A composite material and its use as a co-reactant promoter for a luminol electrochemiluminescence system

Abstract

The application discloses a composite material and application of the composite material as a co-reaction promoter of a luminol electrochemical luminescence system. The composite material is synthesized by a solvothermal method from tungsten chloride, nickel chloride and a carbon nanomaterial. The application is an application in establishing a luminol electrochemical luminescence system. The application effectively improves the stability of a luminol-hydroxyl electrochemical luminescence system by using carbon-modified doped nickel tungsten oxide, can effectively generate a large amount of active oxygen under a low excitation voltage, effectively excites luminol to generate a strong electrochemical luminescence signal. The electrochemical luminescence system can also be used for detecting uric acid. According to the characteristics of uric acid in clearing free radicals, the free radical competition reaction of uric acid and luminol is utilized, and the electrochemical luminescence system has the remarkable advantages of high accuracy, strong specificity, good reproducibility and a large number of repeated times, is suitable for detecting various actual biological samples, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of electrochemistry, and more specifically, to a composite material and its application as a co-reaction promoter in a luminol electrochemiluminescence system. Background Technology

[0002] Electrochemiluminescence (ECL) is a highly sensitive and reliable luminescent detection technology. It involves injecting electrons or holes into luminescent groups or co-reactants, resulting in electron transfer reactions between active intermediates and free radicals generated on the electrode surface. Among various electrochemiluminescent luminescent groups, luminol has attracted widespread attention in ECL systems due to its advantages such as high emission efficiency, low oxidation potential, non-toxicity, and cost-effectiveness.

[0003] In traditional luminol electrochemiluminescence systems, hydrogen peroxide and dissolved oxygen are typically used as co-reactants to generate reactive oxygen species (ROS) for electrochemiluminescence. Hydrogen peroxide often reacts with luminol anions by generating ROS (such as singlet oxygen, hydroxyl radicals, and superoxide), leading to electrochemiluminescence emission. Antioxidants such as uric acid can compete with luminol for ROS; therefore, luminol electrochemiluminescence systems can be used to construct non-uricase biosensors for antioxidants such as uric acid.

[0004] However, hydrogen peroxide is unstable and prone to self-decomposition; while dissolved oxygen, although possessing high stability and non-toxicity, has limited solubility in electrolytes and is affected by atmospheric pressure and temperature. Therefore, it is often necessary to introduce various substances such as nanozymes, single-atom catalysts, and nanomaterials to assist in the catalytic generation of reactive oxygen species and the electrochemical oxidation of luminol. These factors inevitably limit the luminescence efficiency and stability of the luminol electrochemiluminescence system, hindering the practical application of the luminol-H₂O₂ and luminol-dissolved oxygen electrochemiluminescence systems. Existing technology (“Graphene-Modified Electrode Electrochemiluminescence Analysis for the Determination of Methimazole”) discloses a luminol electrochemiluminescence method based on an alkaline medium. The graphene primarily enhances electrode conductivity, while the hydroxide ions in the luminol system hardly participate in the reaction, failing to practically solve the problem of improving the electrochemical oxidation performance of luminol. Therefore, it is necessary to explore highly stable co-reaction promoters to enhance the performance of the luminol electrochemiluminescence system. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention explores co-reactants with high stability and co-reaction promoters with high catalytic activity to construct an electrochemiluminescence system with high stability and high luminescence intensity, and provides a composite material and its application as a co-reaction promoter in a luminol electrochemiluminescence system.

[0006] The first objective of this invention is to provide a composite material.

[0007] A second objective of this invention is to provide the application of the composite material as a co-reaction promoter in the detection of antioxidants in a luminol electrochemiluminescence system.

[0008] A third objective of this invention is to provide an electrochemiluminescence system for detecting antioxidants.

[0009] A fourth object of the present invention is to provide the application of the electrochemiluminescence system in the detection of antioxidants.

[0010] The fifth object of the present invention is to provide a method for detecting antioxidant content.

[0011] To achieve the above objectives, the present invention is implemented through the following solution:

[0012] Tungsten can form high-oxidation-state oxides in oxidation reactions, which gives it high oxidation activity and promotes the oxygen evolution reaction. Furthermore, its strong synergistic effect with other metals enhances their catalytic activity, making tungsten oxides excellent catalysts.

[0013] Based on the reactive oxygen species-mediated electrochemiluminescence mechanism, this invention proposes a novel method to improve the electrochemiluminescence performance of luminol-based electrochemiluminescence systems. A luminol-hydroxide electrochemiluminescence system is established by using hydroxyl radicals as a co-reactant of luminol. Carbon-modified nickel-doped tungsten oxide (Ni-WOx-CNT) is added as a co-reaction promoter, effectively catalyzing the generation of reactive oxygen species from hydroxyl radicals in a low cyclic voltammetric potential range (0V–0.2V), thereby improving the electrochemiluminescence performance of luminol.

[0014] This invention confirms that reactive oxygen species are formed during the electrochemical oxidation of hydroxide ions, subsequently reacting with luminol anion radicals to trigger a strong electrochemiluminescence signal, which maintains extremely high stability in 1300 consecutive tests. This invention also combines in-situ Raman spectroscopy, EPR spectroscopy, and radical scavenging tests to trace the intermediate process of luminol electrochemiluminescence emission.

[0015] A composite material is synthesized from tungsten chloride, nickel chloride, and carbon nanomaterials via a solvothermal method; the mass ratio of the tungsten chloride, nickel chloride, and carbon nanomaterials is (1-7):(1-7):(0.3-1.6). The CAS number of the tungsten chloride is 13283-01-7. The CAS number of the nickel chloride is 7791-20-0.

[0016] Preferably, the mass ratio of tungsten chloride, nickel chloride and carbon nanomaterials is (1-7):(3-5):(1-1.3).

[0017] More preferably, the mass ratio of tungsten chloride, nickel chloride and carbon nanomaterials is 3:3:1.

[0018] Preferably, the reaction solvent in the solvothermal method is anhydrous ethanol.

[0019] More preferably, the solvothermal method includes the following steps: thoroughly mixing tungsten chloride, nickel chloride, carbon nanomaterials and reaction solvent, and then heating and reacting them to obtain the final product.

[0020] More preferably, tungsten chloride and nickel chloride are thoroughly mixed with the reaction solvent, and then thoroughly mixed with carbon nanomaterials to obtain a uniform suspension.

[0021] More preferably, the reaction is carried out under full heating in a high-pressure hydrothermal reactor.

[0022] More preferably, the heating conditions include heating at 140°C to 160°C for 10 to 15 hours.

[0023] More preferably, the heating conditions include heating at 150°C for 12 hours.

[0024] More preferably, after being fully heated and reacted, the mixture is also washed and dried.

[0025] Preferably, the carbon nanomaterial is a carbon nanotube or graphene.

[0026] More preferably, the carbon nanotubes have a length of 4 nm to 6 nm and a purity of >98%.

