ZIF-67 (at) PDA (at) PtCu nano-enzyme, electrochemical immunosensor thereof and method for detecting AQP4-IgG

Abstract

The invention provides a ZIF-67 (at) PDA (at) PtCu nano enzyme, an electrochemical immunosensor thereof, and a method for detecting AQP4-IgG, and belongs to the technical field of electrochemical immunosensing. The ZIF-67 (at) PDA (at) PtCu nano-enzyme and the electrochemical immunosensor are fused, a breakthrough strategy is provided for AQP4-IgG detection, specifically, the electrochemical immunosensor based on the ZIF-67 (at) PDA (at) PtCu nano-enzyme has efficient electrochemical signals, has high specificity and excellent repeatability and stability for AQP4-IgG detection, and is a novel electrochemical immunosensor for AQP4-IgG detection. The AQP4-IgG detection kit has the advantages of high sensitivity, high sensitivity, low detection limit, wide detection range, extremely low detection limit, realization of sensitive, rapid and low-cost detection of AQP4-IgG, and high guidance value for detection of neuromyelitis optica.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical immunosensing, and in particular to a ZIF-67@PDA@PtCu nanozyme and an electrochemical immunosensor thereof, and a method for detecting AQP4-IgG. Background Art

[0002] Neuromyelitis optica spectrum disease (NMOSD) is a group of autoimmune-mediated inflammatory demyelinating diseases of the central nervous system that can lead to severe visual impairment and paralysis. NMOSD is considered a distinct disease due to the discovery of antibodies targeting aquaporin-4 (AQP4) in the membranes of central nervous system astrocytes. These autoantibodies, known as AQP4-IgG, play a central role in the pathophysiology of NMOSD by binding to AQP4 channels, leading to inflammation, cell damage, and neuronal death.

[0003] At present, the methods used for clinical detection of AQP4-IgG include tissue indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA), and cell-based assay (CBA). CBA is specific, reliable, and highly sensitive, and is the gold standard for AQP4-IgG detection. However, the implementation of CBA relies on professional operators, which is time-consuming and labor-intensive, making it difficult to implement in most hospitals. ELISA is currently a widely used AQP4-IgG detection technology due to its strong quantitative ability. It is relatively simple to perform, can handle large sample sizes, and provides quantitative results. However, since NMOSD is a sporadic disease with a relatively low incidence, ELISA testing of a single sample is expensive. Tissue-based IIF has a low sensitivity in the diagnosis of neuromyelitis optica, which may be related to the use of mouse or primate brain tissue as a detection substrate.

[0004] In summary, there is growing interest in developing faster, more cost-effective, and portable AQP4-IgG detection protocols. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a ZIF-67@PDA@PtCu nanozyme and an electrochemical immunosensor thereof, and a method for detecting AQP4-IgG. The electrochemical immunosensor constructed based on the ZIF-67@PDA@PtCu nanozyme of the present invention can realize sensitive, rapid and low-cost detection of AQP4-IgG.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a ZIF-67@PDA@PtCu nanozyme, comprising ZIF-67, and polydopamine and PtCu alloy nanoflowers loaded on the surface of the ZIF-67.

[0007] Preferably, the particle size of the ZIF-67 is 150-200 nm, and the particle size of the PtCu alloy nanoflower is 20-30 nm.

[0008] The present invention provides a method for preparing the above-mentioned ZIF-67@PDA@PtCu nanozyme, comprising the following steps: The ZIF-67 solution, the buffer solution and the dopamine hydrochloride solution were mixed for the first assembly to obtain ZIF-67@PDA; The alcohol solution of the ZIF-67@PDA is mixed with the alcohol solution of the PtCu alloy nanoflowers, and a second assembly is performed to obtain the ZIF-67@PDA@PtCu nanozyme.

[0009] The present invention provides application of the above ZIF-67@PDA@PtCu nanozyme in the field of electrochemical biosensing.

[0010] The present invention provides an AQP4-IgG detection probe, comprising the above-mentioned ZIF-67@PDA@PtCu nanozyme, and a secondary antibody chemically bound to the ZIF-67@PDA@PtCu nanozyme.

[0011] The present invention provides an electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme, comprising an electrochemical immunosensor matrix and the above-mentioned AQP4-IgG detection probe; The electrochemical immunosensor matrix comprises a substrate electrode, a graphene oxide-polydopamine complex attached to the surface of the substrate electrode, a nanogold film deposited on the surfaces of the substrate electrode and the graphene oxide-polydopamine complex, AQP4 modified on the surface of the nanogold film, and bovine serum albumin modified on the surface of the nanogold film for blocking unbound sites.

[0012] Preferably, the method for preparing the electrochemical immunosensor matrix comprises the following steps: The graphene oxide-polydopamine complex solution is loaded onto the surface of the substrate electrode, and after drying, a substrate electrode modified with the graphene oxide-polydopamine complex is obtained; Electrodepositing a nano-gold film on the surface of the substrate electrode modified with the graphene oxide-polydopamine complex to obtain a substrate electrode deposited with the nano-gold film; AQP4 solution is loaded on the surface of the substrate electrode on which the nano-gold film is deposited, and incubated to obtain a substrate electrode modified with AQP4; A bovine serum albumin solution is loaded onto the surface of the substrate electrode modified with AQP4, and incubated to obtain an electrochemical immunosensor matrix.

[0013] The present invention provides the use of the electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme in the preparation of a neuromyelitis optica detection kit.

