The technical solution of the present invention is as follows: 1. A method for preparing a novel electrochemiluminescent biosensor for detecting MicroRNA-126 using a novel nanoconfined reactor WS2@COF-LuCDs, characterized in that it comprises the following steps: 1) The detachable glassy carbon electrode (GCE) was polished on the suede surface with Al2O3 powder until it was smooth, and then ultrasonically cleaned with ultrapure water and anhydrous ethanol for 0.5 h, and dried under nitrogen; 2) 10 µL of WS2@COF-LuCDs nanoparticle solution was dropped onto the electrode surface and stored at room temperature until dry; 3) Continue to drop 8 μL of 1.5 μM DNA S1 on the surface of the glassy carbon electrode, store at room temperature until dry, and wash with ultrapure water; 4) Apply 6 μL of 1% hexanethiol (HT) to block nonspecific active sites, store at room temperature until dry, and wash with ultrapure water; 5) Continue to drop 4~10 μL of a series of different concentrations of Target DNA containing dopamine (DA) obtained by miR-126 with a concentration of 0.1 fM to 10 nM on the electrode surface, store at room temperature until dry, and wash with ultrapure water to obtain an electrochemiluminescent biosensor for detecting miR-126.

[0005] All DNA and RNA solutions were purchased from Shanghai Shenggong Biotechnology Co., Ltd. and diluted with tris buffer (100 mM NaCl, 5 mM MgCl2, pH 7.4).

[0006] The phosphate buffer solution is prepared by mixing 0.1 mol / L disodium hydrogen phosphate solution and 0.1 mol / L potassium dihydrogen phosphate, and the pH value thereof is adjusted to 7.4.

[0007] WS2@COF-LuCDs nanoparticle solution preparation steps: WS2@COF-LuCDs nanoparticles were prepared according to the existing literature. First, 0.2 g of WS2 powder was dissolved in 20 mL of water and ultrasonicated for 1.5 h in an ice bath by an ultrasonic probe. Then, 50 mg of WS2 was mixed with 98 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 42 mg of trimesic acid (BTCA) in an acetonitrile solution. Subsequently, 3.5 mL of acetic acid (HAc) was added to initiate the polymerization reaction, and the mixture was stirred vigorously at room temperature for 6 h. After completion, the mixture was washed with anhydrous ethanol and 1,4-dioxane by centrifugation, and the washing was collected by centrifugation. The obtained WS2@COPs precipitate was redispersed in a round-bottom flask containing 1,4-dioxane (28 mL), trimethylbenzene (7 mL), ultrapure water (5.2 mL) and acetic acid (8 mL). The obtained suspension was sealed and stirred at 70 °C for 48 h. After completion, it was washed several times with tetrahydrofuran and dried in vacuum overnight to obtain WS2@COFs. 0.044 g of luminol and 0.03 g of L-cysteine ​​were dispersed in 30 mL of acetic acid and ultrasonicated uniformly. The precursor was then hydrothermally reacted in a Teflon-lined reactor (180 °C, 10 h) to obtain LuCDs. 0.005 mg of chitosan was dissolved in a mixed solution of 5 mL of water and acetic acid (water: acetic acid = 4.9: 0.1 mL), 1 mg of WS2@COF was ultrasonically dispersed in 5 mL of chitosan solution, and 5 mg of LuCDs were added to the synthetic solution, mixed and shaken at room temperature for 12 h. Then WS2@COF was washed several times, and the WS2@COF-LuCDs composite material was collected after centrifugation at 12,000 rpm for 10 minutes. The precipitate was redispersed in 5~10 mL of PBS and stored at 4°C for later use.

[0008] An electrochemiluminescent biosensor based on WS2@COF-LuCDs prepared by the preparation method according to claim 1, for detecting MicroRNA-126 (miR-126), characterized in that the detection steps are as follows: The biosensor detection adopts a three-electrode system, in which the platinum wire is the counter electrode, the Ag / AgCl electrode is the reference electrode, the biosensor is the working electrode, the MPI-F signal detector is the ECL signal acquisition system, and the electrolyte is 10 mL PBS (pH7.4) containing 50 μL H2O2 (10 mol / L). The photomultiplier tube (PMT) is set to 700 V. The scanning voltage is set to -0.6 ~0.7 V, and the scanning rate is 250 mv / s.