[0027] Specifically, the solvothermal method includes the following steps:

[0028] Dissolve 90 mg of tungsten chloride (CAS No.: 13283-01-7) in 20 mL of anhydrous ethanol to form a clear solution, yielding a tungsten chloride ethanol solution with a concentration of 4.5 mg / mL. Dissolve 90 mg of nickel chloride (CAS No.: 7791-20-0) in 10 mL of anhydrous ethanol to form a nickel chloride ethanol solution with a concentration of 9 mg / mL.

[0029] 20 mL of a 4.5 mg / mL tungsten chloride ethanol solution and 10 mL of a 9 mg / mL nickel chloride ethanol solution were mixed and stirred for 15 minutes until fully mixed. Then, 30 mg of carbon nanotubes (purity >98%, length 4 nm–6 nm, final concentration 1 mg / mL) were added and stirred for 10 minutes to form a homogeneous suspension. The resulting mixture was transferred to the lining of a 50 mL high-pressure hydrothermal reactor and heated at 150 °C for 12 hours. The powdered reactant was collected, washed three times with water, then three times with ethanol, and finally dried in a vacuum oven at 70 °C for 12 hours to obtain the final product.

[0030] The application of any of the aforementioned composite materials as a co-reaction promoter in the detection of antioxidants in the luminol electrochemiluminescence system should also be within the scope of protection of this invention.

[0031] The luminol-hydroxyl electrochemiluminescence system provided by this invention, in electrochemical testing, shows that hydroxyl groups are adsorbed onto Ni-WO3. x -CNT surface. Then, in a forward electrochemical cyclic voltammetry scan, hydroxyl groups were reacted with Ni-WO3 at approximately 0.2 V. x -CNTs are catalytically oxidized to generate reactive oxygen species (Formula 1), which are then reacted with luminol anions (L... 2- ) Luminol anion free radicals generated by electrochemical oxidation (L ·2- Adsorption forms an intermediate (Formula 2). Simultaneously, a small amount of luminol anion radicals react with surface-adsorbed reactive oxygen species to form excited-state 3-aminophthalate anions (AP). 2-* (Formula 3). Subsequently, AP 2-* Relaxing to the ground state, a weak electrochemiluminescence signal is generated (Equation 4). During this process, a large number of intermediates undergo reduction reactions, producing a large amount of AP. 2-* (Formula 3), which ultimately leads to the generation of a strong ECL signal (Formula 4). The specific details of Formulas 1-4 above are as follows:

[0032] Formula 1: OH - →ROS+O2;

[0033] Formula 2: L 2- →L ·2- ;

[0034] Formula 3: L ·2- +ROS→AP 2-* +N2;

[0035] Formula 4: AP 2-* →AP 2- +hv.

[0036] Therefore, the luminol-hydroxyl electrochemiluminescence system provided by the present invention can detect antioxidants that exist in strong alkaline solutions and consume ROS, including but not limited to: uric acid, cysteine ​​and superoxide dismutase.

[0037] This invention claims protection for an electrochemiluminescence system for detecting antioxidants, comprising a three-electrode system, luminol, and an electrolyte; the working electrode of the three-electrode system is modified with a co-reaction promoter for catalyzing the oxidation of hydroxide ions to generate reactive oxygen species; the co-reaction promoter is the composite material; the luminol is used to compete with the antioxidant for binding to the reactive oxygen species; the electrolyte is a solution containing hydroxide ions with a pH of 12-14.

[0038] Preferably, the pH of the electrolyte is 13.5.

[0039] Preferably, the solution containing hydroxide ions is a potassium hydroxide solution.

[0040] More preferably, the concentration of the potassium hydroxide solution is 0.05M to 0.2M.

[0041] More preferably, the concentration of the potassium hydroxide solution is 0.1M.

[0042] Preferably, the working concentration of luminol is 1 μM to 20 μM.

[0043] More preferably, the working concentration of the luminol is 10 μM.

[0044] Preferably, the electrolyte contains the luminol.

[0045] Preferably, the temperature of the electrolyte is 10°C to 50°C.

[0046] More preferably, the temperature of the electrolyte is 20°C to 40°C.

[0047] Preferably, the working electrode of the three-electrode system is a glassy carbon electrode modified with the co-reaction promoter.

[0048] More preferably, the modification method includes the following steps: fully covering the detection end of the working electrode with an aqueous solution of the co-reaction promoter, and then drying.

[0049] More preferably, the inner diameter of the glassy carbon electrode is 8 mm.

[0050] More preferably, the inner diameter of the glassy carbon electrode is 8 mm, and the amount of the co-reaction promoter is 40 μg to 60 μg.

[0051] More preferably, the amount of the co-reaction promoter is 50 μg.

[0052] More preferably, the aqueous solution contains 0.04% wt to 0.06% wt of perfluorosulfonic acid resin.

[0053] More preferably, the aqueous solution contains 0.05% wt perfluorosulfonic acid resin.

[0054] Preferably, the reference electrode of the three-electrode system is a silver / silver chloride electrode.

[0055] Preferably, the counter electrode of the three-electrode system is a platinum wire electrode.

[0056] Preferably, the electrochemiluminescence system further includes an electrochemical workstation connected to the three-electrode system.

[0057] More preferably, the electrochemical workstation includes an ultra-weak luminescence analyzer.

[0058] More preferably, the settings parameters of the electrochemical workstation include: a voltage range of 0 to (0.2V to 0.4V) for cyclic voltammetry scanning; and a scan rate of 30 mV / s. -1 ~50mV s -1 .

[0059] More preferably, the voltage range of the cyclic voltammetric scan is 0V to 0.4V.

[0060] More preferably, the scan rate is 50 mV / s. -1 .

[0061] Preferably, the electrochemiluminescence system further includes a reaction cell, in which the electrolyte is disposed.

[0062] More preferably, the reaction vessel is a quartz beaker.

[0063] This invention utilizes the excellent electrochemiluminescence performance of the established luminol-hydroxyl system. Based on the competitive reaction of uric acid and luminol against reactive oxygen species, and combined with the free radical scavenging properties of uric acid, a uric acid electrochemiluminescence biosensor was prepared. This sensor exhibits a linear relationship in the range of 0.01 μM to 10 μM, has good anti-interference ability, and achieves satisfactory sensitivity and reproducibility in the analysis of uric acid in real samples (human blood, sweat, and urine). Furthermore, the working electrode is reusable, greatly reducing the detection cost.

[0064] Preferably, the antioxidant is uric acid.

[0065] The application of any of the described electrochemiluminescence systems in the detection of antioxidants should also be within the scope of protection of this invention.

[0066] The application of any of the described electrochemiluminescence systems in the preparation of products for detecting antioxidants should also be within the scope of protection of this invention.

[0067] A method for detecting antioxidant content, using any of the electrochemiluminescence systems described above to detect the sample, includes the following steps:

[0068] The sample to be tested, luminol, and electrolyte are thoroughly mixed to obtain a reaction solution; the luminescence intensity of the reaction solution is detected using the three-electrode system, and the results are obtained through analysis.

[0069] Preferably, it includes the following steps:

[0070] The sample to be tested, luminol, and electrolyte are thoroughly mixed to obtain reaction solution 1. The standard of the antioxidant, luminol, and electrolyte are thoroughly mixed to obtain reaction solution 2. The luminescence intensity of reaction solution 1 and reaction solution 2 are detected by the three-electrode system, and the detection results are analyzed to obtain the antioxidant content of the sample to be tested.

