A ZIF-67@PDA@PtCu nanozyme, its electrochemical immunosensor, and a method for detecting AQP4-IgG

Abstract

The present invention provides a ZIF-67@PDA@PtCu nanozyme, an electrochemical immunosensor thereof, and a method for detecting AQP4-IgG, belonging to the technical field of electrochemical immunosensing. The present invention combines the ZIF-67@PDA@PtCu nanozyme with the electrochemical immunosensor, providing a breakthrough strategy for the detection of AQP4-IgG. Specifically, the electrochemical immunosensor based on the ZIF-67@PDA@PtCu nanozyme constructed in the present invention has efficient electrochemical signals, high specificity for the detection of AQP4-IgG, excellent repeatability and stability, a wide detection range, and an extremely low detection limit, and can achieve sensitive, rapid, and low-cost detection of AQP4-IgG, having high guiding value for the detection of neuromyelitis optica.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical immunosensing, and specifically relates to a ZIF-67@PDA@PtCu nanozyme, 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 central nervous system inflammatory demyelinating diseases that can cause 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 the AQP4 channels, leading to inflammation, cell damage, and neuronal death.

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

[0004] In summary, there is an increasing interest in developing faster, more cost-effective, and portable AQP4-IgG detection schemes. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a ZIF-67@PDA@PtCu nanozyme, 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 achieve sensitive, rapid, and low-cost detection of AQP4-IgG.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a ZIF-67@PDA@PtCu nanozyme, which includes ZIF-67, and polydopamine and PtCu alloy nanoflowers loaded on the surface of the ZIF-67.

[0008] Preferably, the particle size of the ZIF-67 is 150-200 nm, and the particle size of the PtCu alloy nanoflowers is 20-30 nm.

[0009] The present invention provides a preparation method of the above-mentioned ZIF-67@PDA@PtCu nanozyme, which includes the following steps:

[0010] Mix a ZIF-67 solution, a buffer solution and a dopamine hydrochloride solution, and perform the first assembly to obtain ZIF-67@PDA;

[0011] Mix the alcohol solution of the ZIF-67@PDA and the alcohol solution of the PtCu alloy nanoflowers, and perform the second assembly to obtain the ZIF-67@PDA@PtCu nanozyme.

[0012] The present invention provides the application of the above-mentioned ZIF-67@PDA@PtCu nanozyme in the field of electrochemical biosensing.

[0013] The present invention provides an AQP4-IgG detection probe, which includes the above-mentioned ZIF-67@PDA@PtCu nanozyme, and a secondary antibody chemically bound to the ZIF-67@PDA@PtCu nanozyme.

[0014] The present invention provides an electrochemical immunosensor based on the ZIF-67@PDA@PtCu nanozyme, which includes an electrochemical immunosensor matrix and the above-mentioned AQP4-IgG detection probe;

[0015] The electrochemical immunosensor matrix includes a substrate electrode, a graphene oxide-polydopamine composite attached to the surface of the substrate electrode, a nano-gold film deposited on the surface of the substrate electrode and the graphene oxide-polydopamine composite, AQP4 modified on the surface of the nano-gold film, and bovine serum albumin modified on the surface of the nano-gold film for blocking unbound sites.

[0016] Preferably, the preparation method of the electrochemical immunosensor matrix includes the following steps:

[0017] Load a graphene oxide-polydopamine composite solution onto the surface of the substrate electrode, and after drying, obtain a substrate electrode modified with the graphene oxide-polydopamine composite;

[0018] Electrodeposit a nano-gold film on the surface of the substrate electrode modified with the graphene oxide-polydopamine composite to obtain a substrate electrode deposited with the nano-gold film;

[0019] Load an AQP4 solution onto the surface of the substrate electrode deposited with the nano-gold thin film and incubate to obtain a substrate electrode modified with AQP4;

[0020] Load a bovine serum albumin solution onto the surface of the substrate electrode modified with AQP4 and incubate to obtain the matrix of the electrochemical immunosensor.

[0021] The present invention provides the application of the above-mentioned electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme in the preparation of a detection kit for neuromyelitis optica.

[0022] The present invention provides a method for detecting AQP4-IgG for non-diagnostic purposes, comprising the following steps:

[0023] Load a sample to be tested onto the surface of the matrix of the electrochemical immunosensor and perform a first incubation to obtain a first incubation product;

[0024] Load an AQP4-IgG detection probe solution onto the surface of the first incubation product and perform a second incubation to obtain a test electrode;

[0025] Place the test electrode in a buffer solution containing H2O2, perform differential pulse voltammetry testing to obtain the peak value of the current signal, and obtain the content of AQP4-IgG in the sample to be tested according to the peak value of the current signal and a predetermined standard curve;

[0026] The standard curve is a linear relationship curve between the logarithmic concentration of AQP4-IgG and the peak value of the current signal;

[0027] The matrix of the electrochemical immunosensor and the AQP4-IgG detection probe are the matrix of the electrochemical immunosensor and the AQP4-IgG detection probe in the above-mentioned electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme.

[0028] Preferably, the scanning potential range of the differential pulse voltammetry testing 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.