[0014] The present invention provides a method for detecting AQP4-IgG for non-diagnostic purposes, comprising the following steps: Loading the sample to be tested onto the surface of the electrochemical immunosensor matrix, performing a first incubation, and obtaining a first incubation product; Loading an AQP4-IgG detection probe solution onto the surface of the first incubation product, performing a second incubation, and obtaining an electrode to be tested; The electrode to be tested is placed in a H 2 O 2 A differential pulse voltammetry test is performed in a buffer solution to obtain a current signal peak value, and the content of AQP4-IgG in the sample to be tested is obtained according to the current signal peak value and a predetermined standard curve; The standard curve is a linear relationship curve between the logarithmic concentration of AQP4-IgG and the peak value of the current signal; The electrochemical immunosensor matrix and the AQP4-IgG detection probe are the electrochemical immunosensor matrix and the AQP4-IgG detection probe in the above-mentioned electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme.

[0015] Preferably, the scanning potential range of the differential pulse voltammetry test is 0 V to -0.4 V, the amplitude is 0.07 V, the pulse width is 50 ms, and the pulse period is 200 ms.

[0016] The present invention provides a ZIF-67@PDA@PtCu nanozyme, comprising ZIF-67, and polydopamine (PDA) and PtCu alloy nanoflowers loaded on the surface of the ZIF-67. In the present invention, the PtCu alloy, especially the PtCu alloy in the form of nanoflowers, exhibits superior catalytic performance relative to pure Pt or Cu nanoparticles. The interaction between Pt and Cu changes the electronic properties of the metal, enhances the adsorption of reaction intermediates, reduces the activation energy required for the catalytic reaction, and the PtCu alloy nanoflower structure provides a large number of active sites on its surface. However, the catalytic reduction ability of the PtCu alloy alone is limited. The present invention adopts the method of introducing ZIF-67, wherein ZIF-67 has excellent stability and high specific surface area, and when loaded with PtCu alloy nanoflowers, its catalytic performance can be significantly improved. In addition, the present invention loads a layer of polydopamine (PDA) on the surface of ZIF-67, which enhances the interaction between the unsaturated edge sites of ZIF-67, thereby enhancing the mechanical and chemical stability and functionality of ZIF-67, and introduces a large number of functional groups, especially amino groups, on its surface. These amino groups provide the best binding sites for the subsequent attachment of platinum (Pt). In the present invention, there is a strong interaction between PDA-modified ZIF-67 and Pt, ensuring the stable and uniform distribution of PtCu alloy nanoflowers on the surface of ZIF-67, which is crucial for maximizing catalytic efficiency.

[0017] The ZIF-67@PDA@PtCu nanozyme provided by the present invention has a high specific surface area and porosity, has excellent catalytic performance, and can significantly amplify electrochemical signals when used for electrochemical biosensing.

[0018] The present invention provides an AQP4-IgG detection probe, comprising the above-mentioned ZIF-67@PDA@PtCu nanozyme, and a secondary antibody chemically bound to the ZIF-67@PDA@PtCu nanozyme, denoted as ZIF-67@PDA@PtCu@Ab probe. The AQP4-IgG detection probe provided by the present invention can be used for electrochemical biosensor detection of AQP4-IgG.

[0019] The present invention provides an electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme, comprising an electrochemical immunosensor matrix and the above-mentioned AQP4-IgG detection probe. In the present invention, the structure of the electrochemical immunosensor is BSA / AQP4 / GO@PDA / GCE, wherein the graphene oxide-polydopamine complex has a significant effect on the charge transfer kinetics of the electrode surface and can enhance the conductivity of the electrode; the role of polydopamine is to enhance the conductivity effect. The present invention combines highly catalytic composite nanoparticles (ZIF-67@PDA@PtCu nanozymes) with electrochemical immunosensors, providing a breakthrough strategy for AQP4-IgG detection. Specifically, the electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozymes constructed by the present invention has an efficient electrochemical (EC) signal, is highly specific for the detection of AQP4-IgG, and has excellent repeatability and stability, and has a wide detection range and an extremely low detection limit. It can achieve sensitive, rapid, and low-cost detection of AQP4-IgG, and has a high clinical application value for the detection of neuromyelitis optica. The results of the embodiment show that the detection range of the electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozymes of the present invention is 0.1 ~ 80 U mL -1 , the detection limit is 0.05 UmL -1 .

[0020] The present invention provides a method for detecting AQP4-IgG, which is simple, efficient, portable, does not require large facilities and professional operators, and has higher cost-effectiveness. This method can detect AQP4-IgG in non-invasive samples (such as serum) and is suitable for detection in clinical environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The structural and compositional analysis results of GO@PDA and ZIF-67@PDA@PtCu NPs; Figure 1 In the figure, A is the TEM image of GO@PDA; B is the TEM image of ZIF-67@PDA; C is the TEM image of PtCu NPs; D is the TEM image of ZIF-67@PDA@PtCu; E is the HAADF-STEM image of ZIF-67@PDA@PtCu; F~K are the HAADF-TEM-EDS mappings of ZIF-67@PDA@PtCu; L is the XRD pattern of ZIF-67@PDA@PtCu; M is the XPS pattern of ZIF-67@PDA@PtCu; N is the FT-IR pattern of ZIF-67@PDA@PtCu; Figure 2 Optimization results for experimental variables of biosensing strategies; Figure 2Where A is the optimized ZIF-67@PDA@PtCu concentration, mg mL -1 ; B is the optimized GO@PDA concentration, mg mL -1 ; C is the optimized pH value of the detection solution, D is the optimized AQP4-IgG incubation time, min; Figure 3 The test results of the immunosensor detecting different concentrations of AQP4-IgG protein; Figure 3 In the figure, A is the target AQP4-IgG at different concentrations (a→g: 0.1, 1, 5, 10, 20, 40, 60 and 80 U mL -1 ) is the DPV value of the electrochemical immunosensor designed under the condition of ; B is the standard curve of peak current and logarithm of AQP4-IgG concentration; Figure 4 The selectivity, reproducibility and stability test results of the electrochemical immunosensor; Figure 4 In the figure, A is the selectivity test result; B is the reproducibility test result, and C is the stability test result. DETAILED DESCRIPTION

[0022] The invention provides a ZIF-67@PDA@PtCu nanozyme, comprising ZIF-67, and polydopamine and PtCu alloy nanoflowers loaded on the surface of the ZIF-67.