[0071] More preferably, the antioxidant standards of different concentrations are thoroughly mixed with luminol and electrolyte to obtain several reaction solutions 2. The luminescence intensity of the reaction solutions 2 is detected by the three-electrode system to construct a standard curve. The luminescence intensity of the reaction solutions 1 is then substituted into the linear regression equation of the standard curve to obtain the antioxidant content of the sample to be tested.

[0072] Preferably, the conditions for detecting the luminescence intensity include: a cyclic voltammetric scan voltage range of 0 to (0.2V to 0.4V); and a scan rate of 30 mV / s. -1 ~50mV s -1 .

[0073] More preferably, the voltage range of the cyclic voltammetric scan is 0V to 0.4V.

[0074] More preferably, the scan rate is 50 mV / s -1 .

[0075] Preferably, the temperature of the electrolyte is 10°C to 50°C.

[0076] More preferably, the temperature of the electrolyte is 20°C to 40°C.

[0077] Preferably, the antioxidant is uric acid.

[0078] Compared with the prior art, the present invention has the following beneficial effects:

[0079] This invention uses hydroxide ions as a co-reactant in luminol electrochemiluminescence and carbon-modified nickel-doped tungsten oxide as a co-reaction promoter, effectively improving the signal intensity and stability of the luminol electrochemiluminescence system. This system can effectively generate a large amount of reactive oxygen species at low excitation voltage, effectively exciting luminol to produce a strong electrochemiluminescence signal, reducing side reactions during testing, and improving detection accuracy. This electrochemiluminescence system can also be used to detect uric acid. Based on the free radical scavenging properties of uric acid, it utilizes the free radical competition reaction between uric acid and luminol, exhibiting significant advantages such as high accuracy, strong specificity, good reproducibility, and high repeatability. It is suitable for the detection of various real biological samples and has broad application prospects. Attached Figure Description

[0080] Figure 1 for (A)Ni-WO xX-ray powder diffraction pattern of CNT; (B) Ni-WO x Scanning electron microscopy image of CNTs; (C)Ni-WO x - High-resolution transmission electron microscopy image of CNTs, and energy dispersive spectroscopy (EDS) spectra of nickel, carbon, tungsten, and oxygen.

[0081] Figure 2 X-ray energy dispersive spectroscopy for comparison of nickel and tungsten content.

[0082] Figure 3 X-ray photoelectron spectroscopy (XPS) of (A) nickel phosphide (Ni2p); (B) tungsten (W4f); (C) oxygen (O1s); (D) Ni-WO x X-ray photoelectron spectroscopy of CNTs.

[0083] Figure 4 For (A) Ni-WO x Cyclic voltammetry curves and (B) electrochemiluminescence curves of the luminol-hydroxyl electrochemiluminescence system with CNT as a co-reaction promoter; (C) Ni-WO x - Electron paramagnetic resonance spectrum of CNTs.

[0084] Figure 5 (A) ECL curves of the luminol-hydroxyl electrochemiluminescence system in saturated solutions of air, nitrogen, and oxygen; (B) ECL curves of the luminol-hydroxyl electrochemiluminescence system in 0.1 mol L⁻¹ solutions. -1 KCl solution and containing 10 μmol L -1 Ni-WO3 is used in luminol KOH solution x Electrochemiluminescence curves of the CNT-based co-reactant / promoter system.

[0085] Figure 6 The effects of (A) thiourea (hydroxyl radical scavenger), benzoquinone (superoxide radical scavenger), and tryptophan (singlet oxygen scavenger) on the quenching of the luminol-hydroxyl electrochemiluminescence system; (B) Ni-WO x -In-situ Raman spectra of CNTs.

[0086] Figure 7 For Ni-WO x A schematic diagram of the principle of the luminol-hydroxyl electrochemiluminescence system with -CNT as a co-reaction promoter.

[0087] Figure 8 For WO x X-ray powder diffraction pattern of CNT.

[0088] Figure 9The effect of different amounts of co-reactant promoters (A) nickel chloride and (B) carbon nanotubes on the electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system.

[0089] Figure 10 for(A)WO x -CNT and (B)Ni-WO x The scanning electron microscope image.

[0090] Figure 11 The electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system when carbon nanotubes are used as a co-reaction promoter.

[0091] Figure 12 forNi-WO x The modified glassy carbon electrode before (A) and after (B) the test, and the corresponding electrolyte before (C) and after (D) the test.

[0092] Figure 13 forNi-WO x - CNT-modified glassy carbon electrode before (A) and after (B) testing, and the corresponding electrolyte before (C) and after (D) testing.

[0093] Figure 14 for (A)Ni-WO x -CNT and Ni-WO x Fourier transform infrared spectrum; (B) Ni-WO x -CNT-modified glassy carbon electrode and Ni-WO x Comparison of electrochemical impedance results for modified glassy carbon electrodes.

[0094] Figure 15 For Ni-WO x -Stability test results of the graphene-luminol-hydroxyl electrochemiluminescence system.

[0095] Figure 16 For different temperatures, (A) the electrochemiluminescence curves of the luminol-hydrogen peroxide system, (B) the dissolved oxygen content in water of the luminol-hydrogen peroxide system, and (C) the Ni-WO3-based... x Electrochemiluminescence curves of the luminol-hydroxyl electrochemiluminescence system of -CNT.

[0096] Figure 17 For Ni-WO x Electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system of -CNT at different electrochemical cyclic voltammetry tests at (A) voltage range and (B) scan rate.

[0097] Figure 18Ni-WO3 under different electrochemical cyclic voltage ranges x -CNT modification of the bubble situation on the surface of glassy carbon electrode.

[0098] Figure 19 The electrochemiluminescence curves (A) and electrochemical cyclic voltammetry curves (B) of the system under different voltage ranges after bubble removal are shown.

[0099] Figure 20 For Ni-WO x Long-term stability test results of the luminol-hydroxyl electrochemiluminescence system of -CNT.

[0100] Figure 21 (A) Schematic diagram of the luminol-hydroxyl electrochemiluminescence system for detecting uric acid; (B) luminol-hydroxyl system in the range of 0.1–10 μmol / L -1 Electrochemiluminescence intensity under uric acid; (C)Ni-WO x - Detection results of using CNT-modified glassy carbon electrode as working electrode for 6 consecutive times; (D) Specificity test results of uric acid detection using the constructed luminol-hydroxyl electrochemiluminescence system.

[0101] Figure 22 0.1–10 μmol L -1 Electrochemiluminescence curves of the luminol-hydroxyl electrochemiluminescence system under uric acid.

[0102] Figure 23 For (A) ascorbic acid and (B) dopamine, respectively at 0.1 mol / L -1 Electrochemical cyclic voltammetry curves of KOH solution (pH=13.5) and PBS (pH=7.4).