[0009] Beneficial results of the present invention 1. The present invention uses WS2@COF-LuCDs nanoconfinement reactor to construct ECL sensing model. This enhanced performance is attributed to the confined pore environment of the COF domain, where WS2@COF exhibits more negative adsorption energy for the co-reactant H2O2, effectively enriching H2O2 at the catalytic edge of WS2. The internal electric field at the interface of WS2 and COF accelerates the electron flow, improves the catalytic activity of WS2, and realizes the confined catalytic enhancement of ECL; 2. The present invention prepares an electrochemiluminescent biosensor for the detection of MicroRNA-126 (miR-126), which has a short response time, a low detection limit, a wide linear range, and good stability. It can achieve simple, rapid, highly sensitive and specific detection, and provides a new analytical method for the early clinical detection of miR-126. Specific implementation plan Example 1 Preparation method of a novel nanoconfined reactor WS2@COF-LuCDs electrochemiluminescent biosensor for detecting MicroRNA-126: 1) The detachable glassy carbon electrode (GCE) was polished on the suede surface with Al2O3 powder until it was smooth, and then ultrasonically cleaned with ultrapure water and anhydrous ethanol for 0.5 h, and dried under nitrogen; 2) Apply 5~10 µL of WS2@COF-LuCDs nanoparticle solution to the electrode surface and store at room temperature until dry; 3) Continue to drop 8 μL of 1.5 μM DNA S1 on the surface of the glassy carbon electrode, store at room temperature until dry, and wash with ultrapure water; 4) Apply 6 μL of 1% hexanethiol (HT) to block nonspecific active sites, store at room temperature until dry, and wash with ultrapure water; 5) Continue to drop 6 μL of a series of different concentrations of Target DNA containing dopamine (DA) obtained by MicroRNA-126 (miR-126) with a concentration of 0.1 fM to 10 nM on the electrode surface, store at room temperature until dry, and wash with ultrapure water to obtain an electrochemiluminescent biosensor for detecting miR-126.

[0010] Example 2 Preparation method of a novel nanoconfined reactor WS2@COF-LuCDs electrochemiluminescent biosensor for detecting MicroRNA-126: 1) The detachable glassy carbon electrode (GCE) was polished on the suede surface with Al2O3 powder until it was smooth, and then ultrasonically cleaned with ultrapure water and anhydrous ethanol for 0.5 h, and dried under nitrogen; 2) Apply 5~10 µL of WS2@COF-LuCDs nanoparticle solution to the electrode surface and store at room temperature until dry; 3) Continue to drop 8 μL of 1.5 μM DNA S1 on the surface of the glassy carbon electrode, store at room temperature until dry, and wash with ultrapure water; 4) Apply 6 μL of 1% hexanethiol (HT) to block nonspecific active sites, store at room temperature until dry, and wash with ultrapure water; 5) Continue to drop 6 μL of a series of different concentrations of Target DNA containing dopamine (DA) obtained by miR-126 with a concentration of 0.1 nM on the electrode surface, store at room temperature until dry, and wash with ultrapure water to prepare an electrochemiluminescent biosensor for detecting miR-126.

[0011] Example 3 Preparation method of a novel nanoconfined reactor WS2@COF-LuCDs electrochemiluminescent biosensor for detecting MicroRNA-126: 1) The detachable glassy carbon electrode (GCE) was polished on the suede surface with Al2O3 powder until it was smooth, and then ultrasonically cleaned with ultrapure water and anhydrous ethanol for 0.5 h, and dried under nitrogen; 2) 10 µL of WS2@COF-LuCDs nanoparticle solution was dropped onto the electrode surface and stored at room temperature until dry; 3) Continue to drop 8 μL of 1.5 μM DNA S1 on the surface of the glassy carbon electrode, store at room temperature until dry, and wash with ultrapure water; 4) Apply 6 μL of 1% hexanethiol (HT) to block nonspecific active sites, store at room temperature until dry, and wash with ultrapure water; 5) Continue to drop 6 μL of a series of different concentrations of Target DNA containing dopamine (DA) obtained by miR-126 with a concentration of 0.1 nM on the electrode surface, store at room temperature until dry, and wash with ultrapure water to prepare an electrochemiluminescent biosensor for detecting miR-126.

[0012] Example 4 Preparation of Target DNA Containing Dopamine (DA) for Detection of miR-126 1) Place 0.5 mL of 10 mg mL-1 magnetic beads-streptavidin (Fe3O4-SA) into a 1.5 mL centrifuge tube. Then add 0.5 mL of 2X B&W buffer solution (containing 10 mmol L -1The mixture was then vibrated to form a homogeneous solution. Thereafter, the magnetic beads could be separated from the solution under the action of an external magnetic field. Meanwhile, the supernatant containing some impurities was discarded. After repeating the washing for 5 times, the obtained magnetic beads were dispersed in 0.5 mL Tris–HCl. 2) Add 6 μL of biotin-labeled S-DNA and PW DNA, incubate at 37°C for 15 min, and wash with 2X B&W buffer solution; 3) Add 2 μL of miR-126 at different concentrations. In order to photo-cut the binding of DNA to magnetic beads in the buffer solution, irradiate the sample with ultraviolet light (365 nm) for 5 min in the buffer solution and incubate for 1.5 h. Then, add 5 U of Nt.BsmAI endonuclease and cut at 37°C for 2 h, and then magnetically separate to obtain the target DNA.