[0023] In the present invention, the polydopamine and PtCu alloy nanoflowers are preferably loaded on the surface of ZIF-67 in sequence; the polydopamine is preferably wrapped on the surface of ZIF-67 in the form of a polydopamine layer, and the PtCu alloy nanoflowers are distributed on the surface of ZIF-67 wrapped with the polydopamine layer.

[0024] In the present invention, the particle size of the ZIF-67 is preferably 150-200 nm; the particle size of the PtCu alloy nanoflower is preferably 20-30 nm, more preferably 24 nm; in the PtCu alloy nanoflower, the mass ratio of Pt to Cu elements is preferably 1-2:1. In the present invention, in the ZIF-67@PDA@PtCu nanozyme, the mass ratio of ZIF-67 to PtCu alloy nanoflower is preferably 5-10:1, more preferably 6-8:1.

[0025] The present invention has no special requirements on the source of the ZIF-67, and the ZIF-67 conventionally available on the market in the art or prepared by oneself can be used.

[0026] In the present invention, the method for preparing the PtCu alloy nanoflowers preferably comprises the following steps: K 2 PtCl 4 , CuCl2 , a dispersant and water are ultrasonically mixed, and an aqueous ascorbic acid solution is added during the ultrasonic mixing to obtain a uniform mixed solution; The uniform mixed solution is placed in a heating environment to perform a reduction reaction to obtain PtCu alloy nanoflowers.

[0027] In the present invention, the dispersant is preferably CTAC. 2 PtCl 4 , CuCl 2 The mass ratio of the water to the dispersant is preferably 38.19:12.37:640. In the present invention, the water is preferably deionized water, and the K 2 PtCl 4 The preferred dosage ratio of K to water is 38.19 mg: 40 mL. 2 PtCl 4 , CuCl 2 After the addition of , dispersant and water is completed, a flocculated mixture is formed.

[0028] In the present invention, the power of the ultrasonic mixing is preferably 100 W, and the time is preferably 10 min. In the present invention, the amount of ascorbic acid used is preferably excessive.

[0029] In the present invention, the temperature of the reduction reaction is preferably 90°C, and the time is preferably 5 minutes. During the reaction, the appearance of the solution changes from white to transparent brown within 5 minutes, and finally to opaque black. After the reduction reaction, the present invention preferably cools the obtained reduction reaction liquid to room temperature, and sequentially performs solid-liquid separation, washing and drying. In the present invention, the solid-liquid separation is preferably centrifugation, the washing is preferably deionized water washing, and the number of washings is preferably three times; the drying is preferably vacuum drying, the drying temperature is preferably 40°C, and the time is preferably overnight.

[0030] In the present invention, the specific surface area of ​​the ZIF-67@PDA@PtCu nanozyme is preferably 1069.15 m 2 g -1 The average pore size is preferably 1.89 nm, and the pore volume is preferably 0.50 cm 3 g -1 .

[0031] In the present invention, the preparation method of the ZIF-67@PDA@PtCu nanozyme comprises the following steps: The ZIF-67 solution, the buffer solution and the dopamine hydrochloride solution were mixed for the first assembly to obtain ZIF-67@PDA; The alcohol solution of the ZIF-67@PDA is mixed with the alcohol solution of the PtCu alloy nanoflowers, and a second assembly is performed to obtain the ZIF-67@PDA@PtCu nanozyme.

[0032] The present invention mixes a ZIF-67 solution, a buffer solution and a dopamine hydrochloride solution, and performs a first assembly to obtain ZIF-67@PDA. In the present invention, the solvent of the ZIF-67 solution is preferably a mixture of ethanol and water, and the volume ratio of the ethanol to water is preferably 3:4. In the present invention, the mass concentration of ZIF-67 in the ZIF-67 solution is preferably 20 mg mL -1 .

[0033] In the present invention, the buffer is preferably a Tris-HCl buffer, the concentration of the Tris-HCl buffer is preferably 10 mM, and the pH value is preferably 8.5. In the present invention, the concentration of the dopamine hydrochloride solution is preferably 2 mg mL -1 .

[0034] In the present invention, the dosage ratio of the ZIF-67, buffer solution and dopamine hydrochloride solution is preferably 100 mg: 5 mL: 20 µL.

[0035] In the present invention, the first assembly is preferably performed under stirring, the temperature of the first assembly is preferably room temperature, and the time is preferably 6 hours. After the first assembly, the present invention preferably centrifuges and washes the obtained mixed solution with methanol in sequence, and disperses the obtained solid in methanol for storage.

[0036] After obtaining the ZIF-67@PDA, the present invention mixes the alcohol solution of the ZIF-67@PDA with the alcohol solution of the PtCu alloy nanoflowers for a second assembly to obtain the ZIF-67@PDA@PtCu nanozyme. In the present invention, the solvent of the alcohol solution of the ZIF-67@PDA is preferably methanol, and the concentration of the alcohol solution of the ZIF-67@PDA is preferably 10-20 mg mL -1 The solvent of the alcohol solution of the PtCu alloy nanoflowers is preferably methanol, and the concentration of the alcohol solution of the PtCu alloy nanoflowers is preferably 10 to 20 mg mL -1 In the present invention, the volume ratio of the alcohol solution of ZIF-67@PDA to the alcohol solution of PtCu alloy nanoflowers is preferably 1-2:1-2.