[0103] Figure 24 forNi-WO x The results of (A) long-term stability test and (B) reproducibility test of the CNT-modified glassy carbon electrode as the working electrode. Detailed Implementation

[0104] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0105] Example 1: Establishment of a luminol-hydroxyl electrochemiluminescence system

[0106] I. Experimental Methods

[0107] 1. Preparation of co-reaction accelerators

[0108] This invention employs a solvothermal method to synthesize carbon-modified nickel-doped tungsten oxide (named Ni-Wo). x -CNT), the specific preparation steps are as follows:

[0109] Dissolve 90 mg of tungsten chloride (CAS No.: 13283-01-7) in 20 mL of anhydrous ethanol to form a clear solution, yielding a tungsten chloride ethanol solution with a concentration of 4.5 mg / mL. Dissolve 90 mg of nickel chloride (CAS No.: 7791-20-0) in 10 mL of anhydrous ethanol to form a nickel chloride ethanol solution with a concentration of 9 mg / mL.

[0110] 20 mL of a 4.5 mg / mL tungsten chloride ethanol solution and 10 mL of a 9 mg / mL nickel chloride ethanol solution were mixed and stirred for 15 minutes until fully mixed. Then, 30 mg of carbon nanotubes (purity >98%, length 4 nm–6 nm, final concentration 1 mg / mL) were added and stirred for 10 minutes to form a homogeneous suspension. The resulting mixture was transferred to the lining of a 50 mL high-pressure hydrothermal reactor and heated at 150 °C for 12 hours. The powdered reactant was collected, washed three times with water, then three times with ethanol, and finally dried in a vacuum oven at 70 °C for 12 hours to obtain the final product.

[0111] 2. Identification of co-reaction accelerators

[0112] Ni-Wo was identified using X-ray powder diffraction (XRD), scanning electron microscopy, high-resolution transmission electron microscopy (HRTEM), X-ray energy dispersive spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS), respectively. x -CNT structure.

[0113] 3. Construction and evaluation of luminol-hydroxyl electrochemiluminescence system

[0114] A classic three-electrode system was used, with an 8mm inner diameter glassy carbon electrode as the working electrode, a silver / silver chloride electrode as the reference electrode, and a platinum wire electrode as the counter electrode. A transparent quartz beaker was used as the reaction cell, and a 0.1M KOH solution (pH = 13.5) containing 10μM luminol was used as the electrolyte (temperature: 25℃). These components constituted the luminol-hydroxyl electrochemiluminescence system.

[0115] The luminescence performance of the luminol-hydroxyl electrochemiluminescence system was detected using an ultra-weak luminescence analyzer.

[0116] During testing, the co-reaction promoter obtained in this embodiment was prepared into a 5 mg / mL suspension using a solvent (an aqueous solution containing 0.05% wt perfluorosulfonic acid resin (Nafion). Then, 10 μL of the above suspension was drop-coated onto the detection end surface of the working electrode and dried at room temperature to obtain the modified working electrode.

[0117] The modified working electrode, reference electrode, and counter electrode were connected to the electrochemical workstation (ultra-weak luminescence analyzer) and placed in a reaction cell containing electrolyte, then transferred to the electrochemiluminescence testing platform. The voltage range for the cyclic voltammetry scan was set to 0V–0.4V, and the scan rate was 50 mV / s. -1 The light signal was collected using an ultra-weak luminescence analyzer with the photomultiplier tube voltage set to 500V. The electrochemiluminescence intensity, cyclic voltammetry curve, and electrochemiluminescence (ECL) curve were recorded. The reaction process was monitored using an electron paramagnetic resonance spectrometer (manufacturer: Bruker (USA); model: ELEXSYS-II E500) and a laser confocal micro Raman spectrometer (manufacturer: Renishaw (UK); model: inVia).

[0118] II. Experimental Results

[0119] 1. Identification results of co-reaction accelerators

[0120] like Figure 1 As shown in Figure A, the X-ray diffraction (XRD) pattern confirms the high purity of tungsten trioxide (WO3, JCPDS#20-1324) and tungsten dioxide (WO2, JCPDS#32-1393). Figure 1 As shown in B, Ni-WO x -CNTs exhibit a unique nanoflower morphology composed of interconnected nanosheets with nanowires between them. This unique structure promotes electron transfer, increases the abundance of active sites, and improves overall stability and catalytic performance. Figure 1 As shown in C, the HRTEM image shows Ni-WO x -CNT and the elemental mappings corresponding to nickel, carbon, tungsten, and oxygen show that WO x It is uniformly doped with Ni, O and C atoms.

[0121] like Figure 2 The X-ray energy dispersive spectroscopy (EDS) showing the nickel-tungsten content comparison, and as shown... Figure 3 The X-ray photoelectron spectroscopy (XPS) spectra of Ni 2p shown in A and D both confirm the presence of Ni-WO3. x - Trace amounts of nickel are present in CNTs. For example... Figure 3 As shown in B and D, Ni-WO xThe W4f spectrum of -CNT can be deconvoluted into three bands. The peak near 35.79 eV belongs to the W4f5 / 2 of WO2, while the peaks near 36.15 and 38.14 eV belong to the W4f of WO3. 7 / 2 and W 4f 5 / 2 High-resolution XPS spectra of O 1s, such as Figure 3 As shown in C and D, the peak near 530.97 eV belongs to the WO bond, the peak near 531.46 eV belongs to the C=O bond, and the peak near 532.65 eV belongs to the OH bond.

[0122] The above characterization indicates that the present invention has successfully synthesized Ni-WO3 with a nanoflower structure mainly composed of nanosheets assembled together. x -CNT.

[0123] 2. Luminescent properties of the luminol-hydroxyl electrochemiluminescence system

[0124] like Figure 4 As shown in Figure A, the cyclic voltammetry curve shows an oxidation peak near 0.22 V and a small reduction peak near 0.13 V; there are two electrochemiluminescence peaks, one of which has an intensity ( e I) is approximately 85563 (au), significantly higher than the electrochemiluminescence intensity of luminol systems disclosed in the prior art (Table 1), while another smaller peak shows an ECL intensity of 2833 (au). For example... Figure 4 As shown in B, during the negative scan of the electrochemical cyclic voltammetry, a cathode reduction current of approximately 0.13 V triggers a superior ECL peak at approximately 0.01 V. (See Figure B.) Figure 4 As shown in C, Ni-WO x Electron paramagnetic resonance spectroscopy of reactive oxygen species in -CNT confirmed the generation of hydroxyl radicals, singlet oxygen, and superoxide radicals.

[0125] like Figure 5 As shown in Figure A, the ECL performance of the luminol-hydroxyl electrochemiluminescence system established in this invention did not change significantly under three different conditions: saturated nitrogen (deoxygenated), oxygen (oxygen-enriched), and air electrolyte solutions. Figure 5 As shown in B, when using 0.1 mol L -1 KCl solution (containing 10 μmol L) -1 When luminol was used as the electrolyte, no obvious electrochemiluminescence peak was observed in the system.