[0013] Example 5 Preparation of Target DNA Containing Dopamine (DA) for Detection of miR-126 1) Place 0.5 mL of 10 mg mL-1 magnetic beads-streptavidin (Fe3O4-SA) into a 1.5 mL centrifuge tube. Then add 0.5 mL of 2X B&W buffer solution (containing 10 mmol L -1 The mixture was then vibrated to form a homogeneous solution. Thereafter, the magnetic beads could be separated from the solution under the action of an external magnetic field. Meanwhile, the supernatant containing some impurities was discarded. After repeating the washing for 5 times, the obtained magnetic beads were dispersed in 0.5 mL Tris–HCl. 2) Add 6 μL of biotin-labeled S-DNA and PW DNA, incubate at 37°C for 15 min, and wash with 2X B&W buffer solution; 3) 1 μL of miR-126 at different concentrations was added. In order to photo-cleave the binding of DNA to magnetic beads in the buffer solution, the sample was irradiated with UV light (365 nm) for 5 min in the buffer solution and then incubated for 1.5 h. Subsequently, 5 U of Nt.BsmAI endonuclease was added to cut at 37°C for 2 h, and then the target DNA was obtained by magnetic separation.

[0014] Example 6 Detection of miR-126: The biosensor detection used a three-electrode system, in which a platinum wire was used as the counter electrode, an Ag / AgCl electrode was used as the reference electrode, a biosensor constructed with a known concentration of the miR-126 to be tested was used as the working electrode, the MPI-F signal detector was used as the ECL signal acquisition system, and the electrolyte was 10 mL PBS (pH 6.0) containing 50 μL H2O2 (10 mol / L). The photomultiplier tube (PMT) was set to 700 V. The scanning voltage was set to -0.6 ~ 0.7 V, and the scanning rate was 250 mv / s.

[0015] Example 7 Detection of miR-126: The biosensor detection used a three-electrode system, in which a platinum wire was used as the counter electrode, an Ag / AgCl electrode was used as the reference electrode, a biosensor constructed with a known concentration of the miR-126 to be tested was used as the working electrode, the MPI-F signal detector was used as the ECL signal acquisition system, and the electrolyte was 10 mL PBS (pH 7.4) containing 20 μL H2O2 (10 mol / L). The photomultiplier tube (PMT) was set to 700 V. The scanning voltage was set to -0.6 ~ 0.7 V, and the scanning rate was 250 mv / s.

[0016] Example 8 Detection of miR-126: The biosensor detection used a three-electrode system, in which the platinum wire was the counter electrode, the Ag / AgCl electrode was the reference electrode, the biosensor constructed with a known concentration of the miR-126 to be tested was the working electrode, the MPI-F signal detector was the ECL signal acquisition system, and the electrolyte was 10 mL PBS (pH 7.4) containing 50 μL H2O2 (10 mol / L). The photomultiplier tube (PMT) was set to 700 V. The scanning voltage was set to -0.6 ~ 0.7 V, and the scanning rate was 100 mv / s.

Claims

1. A method for preparing a novel electrochemiluminescent biosensor for detecting MicroRNA-126 (miR-126) using a nanoconfined reactor, characterized in that: The following steps are involved: 1) The detachable glassy carbon electrode (GCE) was polished on the suede surface with Al2O3 powder until it was smooth, and then ultrasonically cleaned with ultrapure water and anhydrous ethanol for 0.5 h, and dried under nitrogen; 2) 10 µL of WS2@COF-LuCDs nanoparticle solution was dropped onto the electrode surface and stored at room temperature until dry; 3) Continue to drop 8 μL of 1.5 μM DNA S1 on the surface of the glassy carbon electrode, store at room temperature until dry, and wash with ultrapure water; 4) Apply 6 μL of 1% hexanethiol (HT) to block nonspecific active sites, store at room temperature until dry, and wash with ultrapure water; 5) Continue to drop 6 μL of a series of different concentrations of Target DNA containing dopamine (DA) obtained by miR-126 with a concentration of 0.1 fM to 10 nM on the electrode surface, store at room temperature until dry, and wash with ultrapure water to obtain an electrochemiluminescent biosensor for detecting miR-126.