[0037] In the present invention, the second assembly is preferably carried out under stirring, the temperature of the second assembly is preferably room temperature, and the time is preferably 8 hours. After the second assembly, the present invention preferably centrifuges and washes the obtained mixed solution with methanol in sequence, and disperses the obtained solid in deionized water for storage.

[0038] The present invention provides the application of the above-mentioned ZIF-67@PDA@PtCu nanozyme in the field of electrochemical biosensing.

[0039] The present invention provides an AQP4-IgG detection probe, comprising the above-mentioned ZIF-67@PDA@PtCu nanozyme, and a secondary antibody chemically bound to the ZIF-67@PDA@PtCu nanozyme. In the present invention, the secondary antibody is preferably a mouse anti-human IgG labeled antibody. In the present invention, the carboxyl group of the secondary antibody is chemically connected to the amino group in the ZIF-67@PDA@PtCu nanozyme, and the amino group is derived from polydopamine.

[0040] In the present invention, the method for preparing the AQP4-IgG detection probe preferably comprises the following steps: The ZIF-67@PDA@PtCu nanozyme was mixed with the buffer solution, and EDC·HCl and secondary antibody were added to perform the coupling reaction; After the coupling reaction, BSA solution was added to block the unreacted sites.

[0041] In the present invention, the buffer solution is preferably a boric acid buffer solution (BBS), the concentration of the boric acid buffer solution is preferably 0.2 M, and the pH value is preferably 7.4. In the present invention, after the mass of the ZIF-67@PDA@PtCu nanozyme is mixed with the buffer solution, the concentration of the ZIF-67@PDA@PtCu nanozyme in the resulting mixed solution is preferably 0.5~3 mg·mL -1 , more preferably 2 mg·mL -1 .

[0042] In the present invention, the mass ratio of the ZIF-67@PDA@PtCu nanozyme to the buffer solution, EDC·HCl and the secondary antibody is preferably 2:0.4:0.2. In the present invention, the temperature of the coupling reaction is preferably room temperature, and the time is preferably 2.5h.

[0043] In the present invention, the mass concentration of the BSA solution is preferably 10%; and the time for blocking the unreacted sites is preferably 2 hours.

[0044] The present invention provides an electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme, comprising an electrochemical immunosensor matrix and the above-mentioned AQP4-IgG detection probe.

[0045] In the present invention, the electrochemical immunosensor matrix includes a base electrode, a graphene oxide-polydopamine complex attached to the surface of the base electrode, a nanogold film deposited on the surface of the base electrode and the graphene oxide-polydopamine complex, AQP4 modified on the surface of the nanogold film, and bovine serum albumin modified on the surface of the nanogold film for blocking unbound sites.

[0046] In the present invention, the graphene oxide-polydopamine composite comprises graphene oxide and polydopamine wrapped on the surface of the graphene oxide. In the present invention, the preparation method of the graphene oxide-polydopamine composite (GO@PDA) preferably comprises the following steps: The graphene oxide was ultrasonically mixed with the solvent, and dopamine hydrochloride and Tris-HCl buffer were added in sequence to carry out a loading reaction.

[0047] In the present invention, the solvent is preferably a mixture of ethanol and water, and the volume ratio of ethanol to water is preferably 3:4. In the present invention, the amount ratio of graphene oxide to solvent is preferably 80 mg:140 mL. In the present invention, the ultrasonic mixing time is preferably 5 min.

[0048] In the present invention, the mass ratio of the graphene oxide to dopamine hydrochloride is preferably 80:1 to 2. In the present invention, the concentration of the Tris-HCl buffer is preferably 10 mM, and the pH value is preferably 8.5.

[0049] In the present invention, the loading reaction is preferably carried out under stirring conditions, and the time is preferably overnight. After the loading reaction, the present invention preferably centrifuges the obtained loading reaction solution, and washes and dries the obtained solid. In the present invention, the washing solution used for the washing is preferably ethanol and deionized water; the drying temperature is preferably 50°C.

[0050] In the present invention, the method for preparing the electrochemical immunosensor matrix comprises the following steps: The graphene oxide-polydopamine complex solution is loaded onto the surface of the substrate electrode, and after drying, a substrate electrode modified with the graphene oxide-polydopamine complex is obtained; Electrodepositing a nano-gold film on the surface of the substrate electrode modified with the graphene oxide-polydopamine complex to obtain a substrate electrode deposited with the nano-gold film; AQP4 solution is loaded on the surface of the substrate electrode on which the nano-gold film is deposited, and the substrate electrode is incubated to obtain a substrate electrode modified with AQP4; A bovine serum albumin solution is loaded onto the surface of the substrate electrode modified with AQP4, and incubated to obtain an electrochemical immunosensor matrix.

[0051] The present invention loads the graphene oxide-polydopamine complex solution onto the surface of the substrate electrode, and after drying, obtains a substrate electrode modified with the graphene oxide-polydopamine complex. In the present invention, the substrate electrode is preferably a glassy carbon electrode (GCE). Before use, the present invention preferably performs a pretreatment on the substrate electrode, and the pretreatment preferably includes: polishing, cleaning and drying the substrate electrode in sequence. In the present invention, the polishing agent used for polishing is preferably Al 2 O 3 The polishing time is preferably 8 minutes.