[0126] like Figure 6As shown in Figure A, the addition of thiourea, benzoquinone, and tryptophan quenched the electrochemiluminescence intensity by 45.9%, 62.3%, and 65%, respectively, further demonstrating the presence of hydroxyl radicals, singlet oxygen, and superoxide radicals in the luminol-hydroxyl electrochemiluminescence system of this invention. Figure 6 As shown in B, the intermediate substances in the electrochemiluminescence process were monitored by in-situ Raman spectroscopy at 1140 cm⁻¹. -1 The nearby peak can be attributed to the OO stretching vibration of the superoxide radical, indicating that the electrochemical reduction of hydroxide ions to generate reactive oxygen species occurs at 0.2 V during the positive electrochemical cyclic voltammetry scan.

[0127] The above results indicate that, unlike other ECL systems based on dissolved oxygen, the active oxygen source of the luminol-hydroxyl electrochemiluminescence system established in this invention is hydroxyl, and is unrelated to dissolved oxygen in the solution.

[0128] 3. Luminescence mechanism of the luminol-hydroxyl electrochemiluminescence system

[0129] Based on the above results, a schematic diagram of the electrochemiluminescence process of the luminol-hydroxyl electrochemiluminescence system is shown below. Figure 7 As shown, the details are as follows:

[0130] First, in the electrochemical test, hydroxide ions were adsorbed onto Ni-WO3. x -CNT surface. Then, in a forward electrochemical cyclic voltammetry scan, hydroxyl groups were reacted with Ni-WO3 at approximately 0.2 V. x -CNTs are catalytically oxidized to generate reactive oxygen species (Formula 1), which are then reacted with luminol anions (L-16N) in the KOH solution. 2- ) Luminol anion free radicals generated by electrochemical oxidation (L ·2- Adsorption forms an intermediate (Formula 2). Simultaneously, a small amount of luminol anion radicals react with surface-adsorbed reactive oxygen species to form excited-state 3-aminophthalate anions (AP). 2-* (Formula 3). Subsequently, AP 2-* Relaxing to the ground state, a weak electrochemiluminescence signal is generated (Equation 4). During this process, a large number of intermediates undergo reduction reactions, producing a large amount of AP. 2-* (Formula 3), which ultimately leads to the generation of a strong ECL signal (Formula 4). The specific details of Formulas 1-4 above are as follows:

[0131] Formula 1: OH - →ROS+O2;

[0132] Formula 2: L 2- →L ·2- ;

[0133] Formula 3: L ·2-+ROS→AP 2-* +N2;

[0134] Formula 4: AP 2-* →AP 2- +hv.

[0135] Example 2: Effect of the luminol-hydroxyl electrochemiluminescence system

[0136] I. Experimental Methods

[0137] 1. Co-reaction accelerator

[0138] (1) Dosage of nickel chloride

[0139] The method is the same as step 1 of Example 1, “Preparation of Co-reaction Promoter”, except that the final concentration of nickel chloride in the suspension is 0 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL or 7 mg / mL.

[0140] (2) Amount of carbon nanotubes

[0141] The method is the same as step 1 of Example 1, “Preparation of Co-reaction Promoter”, except that the mass of carbon nanotubes added is 0 mg, 9 mg, 18 mg, 30 mg, 39 mg or 48 mg, that is, the concentration of carbon nanotubes in the mixture is 0 mg / mL, 0.3 mg / mL, 0.6 mg / mL, 1 mg / mL, 1.3 mg / mL and 1.6 mg / mL.

[0142] (3) Graphene modification

[0143] The method is the same as step 1 of Example 1, “Preparation of Co-reaction Promoter”, except that 30 mg of carbon nanotubes is replaced with 30 mg of graphene.

[0144] The co-reaction promoter obtained after adjusting the amount of nickel chloride, carbon nanotubes, and graphene was used in subsequent experiments according to step 3 of Example 1, "Construction and luminescence performance evaluation of luminol-hydroxyl electrochemiluminescence system".

[0145] 2. Temperature

[0146] The co-reaction accelerator Ni-Wo prepared in Example 1 x -CNT, follow the steps of Example 1, step 3, "Construction and Evaluation of Luminol-Hydroxide Electrochemiluminescence System", for subsequent experiments, the only difference being that the temperature of the electrolyte in the reaction cell is set to 20℃, 30℃ or 40℃ respectively.

[0147] 3. Parameters of electrochemical cyclic voltammetry test

[0148] (1) Voltage

[0149] The co-reaction accelerator Ni-Wo prepared in Example 1 x -CNT, follow the steps of Example 1, step 3, "Construction and Evaluation of Luminol-Hydroxide Electrochemiluminescence System", for subsequent experiments, with the only difference being that the voltage range of the cyclic voltammetry scan is set to 0V~0.2V, 0V~0.3V, 0V~0.4V or 0V~0.5V.

[0150] (2) Scan rate

[0151] The co-reaction accelerator Ni-Wo prepared in Example 1 x -CNT, follow the steps in Example 1, step 3, "Construction and Evaluation of Luminol-Hydroxide Electrochemiluminescence System", for subsequent experiments, the only difference being that the scan rate was set to 30 mVs. -1 40mV s -1 50mV s -1 60mV s -1 or 70mV s -1 .

[0152] 4. Number of tests

[0153] The co-reaction accelerator Ni-Wo prepared in Example 1 x -CNT, according to step 3 of Example 1, “Construction and Evaluation of Luminol-Hydroxyl Electrochemiluminescence System”, continuously tested for 30,000 seconds, which is equivalent to about 1,300 continuous luminescence tests.

[0154] II. Experimental Results

[0155] 1. Co-reaction accelerator

[0156] (1) Dosage of nickel chloride

[0157] like Figure 8 As shown, in the absence of nickel chloride (i.e., the concentration of the nickel chloride ethanol solution is 0 mg / mL), the synthesized co-reactant accelerator (denoted as WO) x The peaks of -CNT are attributed to WO2 (JCPDS#32-1393) and WO3 (JCPDS#32-1394). For example... Figure 9 As shown in A, when nickel chloride is not used, the synthesized co-reactant accelerator (denoted as WO) x -CNT), ECL strength as low as 8500 (au). Figure 10 The A in WO x -Scanning electron microscope images of CNTs, from which WO can be seen x -CNTs have uneven particle morphology.

[0158] like Figure 9 As shown in Figure A, the electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system gradually increased as the final concentration of nickel chloride increased from 1 mg / mL to 3 mg / mL. This indicates that the addition of nickel enhanced the synergistic effect between nickel and tungsten, thereby strengthening the Ni-WO3 electrochemiluminescence system. x The catalytic activity of -CNTs enhances the electrochemiluminescence performance of the system; with the synthesis of Ni-WO3... x With further increases in the amount of nickel chloride used in CNT (i.e., increasing the final concentration of nickel chloride from 3 mg / mL to 7 mg / mL), the electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system decreased slightly, but the overall luminescence intensity remained above 32054 (au) and did not show significant decay.

[0159] Therefore, overall, the final concentration of nickel chloride in the synthesized Ni-WO3 is 1 mg / mL to 7 mg / mL. x -CNTs can all be used as co-reactants and promoters in the luminol-hydroxyl electrochemiluminescence system of this invention, wherein the final concentration of nickel chloride is 3 mg / mL for the synthesis of Ni-Wo. x -CNTs have the best effect in promoting the reaction.