2. The preparation method of the electrochemiluminescent biosensor for detecting MicroRNA-126 (miR-126) using a novel nanoconfined reactor WS2@COF-LuCDs as claimed in claim 1, wherein the WS2@COF-LuCDs nanoparticle solution is characterized in that: The preparation steps are as follows: WS2@COF-LuCDs nanoparticles are prepared according to the existing relevant literature. First, 0.2 g WS2 powder was dissolved in 20 mL water and ultrasonicated by an ultrasonic probe for 1.5 h in an ice bath. Then, 50 mg WS2 was mixed with 98 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 42 mg of trimesic acid (BTCA) in an acetonitrile solution. Subsequently, 3.5 mL of acetic acid (HAc) was added to initiate the polymerization reaction and stirred vigorously at room temperature for 6 h. After completion, the WS2@COPs precipitate was washed with anhydrous ethanol and 1,4-dioxane by centrifugation, and the washing was collected by centrifugation. The obtained WS2@COPs precipitate was redispersed in a round-bottom flask containing 1,4-dioxane (28 mL), trimethylbenzene (7 mL), ultrapure water (5.2 mL) and acetic acid (8 mL). The obtained suspension was sealed and stirred at 70 °C for 48 h. After completion, it was washed several times with tetrahydrofuran and vacuum dried overnight to obtain WS2@COFs.

3. Disperse 0.044 g of luminol and 0.03 g of L-cysteine ​​in 30 mL of acetic acid and ultrasonicate evenly. Then the precursor was hydrothermally reacted in a Teflon-lined reactor (180 °C, 10 h) to obtain LuCDs. 0.005 mg of chitosan was dissolved in a mixed solution of 5 mL of water and acetic acid (water: acetic acid = 4.9: 0.1 mL), 1 mg of WS2@COF was ultrasonically dispersed in 5 mL of chitosan solution, and 5 mg of LuCDs was added to the synthetic solution, mixed and shaken at room temperature for 12 h. Then WS2@COF was washed several times, and the WS2@COF-LuCDs composite material was collected after centrifugation at 12,000 rpm for 10 minutes. The precipitate was redispersed in 5 mL of PBS and stored at 4 °C for later use.

4. The preparation method of the electrochemiluminescent biosensor for detecting MicroRNA-126 (miR-126) using a novel nanoconfined reactor WS2@COF-LuCDs as claimed in claim 1, wherein the Target DNA containing dopamine (DA) for detecting miR-126 is characterized in that: The preparation steps are as follows: 1) Place 0.5 mL of 10 mg mL-1 magnetic beads-streptavidin (Fe3O4-SA) into a 1.5 mL centrifuge tube. Then add 0.5 mL of 2X B&W buffer solution (containing 10 mmol L -1 Tris–HCl buffer solution with a pH of 7.6; 1.0mmol L-1 EDTA; 2.0 mol L-1 NaCl). Subsequently, the mixture was vibrated to form a homogeneous solution. Thereafter, the magnetic beads could be separated from the solution under the action of an external magnetic field. At the same time, the supernatant containing some impurities was discarded. After repeating the washing for 5 times, the obtained magnetic beads were dispersed in 0.5 mL Tris–HCl. 5.2) Add 6 μL of biotinylated S-DNA and PW DNA, incubate at 37°C for 15 min, and wash with 2X B&W buffer. 6.3) Add 1 μL of miR-126 at different concentrations. In order to photo-cleave the binding of DNA to magnetic beads in the buffer solution, irradiate the sample with ultraviolet light (365 nm) for 5 min in the buffer solution and incubate for 1.5 h. Then, add 5U of Nt.BsmAI endonuclease and cut at 37°C for 2 h. Then, magnetic separation is performed to obtain the target DNA.

7. A method for preparing an electrochemiluminescent biosensor for detecting MicroRNA-126 (miR-126) using a novel nanoconfined reactor WS2@COF-LuCDs as described in claim 1, wherein the standard solutions of MicroRNA-126 (miR-126) of different concentrations are prepared by diluting 1 µM MicroRNA-126 (miR-126) with 10 mM tris buffer (100 mM NaCl, 5 mMMgCl2, pH 7.4) solution.

8. A novel nanoconfined reactor WS2@COF-LuCDs electrochemiluminescent biosensor detection method for MicroRNA-126 (miR-126), characterized in that: The detection steps are as follows: The biosensor detection used a three-electrode system, in which the platinum wire was the counter electrode, the Ag / AgCl electrode was the reference electrode, the biosensor was the working electrode, the MPI-F signal detector was the ECL signal acquisition system, and the electrolyte was 10 mL PBS (pH 7.4) containing 50 μL H2O2 (10 mol / L). The photomultiplier tube (PMT) was set to 700 V. The scanning voltage was set to -0.6 ~ 0.7 V, and the scanning rate was 250 mv / s.