[0052] In the present invention, the concentration of the graphene oxide-polydopamine composite solution is preferably 1-6 mg ml -1 The dosage is preferably 10 μL; the drying time is preferably 37°C.

[0053] The present invention electro-deposit nano-gold thin film on the surface of the substrate electrode modified with the graphene oxide-polydopamine complex to obtain a substrate electrode deposited with the nano-gold thin film. In the present invention, the electro-deposition is preferably carried out on HAuCl 4 In solution, the HAuCl 4 The mass concentration of the solution is preferably 1.0%. In the present invention, the potential of the electrodeposition is preferably -0.2V, and the time is preferably 30s. After the electrodeposition, the present invention preferably uses deionized water to rinse the electrode and air-dry it at room temperature.

[0054] The present invention loads an AQP4 solution on the surface of the substrate electrode deposited with the nano-gold film, and incubates the solution to obtain a substrate electrode modified with AQP4. In the present invention, the source of the AQP4 solution is commercially available. In the present invention, the concentration of the AQP4 solution is preferably 20 μg mL -1 The loading volume is preferably 10 μL. In the present invention, the incubation temperature is preferably 37° C., and the incubation time is preferably 1 h.

[0055] The present invention loads a bovine serum albumin solution onto the surface of the substrate electrode modified with AQP4 and incubates the solution to obtain an electrochemical immunosensor substrate. In the present invention, the concentration of the bovine serum albumin solution is preferably 1wt%, and the loading amount is preferably 10 μL. In the present invention, the incubation temperature is preferably 37°C, and the time is preferably 1h. After the incubation, the present invention preferably uses a 10 mM, pH 7.4 PBS buffer solution to rinse the electrode to remove any unbound protein.

[0056] In the present invention, when the electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme is used, the AQP4 protein (antigen) in the electrochemical immunosensor matrix combines with the AQP4-IgG (antibody) and anti-AQP4-IgG antibody in the sample to be tested to construct a sandwich-type electrochemical immunosensor.

[0057] The present invention provides the use of the electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme in the preparation of a neuromyelitis optica detection kit.

[0058] The present invention provides a method for detecting AQP4-IgG for non-diagnostic purposes, comprising the following steps: Loading the sample to be tested onto the surface of the electrochemical immunosensor matrix, performing a first incubation, and obtaining a first incubation product; Loading an AQP4-IgG detection probe solution onto the surface of the first incubation product, performing a second incubation, and obtaining an electrode to be tested; The electrode to be tested is placed in a H 2 O 2 A differential pulse voltammetry test is performed in a buffer solution to obtain a current signal peak value, and the content of AQP4-IgG in the sample to be tested is obtained according to the current signal peak value and a predetermined standard curve; The standard curve is a linear relationship curve between the logarithmic concentration of AQP4-IgG and the peak value of the current signal; The electrochemical immunosensor matrix and the AQP4-IgG detection probe are the electrochemical immunosensor matrix and the AQP4-IgG detection probe in the above-mentioned electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme.

[0059] The present invention loads the sample to be tested onto the surface of the electrochemical immunosensor matrix, performs a first incubation, and obtains a first incubation product. In the present invention, the sample to be tested is preferably serum, urine or an aqueous solution. In the present invention, the loading amount of the sample to be tested is preferably 8~10 μL. In the present invention, the temperature of the first incubation is preferably 37°C, and the time is preferably 1h. After the first incubation, the present invention preferably uses a 10 mM, pH 7.4 PBS buffer solution to rinse the electrode to remove any unbound proteins.

[0060] After obtaining the first incubation product, the present invention loads the AQP4-IgG detection probe solution onto the surface of the first incubation product, performs a second incubation, and obtains the electrode to be tested. In the present invention, the concentration of the AQP4-IgG detection probe solution is preferably 0.5-3 mg mL -1, the loading amount is preferably 8~10 μL. In the present invention, the temperature of the second incubation is preferably 37°C, and the time is preferably 1h. After the second incubation, the present invention preferably uses a 10 mM, pH 7.4 PBS buffer solution to rinse the electrode to remove any unbound protein. In the present invention, the electrode to be tested is preferably stored at 4°C for subsequent analysis.

[0061] After obtaining the electrode to be tested, the present invention places the electrode to be tested in a solution containing H 2 O 2 In the buffer solution containing H, a differential pulse voltammetry test is performed to obtain a current signal peak value. 2 O 2 The buffer solution preferably contains H 2 O 2 10 mM PBS buffer solution, pH 7.4, the H 2 O 2 The concentration is preferably 5 mM.

[0062] In the present invention, the scanning potential range of the differential pulse voltammetry test is preferably 0 V to -0.4 V, the amplitude is preferably 0.07 V, the pulse width is preferably 50 ms, and the pulse period is preferably 200 ms.

[0063] After obtaining the current peak value, the present invention obtains the content of AQP4-IgG in the sample to be tested according to the current signal peak value and a predetermined standard curve; the standard curve is a linear relationship curve between the logarithmic concentration of AQP4-IgG and the current signal peak value. In the present invention, the method for obtaining the standard curve preferably includes the following steps: Provide AQP4-IgG standard solution with known concentrations in gradient; The AQP4-IgG standard solution with known concentration in the gradient is used as the sample to be tested and loaded onto the surface of the electrochemical immunosensor substrate. The AQP4-IgG detection probe solution is loaded in sequence according to the above method and a differential pulse voltammetry test is performed to obtain the current signal peaks corresponding to different concentrations of AQP4-IgG, and a standard curve is drawn according to the AQP4-IgG logarithmic concentration and the current signal peak.

[0064] In the present invention, the source of the AQP4-IgG is commercially available.