[0160] (2) Amount of carbon nanotubes

[0161] like Figure 11 As shown, the electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system using carbon nanotubes alone as a co-reactant promoter is extremely low (only about 470 a.u.), indicating that carbon nanotubes have no significant effect on the electrochemiluminescence performance of the luminol-hydroxyl system.

[0162] like Figure 9 As shown in B, in three consecutive tests, the co-reaction promoter (denoted as Ni-WO) obtained without the addition of carbon nanotubes during synthesis (i.e., the concentration of carbon nanotubes was 0 mg / mL) was used. x At that time, the electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system was as low as 6375 (au). Figure 10 B in the text is Ni-WO x The scanning electron microscope image shows Ni-WO x Its morphology consists of irregularly shaped granules of varying sizes. For example... Figure 12 As shown in A to D, after measurement, Ni-WO x The precipitate from the modified glassy carbon electrode (GCE) surface into the electrolyte is the main reason for the poor stability of the luminol-hydroxyl system.

[0163] like Figure 9As shown in Figure B, the electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system gradually increased with the increase of carbon nanotube concentration from 0.3 mg / mL to 1 mg / mL. This increase was further observed with the synthesis of Ni-WO4. x With further increases in the amount of carbon nanotubes used in CNTs (i.e., the concentration of carbon nanotubes increased from 1 mg / mL to 1.6 mg / mL), the electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system decreased slightly, but the overall luminescence intensity remained above 27667 (au) and did not show significant decay.

[0164] like Figure 13 As shown in A to D, in three consecutive tests, the Ni-WO prepared in Example 1 was used. x -CNTs were used as co-reactant accelerators, but Ni-WO3 was not found. x -CNTs show a clear separation from the surface of the modified glassy carbon electrode, indicating that Ni-WO x -CNTs are firmly fixed to the GCE surface. This indicates that carbon nanotubes can enhance Ni-WO3. x - The adhesion between CNTs and glassy carbon electrodes is improved, thereby enhancing the stability of the luminol-hydroxyl electrochemiluminescence system.

[0165] Therefore, overall, Ni-WO3 synthesized with carbon nanotube concentrations ranging from 0.3 mg / mL to 1.6 mg / mL... x -CNTs can all be used as co-reactants and promoters in the luminol-hydroxyl electrochemiluminescence system of this invention, wherein the concentration of carbon nanotubes is 1 mg / mL when synthesizing Ni-Wo. x -CNTs have the best effect in promoting the reaction.

[0166] like Figure 14 As shown in Figure A, in the Fourier transform infrared spectrum, 3430 cm⁻¹ -1 The peak at that location corresponds to the hydroxide group and is associated with Ni-WO3. x In comparison, Ni-WO x The peak intensity of -CNT was significantly enhanced, indicating that NiWO x -CNTs more effectively promote hydroxide adsorption. For example... Figure 14 As shown in B, Ni-WO x -CNTs have significantly lower resistance than Ni-WO3. x The resistance indicates that the introduction of carbon nanotubes effectively promoted the Ni-WO3 synthesis. x -CNTs enhance electron transfer between the electrolyte and species such as hydroxides and reactive oxygen species, thereby enhancing Ni-WO3. x -CNT catalytic activity.

[0167] (3) Graphene modification

[0168] like Figure 15 As shown, Ni-WO3 synthesized by replacing carbon nanotubes with graphene. x -CNTs, as co-reaction promoters, maintained excellent stability in long-term testing of the luminol-hydroxyl electrochemiluminescence system. With the aid of graphene, Ni-WO x -CNTs can adhere firmly to the glassy carbon electrode, preventing them from falling off.

[0169] 2. Temperature

[0170] like Figure 16 As shown in A and B, the electrochemiluminescence performance of the luminol-hydrogen peroxide system is greatly affected by temperature, and the ECL performance of the system varies significantly at different temperatures. Correspondingly, the dissolved oxygen concentration in the luminol-hydrogen peroxide system changes significantly at different temperatures, with the dissolved oxygen concentration decreasing at higher temperatures. This is the main reason for the reduced stability of the luminol-hydrogen peroxide system.

[0171] On the contrary, such as Figure 16 As shown in C, this invention uses Ni-WO x The luminol-hydroxyl electrochemiluminescence system, with CNTs as a co-reaction promoter, exhibits electrochemiluminescence performance unaffected by temperature and maintains excellent stability at different temperatures.

[0172] 3. Parameters of electrochemical cyclic voltammetry test

[0173] (1) Voltage

[0174] from Figure 17 As can be seen from A, when the voltage range of the electrochemical cyclic voltammetry test increases from 0–0.2V to 0–0.4V, the electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system increases and remains stable; as the voltage range of the electrochemical cyclic voltammetry test further increases to 0–0.5V and 0–0.6V, the luminescence intensity of the luminol-hydroxyl electrochemiluminescence system becomes unstable.

[0175] like Figure 18 As shown, no obvious bubbles appeared on the glassy carbon electrode surface within the electrochemical cyclic voltammetry test voltage range of 0–0.4 V; however, when the electrochemical cyclic voltammetry test voltage range increased to 0–0.6 V, the number of bubbles increased significantly. To explore the effect of bubbles on stability, the electrode was removed and rinsed with electrolyte after one of the three measurements. Figure 19As shown in A and B, after removing bubbles from the working electrode (glassy carbon electrode), the luminol-hydroxyl electrochemiluminescence system maintains good stability in electrochemiluminescence intensity within voltage ranges of 0–0.5 V and 0–0.6 V. Therefore, the applicable voltage range for electrochemical cyclic voltammetry testing of the luminol-hydroxyl electrochemiluminescence system of this invention is 0–(0.2 V–0.4 V), and the optimal voltage range for electrochemical cyclic voltammetry testing is 0–0.4 V.

[0176] The above results indicate that increasing the excitation voltage promotes Ni-WO3 synthesis. x The catalytic reaction of -CNTs enhances ROS generation. High-voltage electrochemical cyclic voltammetry excitation can effectively promote the generation of active oxygen and accelerate the oxygen evolution reaction in water electrolysis. The generated oxygen is adsorbed on the surface of the glassy carbon electrode, hindering the Ni-WO3 reaction. x -The contact between CNTs and hydroxyl and luminol anions reduces the electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system.

[0177] Compared with the prior art (Table 1), the luminol-hydroxyl electrochemiluminescence system of the present invention has a smaller applicable excitation voltage range, which is more conducive to reducing electrochemical side reactions in the measurement.

[0178] (2) Scan rate

[0179] In electrochemical reactions, low scan rates can enhance charge transfer and stability, but lead to slower reaction rates. On the other hand, high scan rates can accelerate reaction rates, but may compromise stability due to the limited diffusion of the electrolyte.

[0180] like Figure 17 As shown in B, with the scan rate of the electrochemical cyclic voltammetry test increasing from 30 mV s... -1 Increase to 50mVs -1 The electrochemiluminescence intensity of the luminol-hydroxyl electrochemiluminescence system gradually increased and remained stable without significant decay during continuous testing; as the scan rate was further increased from 50 mV s... -1 Increased to 70mV s -1 The luminol-hydroxyl electrochemiluminescence intensity increases, but it exhibits significant decay during continuous testing, indicating poor surface stability. Therefore, the appropriate scan rate range for electrochemical cyclic voltammetry testing of the luminol-hydroxyl electrochemiluminescence system of this invention is 30 mV s. -1 ~50mV s -1 The optimal scan rate for electrochemical cyclic voltammetry is 50 mV s. -1 .