[0065] As a specific embodiment of the present invention, the standard curve is preferably I = -27.76 - 21.48lgCAQP4-IgG, R 2 = 0.978, and the limit of detection (LOD) was calculated to be 0.05 U mL -1 .

[0066] In the present invention, the linear detection range of AQP4-IgG is preferably 0.1 U mL -1 ~ 80 U mL -1 .

[0067] The following is a detailed description of a ZIF-67@PDA@PtCu nanozyme and an electrochemical immunosensor thereof, and a method for detecting AQP4-IgG provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1 Preparation of ZIF-67@PDA@PtCu nanozyme using the following steps: (1) Synthesis of PtCu NPs 38.19 mg K 2 PtCl 4 、12.37 mg CuCl 2 640 mg CTAC and 40 mL deionized water were mixed in a flask to form a flocculated mixture, which was sonicated at 100 W for 10 min. While sonicating, 3 mL of freshly prepared ascorbic acid aqueous solution (17.6 mg mL -1 ) was added to the mixture, and the resulting homogeneous solution was vigorously stirred in a 90 °C oil bath for 5 min and then cooled to room temperature. During the reaction, the appearance of the solution changed from white to transparent brown within 5 minutes and finally to opaque black. The resulting product was centrifuged, washed three times with deionized water, and dried in vacuum at 40 °C overnight to obtain PtCu NPs.

[0069] (2) Synthesis of ZIF-67 0.5 mmol Co(NO 3 ) 2 6H 2 O and 5 mmol 2-methylimidazole were dissolved in 10 mL methanol at 37 °C to obtain a salt solution and a ligand solution, respectively. The salt solution was then added to the ligand solution and stirred vigorously. After stirring for 12 minutes, the mixture was allowed to stand for 20 hours. The resulting solid product was collected by centrifugation, washed with methanol three times, and dried under vacuum at 40 °C for 24 hours to obtain ZIF-67.

[0070] (3) Synthesis of ZIF-67@PDA PDA-modified ZIF-67 was synthesized by a one-pot assembly method, as follows: 100 mg ZIF-67 was dissolved in 5 mL of a mixture of ethanol and water (3:4, v / v), and the above solution was mixed with 5 mL of 10 mM Tris-HCl buffer (pH 8.5) under ultrasound. Then, 20 µL of 2 mg mL-1 The dopamine hydrochloride solution was stirred continuously for 6 h. The obtained ZIF-67@PDA was separated by centrifugation at 12,000 rpm for 5 min, washed twice with methanol, and then redispersed in 2 mL of methanol.

[0071] (4) Synthesis of ZIF-67@PDA@PtCu 1 mL of 20 mg mL -1 Add 2 mL of methanol solution of PtCu nanoparticles to a concentration of 10 mg / mL -1 The product was centrifuged, washed three times with methanol, and then dispersed in deionized water for later use.

[0072] Example 2 Construction of electrochemical immunosensor matrix (BSA / AQP4 / GO@PDA / GCE) (1) Construction of GO@PDA First, 80 mg of graphene oxide was ultrasonically dispersed in 140 mL of a mixture of ethanol and water (3:4, v / v) for 5 min. Then, 2 mg of dopamine hydrochloride was added to the solution while stirring at 1000 rpm for 5 min. Subsequently, 100 mL of 10 mM Tris-HCl buffer (pH 8.5) was slowly added and the reaction was stirred continuously overnight. The resulting sediment was then collected by centrifugation and thoroughly washed with ethanol and deionized water until the upper liquid was clear. Finally, the black precipitate was dried at 50 °C.

[0073] (2) Synthesis of ZIF-67@PDA@PtCu@Ab probe 2 mg of ZIF-67@PDA@PtCu nanoparticles were dissolved in 1 mL of 0.2 M boric acid buffer solution (BBS) (pH 7.4). Then 400 µg of EDC·HCl and 200 µg of mouse anti-human IgG labeled antibody were added and shaken for 2.5 hours. To block any unreacted sites, 110 µL of 10 wt% BSA solution was added and then shaken for another 2 hours. Finally, the ZIF-67@PDA@PtCu@Ab probes were collected, centrifuged at 12,000 rpm for 10 minutes, and dispersed in 0.5 mL of fresh BBS solution.

[0074] (3) Construction of BSA / AQP4 / GO@PDA / GCE First, use Al 2 O 3 The bare GCE was slurry polished for 8 min until a mirror-like surface was obtained. It was then ultrasonically cleaned with deionized water for 5 cycles and dried with nitrogen in preparation for modification.-1 The GO@PDA solution was loaded on the cleaned GCE and dried at 37 °C. 4 The solution was kept at a constant potential of -0.2 V for 30 seconds to electro-deposit AuNPs on the GCE surface to form AuNPs / GO@PDA film. The electrode was then rinsed with deionized water and air-dried at room temperature. 10 μL of AQP4 solution (20 μg mL -1 ), and incubated at 37 °C for 1 h. To block nonspecific binding sites, 10 μL of 1 wt.% BSA solution was taken and incubated at 37 °C for 1 h. Finally, the obtained electrochemical immunosensor (BSA / AQP4 / GO@PDA / GCE) was rinsed with 10 mM PBS (pH 7.4) and stored at 4 °C for future use.

[0075] Structural characterization The structure and composition of GO@PDA and ZIF-67@PDA@PtCu NPs were analyzed. Figure 1 As shown. Figure 1 As shown in Figure A, the structure of GO@PDA was observed by transmission electron microscopy (TEM), and the results confirmed the successful synthesis of GO@PDA. Figure 1 As shown in Figure B, the morphology of ZIF-67@PDA is consistent with that of ZIF-67, indicating that PDA has little effect on the morphology of the prepared ZIF-67. Figure 1 As shown in Figure C, the synthesized PtCu nanoparticles (NPs) exhibit a nanoflower shape with an average size of about 24 nm and high monodispersity. High-resolution transmission electron microscopy (HRTEM) and high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) images further revealed that these PtCu NPs formed a nanoflower-like structure (see Figure 1 D and E), PtCu NPs were successfully encapsulated on the surface of ZIF-67. When loaded on ZIF-67, the PtCu NPs on the ZIF-67 nanocomposite exhibited a typical mesoporous morphology. Figure 1 As shown in Figures F~K, Pt and Cu are mainly distributed on the surface of the nanoparticle structure, while Co, N, C and O are evenly distributed inside. The XRD peaks of ZIF-67@PDA@PtCu ( Figure 1 The L in (L) is consistent with that of ZIF-67@PDA, indicating that ZIF-67@PDA@PtCu retains its phase structure. XPS was then used to analyze the elemental composition and chemical bonds of the ZIF-67@PDA@PtCu nanocomposite. Figure 1The XPS spectrum of the nanocomposite material shown in M ​​contains peaks of C 1s, Cu 2p, N 1s, Pt 4f, O 1s and Co 2p. Figure 1 As shown in Figure 1, the FT-IR spectrum shows that ZIF-67@PDA@PtCu has a higher peak at 912 cm-1 than ZIF-67@PDA. -1 A new peak appeared at , indicating that PtCu has been effectively loaded on the surface of ZIF-67.

[0076] Example 3 Electrochemical Detection of AQP4-IgG (1) Take aliquots of AQP4-IgG solutions (serum samples) of different concentrations, 8 μL each, apply them on the surface of BSA / AQP4 / GO@PDA / GCE, and incubate at 37 °C for 1 hour. Then, 10 μL of 2.0 mg mL -1 The ZIF-67@PDA@PtCu@Ab probe was added to the modified electrode and incubated for another 1 h at the same temperature. After each incubation step, the electrode was thoroughly rinsed with 10 mM PBS to remove any unbound proteins and stored at 4 °C for later analysis. Differential pulse voltammetry (DPV) was measured using a scanning potential range of 0 V to -0.4 V, an amplitude of 0.07 V, a pulse width of 50 ms, and a pulse period of 200 ms. The measurements were performed in a solution containing 5 mM H 2 O 2 The PCR products were prepared in 10 mM PBS (pH 7.0).

[0077] (2) Optimization of experimental variables of biosensing strategy To achieve optimal analytical performance, various experimental parameters were systematically investigated (AQP4-IgG concentration was 10 U mL -1 ), the results are as follows Figure 2 shown.

[0078] The first step is to optimize the concentration of ZIF-67@PDA@PtCu to enhance the electrocatalytic activity and reduce the cost of the developed electrochemical immunosensor. Figure 2 As shown in Figure A, the best performance of ZIF-67@PDA@PtCu nanocomposites is achieved at a concentration of 2.0 mg mL -1 , after which the electrochemical current began to decrease. Therefore, it was determined that 2.0 mg mL -1 The ZIF-67@PDA@PtCu concentration was found to be the ideal concentration for the detection of AQP4-IgG. Next, the concentration of GO@PDA was adjusted, given its significant effect on the charge transfer kinetics on the electrode surface. Figure 2As shown in Figure B, the heights of the oxidation peak and the reduction peak are -1 Based on these results, 3 mg mL -1 The pH value may be a key factor affecting the stability of electrochemical immunosensors because it affects both the bioactivity of antigens and antibodies and the electrochemical properties of ZIF-67@PDA@PtCu. Various PBS solutions with pH values ​​ranging from 6.0 to 8.5 were tested. Figure 2 C in Figure 2 shows that as the pH value increases from 6.0 to 7.0, the electrochemical signal gradually increases and reaches a peak at pH 7.0. Thereafter, the signal begins to decrease as the pH value further increases. Finally, the incubation time of AQP4-IgG is another important factor affecting the performance of the entire system. Figure 2 As shown in D, the DPV signal gradually increased with the extension of incubation time and reached the maximum current response at 60 min.

[0079] (3) Under optimized experimental conditions, the electrochemical immunosensor was used to detect different concentrations of AQP4-IgG protein. The results are as follows: Figure 3 shown. Figure 3 Figure A shows the DPV signal values ​​for the quantitative detection of AQP4-IgG, ranging from 0.1 U mL -1 (curve a) to 80 U mL -1 (Curve h). Figure 3 In Figure B, there is an obvious linear relationship between DPV current and the logarithmic concentration of AQP4-IgG (R 2 = 0.978). The linear regression equation obtained was I = -27.76-21.48 lgC AQP4-IgG , the limit of detection (LOD) was calculated to be 0.05 U mL -1 , based on three standard deviations of the blank response. Compared with previous studies, the protease-free electrochemical immunosensor of the present invention has a wide linear range and excellent detection limit, which is due to the ZIF-67@PDA@PtCu nanozyme's ability to detect H 2 O 2 The high catalytic activity and excellent conductivity of GO@PDA.

[0080] Example 4 Specificity and stability analysis The selectivity, reproducibility and stability of the electrochemical immunosensor were evaluated. Figure 4 shown.