[0181] 4. Long-term stability

[0182] Under optimal synthesis conditions (final nickel chloride concentration of 3 mg / mL; carbon nanotube concentration of 1 mg / mL) and electrochemical testing conditions (electrochemical cyclic voltammetry, voltage range of 0–0.4 V, scan rate of 50 mV s), the following conditions were met. -1 Under these conditions, the luminol-hydroxyl electrochemiluminescence system was subjected to 1300 consecutive tests.

[0183] Table 1 Comparison of different luminol-based electrochemiluminescence systems

[0184]

[0185]

[0186] Note: a HEO: High entropy oxide; b Fe-NC: Single-atom iron catalyst; c DO: Dissolved oxygen; d No.: Number of stability tests; e I: Electrochemiluminescence intensity.

[0187] As shown in Table 1 and Figure 20 As shown, the luminol-hydroxyl electrochemiluminescence system of the present invention maintains the initial electrochemiluminescence intensity after 1300 consecutive tests, with a relative standard deviation (RSD) of only 2.76% (less than 5%), and its stability is far superior to that of the luminol system in the prior art.

[0188] Example 3: Application of the luminol-hydroxyl electrochemiluminescence system in the detection of uric acid

[0189] I. Experimental Methods

[0190] 1. Uric acid testing

[0191] The luminol-hydroxyl electrochemiluminescence system described in Example 1 can be used as a sensor to detect the uric acid content in a sample. The detection principle is as follows: Figure 21 As shown in A, specifically: uric acid itself has the property of scavenging free radicals (also called reactive oxygen species, i.e., ROS). When uric acid is added to the luminol-hydroxyl electrochemiluminescence system of the present invention, it will neutralize some of the ROS generated during the reaction, reduce the amount of ROS that reacts with luminol, and thus reduce the ECL signal intensity of the system, thereby realizing the detection of uric acid.

[0192] The detection method is as follows:

[0193] 100 μL of the sample to be tested was added to the reaction cell, and the detection was performed according to step 3 of Example 1, "Construction and Evaluation of Luminol-Hydroxide Electrochemiluminescence System". The voltage range of the cyclic voltammetry scan was set to 0 V to 0.4 V, and the scan rate was 50 mV s.-1 The light signal was collected using an ultra-weak luminescence analyzer with the photomultiplier tube voltage set to 500V. The electrochemiluminescence intensity, cyclic voltammetry curve and electrochemiluminescence curve of each test material were recorded.

[0194] 2. Sample preparation

[0195] The present invention does not have specific limitations on the source of the test samples, including but not limited to bodily fluid samples such as blood, urine, and sweat. Furthermore, bodily fluid samples require no additional pretreatment; 100 μL is directly added to the electrolyte for detection after sampling.

[0196] In this embodiment, uric acid or other substances dissolved in an electrolyte (0.1M KOH solution containing 10μM luminol, pH=13.5) were used as the test samples to evaluate the detection effect.

[0197] 3. Evaluation of the detection effect

[0198] (1) Detection range

[0199] Different masses of uric acid were dissolved in an electrolyte to prepare test samples with concentrations of 0.1 μM, 0.5 μM, 1 μM, 2.5 μM, 4 μM, 5.5 μM, 7 μM, 8.5 μM and 10 μM, respectively, and the samples were tested according to the "Detection Method" in step 1 of this embodiment.

[0200] (2) Stability

[0201] The reference electrode, counter electrode, and modified working electrode from Example 1 were stored in a sealed environment at room temperature and in a dust-free environment for 30 days. The luminol-hydroxyl electrochemiluminescence system from Example 1 was used for testing every 5 days, for a total of 6 tests.

[0202] Each test used a 4 μM sample prepared by dissolving uric acid in electrolyte (prepared fresh each time).

[0203] The stability of the luminol-hydroxyl electrochemiluminescence system of this invention was evaluated by comparing the results of each test.

[0204] (3) Specificity

[0205] The specificity of the luminol-hydroxyl electrochemiluminescence system of this invention for uric acid detection was evaluated by using common reducing substances and metal ions found in human samples as potential interfering factors.

[0206] The specific information of the sample to be tested is as follows:

[0207] The electrolyte is referred to as sample Blank; the 4 μM sample prepared by dissolving uric acid in the electrolyte is sample UA; the 20 μM sample prepared by dissolving uric acid, ascorbic acid, dopamine, glutathione, cystine, glucose, potassium chloride, calcium chloride, and pyruvate in the electrolyte is sample Mixture; the 20 μM sample prepared by dissolving ascorbic acid in the electrolyte is sample AA; the sample prepared by dissolving dopamine in the electrolyte... The sample with a concentration of 20 μM is designated as sample DA; the sample with a concentration of 20 μM prepared by dissolving glutathione in electrolyte is designated as sample GSH; the sample with a concentration of 20 μM prepared by dissolving cystine in electrolyte is designated as sample Gys; the sample with a concentration of 20 μM prepared by dissolving glucose in electrolyte is designated as sample Glucose; and the sample with a concentration of 20 μM prepared by dissolving potassium chloride in electrolyte is designated as sample K. + The sample to be tested, with a concentration of 20 μM, is prepared by dissolving calcium chloride in an electrolyte solution. 2+ The sample PA is prepared by dissolving pyruvate in electrolyte to obtain a concentration of 20 μM.

[0208] (4) Reproducibility

[0209] Following step 3 of Example 1, "Construction and luminescence performance evaluation of the luminol-hydroxyl electrochemiluminescence system", ten Ni-WO3 electrodes were repeatedly prepared. x Using a CNT-modified glassy carbon electrode as the working electrode, the system was used to detect 4 μM uric acid samples prepared by dissolving uric acid in electrolyte. The results of each test were compared to evaluate the reproducibility of the system in detecting uric acid.

[0210] (5) Sample recovery

[0211] Sensor recovery tests were conducted in human serum, urine, and sweat diluted with electrolyte to investigate the effectiveness of the luminol-hydroxyl electrochemiluminescence system in detecting uric acid in real samples.

[0212] II. Experimental Results

[0213] (1) Detection range

[0214] like Figure 21 B and Figure 22 As shown, in the range of 0.1–10 μmol L -1 Within the concentration range, the uric acid content in different solvents showed a strong linear relationship with the ECL intensity of the luminol-hydroxyl electrochemiluminescence system (linear range: 0.1 μmol L). -1 ~10 μmol L -1 The detection limit for uric acid is 0.03 μmol / L.-1 (S / N = 3).

[0215] In this embodiment, the sample was diluted 100-fold before measuring the actual body fluid sample, indicating that the linear range of the luminol-hydroxyl electrochemiluminescence system for detecting uric acid content in body fluid samples is 10 μmol / L. -1 ~1000mol L -1 It can completely cover the concentration range of uric acid in human samples (the normal concentration of uric acid in the human body is below 500 μmol / L). -1 ).