[0081] In order to evaluate the specificity of the electrochemical immunosensor of the present invention, cross-reactivity tests were performed using several interfering substances including bovine serum albumin (BSA), myelin oligodendrocyte glycoprotein IgG (MOG-IgG), anti-acetylcholine receptor (AChR) antibody, N-methyl-D-aspartate receptor (NMDAR) antibody, and a mixture containing AQP4-IgG. The test conditions were similar to those in Example 3. Figure 4 As shown in A, the analytes in A are blank (no analyte), serum from MOG, AChR, NMDAR positive patients, and mixture (10 U mL -1 AQP4-IgG and MOG, AChR, NMDAR). A significant current increase was observed in the presence of AQP4-IgG protein, while the signals of other interfering proteins showed no significant changes compared with the blank group. These results indicate that the electrochemical immunosensor is highly specific for AQP4-IgG, which may be due to the strong affinity of the antibody to the antigen and the minimal nonspecific adsorption of the nanomaterial.

[0082] In clinical applications, repeatability and stability are crucial to the performance of electrochemical immunosensors. To evaluate these factors, the relative standard deviation (RSD) method was used. The reproducibility of electrochemical immunosensors is an important indicator for judging the performance of the assay. Under optimal conditions, five consecutive measurements of 10 U mL -1 AQP4-IgG protein. Figure 4 As shown in B, it can be seen that the relative standard deviation (RSD) of the five times is less than 5%. In addition, by storing the electrochemical immunosensor at 4 °C for 15 days and keeping AQP4-IgG (10 U mL -1 ) concentration was kept constant and its long-term stability was tested. Figure 4 As shown in C, 10 U mL -1 The long-term stability of the developed electrochemical immunosensor was evaluated by AQP4-IgG, and the EC signal gradually decreased, but still retained 88.1% of the initial current. In conclusion, ZIF-67@PDA@PtCu NPs and GO@PDA materials maintained their performance and antibody affinity, ensuring the stability of the electrochemical immunosensor during long-term storage and transportation, making it very suitable for practical applications.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A ZIF-67@PDA@PtCu nanozyme, characterized in that: The invention comprises ZIF-67, and polydopamine and PtCu alloy nanoflowers loaded on the surface of the ZIF-67.

2. The ZIF-67@PDA@PtCu nanozyme according to claim 1, characterized in that The particle size of the ZIF-67 is 150-200 nm, and the particle size of the PtCu alloy nanoflower is 20-30 nm.

3. The method for preparing the ZIF-67@PDA@PtCu nanozyme according to claim 1 or 2, characterized in that: The following steps are involved: The ZIF-67 solution, the buffer solution and the dopamine hydrochloride solution were mixed for the first assembly to obtain ZIF-67@PDA; The alcohol solution of the ZIF-67@PDA is mixed with the alcohol solution of the PtCu alloy nanoflowers, and a second assembly is performed to obtain the ZIF-67@PDA@PtCu nanozyme.

4. Application of the ZIF-67@PDA@PtCu nanozyme described in claim 1 or 2 or the ZIF-67@PDA@PtCu nanozyme prepared by the preparation method described in claim 3 in the field of electrochemical biosensing.

5. An AQP4-IgG detection probe, characterized in that: It comprises the ZIF-67@PDA@PtCu nanozyme described in claim 1 or 2 or the ZIF-67@PDA@PtCu nanozyme prepared by the preparation method described in claim 3, and a secondary antibody chemically bound to the ZIF-67@PDA@PtCu nanozyme.

6. An electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme, characterized in that: It comprises an electrochemical immunosensor matrix and the AQP4-IgG detection probe according to claim 5; The electrochemical immunosensor matrix comprises a substrate electrode, a graphene oxide-polydopamine complex attached to the surface of the substrate electrode, a nanogold film deposited on the surfaces of the substrate electrode and the graphene oxide-polydopamine complex, AQP4 modified on the surface of the nanogold film, and bovine serum albumin modified on the surface of the nanogold film for blocking unbound sites.

7. The electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme according to claim 6, characterized in that: The preparation method of the electrochemical immunosensor matrix comprises the following steps: The graphene oxide-polydopamine complex solution is loaded onto the surface of the substrate electrode, and after drying, a substrate electrode modified with the graphene oxide-polydopamine complex is obtained; Electrodepositing a nano-gold film on the surface of the substrate electrode modified with the graphene oxide-polydopamine complex to obtain a substrate electrode deposited with the nano-gold film; AQP4 solution is loaded on the surface of the substrate electrode on which the nano-gold film is deposited, and the substrate electrode is incubated to obtain a substrate electrode modified with AQP4; A bovine serum albumin solution is loaded onto the surface of the substrate electrode modified with AQP4, and incubated to obtain an electrochemical immunosensor matrix.

8. Use of the electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme according to claim 6 or 7 in the preparation of a neuromyelitis optica detection kit.

9. A method for detecting AQP4-IgG for non-diagnostic purposes, characterized in that: The following steps are involved: Loading the sample to be tested onto the surface of the electrochemical immunosensor matrix, performing a first incubation, and obtaining a first incubation product; Loading an AQP4-IgG detection probe solution onto the surface of the first incubation product, performing a second incubation, and obtaining an electrode to be tested; Placing the electrode to be tested in a buffer solution containing H2O2, performing a differential pulse voltammetry test, obtaining a current signal peak value, and obtaining the AQP4-IgG content in the sample to be tested according to the current signal peak value and a predetermined standard curve; The standard curve is a linear relationship curve between the logarithmic concentration of AQP4-IgG and the peak value of the current signal; The electrochemical immunosensor matrix and AQP4-IgG detection probe are the electrochemical immunosensor matrix and AQP4-IgG detection probe in the electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme according to claim 6 or 7.

10. The detection method according to claim 9, characterized in that: The scanning potential range of the differential pulse voltammetry test is 0 V~-0.4 V, the amplitude is 0.07 V, the pulse width is 50 ms, and the pulse period is 200 ms.

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