[0216] (2) Stability

[0217] like Figure 21 As shown in C, Ni-WO x -CNT-modified glassy carbon electrode (Ni-WO3) x The CNT / GCE and luminol-hydroxyl electrochemiluminescence systems showed no significant difference in six consecutive measurements (RSD = 4.1%, less than 5%), demonstrating significant stability and allowing for repeated use.

[0218] (3) Specificity

[0219] like Figure 21 As shown in D, ascorbic acid, dopamine, glutathione, cystine, glucose, potassium ions, and calcium ions have virtually no effect on the detection results of the luminol-hydroxyl electrochemiluminescence system.

[0220] like Figure 23 As shown in Figures A and B, the interference of ascorbic acid and dopamine on uric acid detection is reduced due to the reaction of large amounts of ascorbic acid and dopamine with potassium hydroxide. Furthermore, glucose, pyruvate, potassium ions, and calcium ions did not show significant interference on uric acid detection, indicating that the luminol-hydroxyl electrochemiluminescence system has high specificity for uric acid detection.

[0221] (4) Reproducibility

[0222] like Figure 24 As shown in Figure A, after the sample was stored at room temperature for 30 days, the electrochemiluminescence intensity was detected by the luminol-hydroxyl electrochemiluminescence system. The results showed that the electrochemiluminescence intensity did not decrease significantly during this period (RSD = 4.9%, less than 5%). Figure 24 As shown in B, by testing 10 independently manufactured Ni-WO x The CNT-modified glassy carbon electrode showed no significant difference in the electrochemiluminescence intensity of uric acid (RSD = 3.2%, less than 5%), indicating that the luminol-hydroxyl electrochemiluminescence system has good reproducibility.

[0223] (5) Sample recovery

[0224] Table 2 Results of spiked recovery tests in serum, urine, and sweat samples.

[0225]

[0226] As shown in Table 2, the recovery rate of uric acid detection by the luminol-hydroxyl electrochemiluminescence system of the present invention is between 96% and 109% in different types of body fluid samples, indicating that the uric acid sensor meets the detection requirements of actual biological samples.

[0227] In summary, the luminol-hydroxyl electrochemiluminescence system established in this invention exhibits excellent stability, selectivity, and reproducibility in the detection of uric acid, and shows promise for improving electrochemiluminescence intensity and its application in biosensing.

[0228] Example 4: A luminol-hydroxyl electrochemiluminescence system for detecting uric acid

[0229] I. Composition

[0230] Based on the established luminol-hydroxyl electrochemiluminescence system, this invention provides an electrochemiluminescence system for detecting uric acid, comprising a three-electrode system, luminol, electrolyte, electrochemical workstation, and reaction cell.

[0231] Three-electrode system: The inner diameter is 8 mm as in Example 1, and the surface of the detection end is modified with Ni-WO₂. x A glassy carbon electrode with CNTs is used as the working electrode, a silver / silver chloride electrode as the reference electrode, and a platinum wire electrode as the counter electrode. A co-reaction promoter is used to catalyze the oxidation of hydroxide ions to generate reactive oxygen species.

[0232] Luminol: Used to competitively bind the reactive oxygen species with antioxidants at a working concentration of 10 μM.

[0233] Electrolyte: 0.1M KOH solution, pH=13.5.

[0234] Reaction chamber: transparent quartz beaker.

[0235] II. Instructions for Use

[0236] (1) Preparation of standard products

[0237] Uric acid standards were dissolved in an electrolyte to prepare standard dilutions with concentrations of 0.1 μM, 0.5 μM, 1 μM, 2.5 μM, 4 μM, 5.5 μM, 7 μM, 8.5 μM and 10 μM.

[0238] (2) Detection

[0239] Take 100 μL of each concentration of standard dilution solution and mix it thoroughly with luminol and electrolyte in the reaction cell. Then, connect the working electrode, reference electrode, and counter electrode to the ultra-weak luminescence analyzer and place them in the reaction cell containing electrolyte. Transfer to the electrochemiluminescence testing platform, set the voltage range of the cyclic voltammetry scan to 0 V to 0.4 V, and the scan rate to 50 mVs. -1 The light signal was collected using an ultra-weak luminescence analyzer with the photomultiplier tube voltage set to 500V. The luminescence intensity of each standard dilution was obtained, a standard curve of concentration-luminescence intensity was plotted, and a linear regression equation was obtained.

[0240] 100 μL of the sample to be tested was mixed thoroughly with luminol and electrolyte in a reaction cell. The working electrode, reference electrode, and counter electrode were then connected to the ultra-weak luminescence analyzer and placed in the reaction cell containing the electrolyte. The mixture was then transferred to the electrochemiluminescence testing platform, and the cyclic voltammetry scan voltage range was set to 0 V–0.4 V, with a scan rate of 50 mV / s. -1 The light signal was collected using an ultra-weak luminescence analyzer, with the photomultiplier tube voltage of the instrument set to 500V, to obtain the luminescence intensity of the sample to be tested.

[0241] The luminescence intensity of the sample to be tested is substituted into the linear regression equation to calculate the uric acid content in the sample.

[0242] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A composite material, characterized by, The tungsten chloride, nickel chloride and carbon nanomaterials are synthesized by a solvothermal method, and the mass ratio of the tungsten chloride, nickel chloride and carbon nanomaterials is (1-7):(1-7):(0.3-1.6).

2. Use of the composite material of claim 1 as a co-reactive promoter in a luminol electrochemiluminescence system for detecting antioxidants.

3. An electrochemiluminescent system for detecting an antioxidant, comprising: The system comprises a three-electrode system, luminol and an electrolyte solution; The working electrode of the three-electrode system is modified with a co-reactive promoter for catalyzing the oxidation of hydroxyl ions to generate active oxygen; the co-reactive promoter is the composite material of claim 1; The luminol is used to compete with antioxidants for binding to the active oxygen; The electrolyte solution is a solution containing hydroxyl ions, and the pH is 12-14.

4. The electrochemiluminescent system of claim 3, wherein, The solution containing hydroxyl ions is a potassium hydroxide solution.

5. The electrochemiluminescent system of claim 4, wherein, The concentration of the potassium hydroxide solution is 0.05M-0.2M.

6. The electrochemiluminescent system of claim 3, wherein, The working concentration of the luminol is 1μM-20μM.

7. The electrochemiluminescent system of claim 3, wherein, The system further comprises an electrochemical workstation connected to the three-electrode system.

8. Use of the electrochemiluminescence system of any one of claims 3-7 for detecting antioxidants.

9. A method of detecting the content of an antioxidant, characterized by, The electrochemiluminescence system of any one of claims 3-7 is used to detect a sample to be tested, comprising the following steps: The sample to be tested, luminol and an electrolyte solution are mixed thoroughly to obtain a reaction solution; the luminescence intensity of the reaction solution is detected by the three-electrode system, and the analysis is completed.

10. The method of claim 9, wherein, The condition for detecting the light emission intensity includes: the voltage range of cyclic voltammetry scanning is 0~(0.2V~0.4V); the scanning rate is 30mV s -1 ~50mV s -1 .

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