[0029] The present invention provides a ZIF-67@PDA@PtCu nanozyme, which includes ZIF-67, as well as 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 compared 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-like structure provides a large number of active sites on its surface. However, the catalytic reduction ability of the single PtCu alloy is limited. The present invention adopts the method of introducing ZIF-67, where ZIF-67 has excellent stability and a high specific surface area. When loading 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, enhancing the interaction between the unsaturated edge sites of ZIF-67, thereby enhancing the mechanical, chemical stability and functionality of ZIF-67, and introducing 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 the catalytic efficiency.

[0030] The ZIF-67@PDA@PtCu nanozyme provided by the present invention has a high specific surface area and porosity, and has excellent catalytic performance, and can significantly amplify the electrochemical signal when used in electrochemical biosensing.

[0031] The present invention provides an AQP4-IgG detection probe, which includes the above-mentioned ZIF-67@PDA@PtCu nanozyme, and a secondary antibody chemically bound to the ZIF-67@PDA@PtCu nanozyme, denoted as the ZIF-67@PDA@PtCu@Ab probe. The AQP4-IgG detection probe provided by the present invention can be used for the electrochemical biosensing detection of AQP4-IgG.

[0032] The present invention provides an electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme, which includes 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, where the graphene oxide-polydopamine composite has a significant impact on the charge transfer kinetics of the electrode surface and can enhance the conductivity of the electrode; the role of polydopamine is to improve the conductive effect. The present invention combines high catalytic composite nanoparticles (ZIF-67@PDA@PtCu nanozyme) with an electrochemical immunosensor, providing a breakthrough strategy for the detection of AQP4-IgG. Specifically, the electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme constructed in the present invention has efficient electrochemical (EC) signals, high specificity for the detection of AQP4-IgG, excellent repeatability and stability, a wide detection range, and an extremely low detection limit, and can achieve sensitive, rapid, and low-cost detection of AQP4-IgG, having high clinical application value for the detection of neuromyelitis optica. The results of the examples show that the detection range of the electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme in the present invention is 0.1 ~ 80 U mL -1 , and the detection limit is 0.05 U mL -1 .

[0033] The present invention provides a method for detecting AQP4-IgG, which is simple, efficient, and portable, does not require relying on 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 a clinical environment. Description of the Drawings

[0034] Figure 1 are the structural and compositional analysis results of GO@PDA and ZIF-67@PDA@PtCu NPs; Figure 1 In, 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;

[0035] Figure 2 are the optimization results of the experimental variables of the biosensing strategy; Figure 2where A is the optimized concentration of ZIF-67@PDA@PtCu, mg mL -1 ; B is the optimized concentration of GO@PDA, mg mL -1 ; C is the optimized pH value of the detection solution, D is the optimized incubation time of AQP4-IgG, min;

[0036] Figure 3 are the test results of the immunosensor for detecting AQP4-IgG protein at different concentrations; Figure 3 where A is the DPV value of the electrochemical immunosensor designed under different concentrations of the target AQP4-IgG (a→g: 0.1, 1, 5, 10, 20, 40, 60, and 80 U mL -1 ); B is the standard curve of the peak current versus the logarithm of the AQP4-IgG concentration;

[0037] Figure 4 are the test results of the selectivity, reproducibility, and stability of the electrochemical immunosensor; Figure 4 where A is the test result of selectivity; B is the test result of reproducibility, C is the test result of stability. Detailed implementation mode

[0038] The present invention provides a ZIF-67@PDA@PtCu nanozyme, including ZIF-67, and polydopamine and PtCu alloy nanoflowers loaded on the surface of the ZIF-67.

[0039] In the present invention, the polydopamine and PtCu alloy nanoflowers are preferably loaded on the surface of the ZIF-67 in sequence; the polydopamine is preferably wrapped on the surface of the ZIF-67 in the form of a polydopamine layer, and the PtCu alloy nanoflowers are distributed on the surface of the ZIF-67 wrapped with the polydopamine layer.

[0040] In the present invention, the particle size of the ZIF-67 is preferably 150-200 nm; the particle size of the PtCu alloy nanoflowers is preferably 20-30 nm, more preferably 24 nm; in the PtCu alloy nanoflowers, the mass ratio of Pt and 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 nanoflowers is preferably 5-10:1, more preferably 6-8:1.

[0041] The present invention has no special requirements for the source of the ZIF-67, and it can be either the conventional commercially available ZIF-67 in the art or prepared by itself.

[0042] In the present invention, the preparation method of the PtCu alloy nanoflowers preferably includes the following steps:

[0043] K2PtCl4, CuCl2, a dispersant and water were ultrasonically mixed, and an ascorbic acid aqueous solution was added while ultrasonically mixing to obtain a uniformly mixed solution;

[0044] The uniformly mixed solution was placed in a heating environment for a reduction reaction to obtain PtCu alloy nanoflowers.

[0045] In the present invention, the dispersant is preferably CTAC. In the present invention, the mass ratio of K2PtCl4, CuCl2, and the dispersant is preferably 38.19:12.37:640. In the present invention, the water is preferably deionized water, and the dosage ratio of K2PtCl4 to water is preferably 38.19 mg:40 mL. After the addition of K2PtCl4, CuCl2, the dispersant, and water is completed in the present invention, a flocculent mixture is formed.

[0046] 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 dosage of ascorbic acid is preferably in excess.

[0047] In the present invention, the temperature of the reduction reaction is preferably 90 °C, and the time is preferably 5 min. During the reaction process, the appearance of the solution changes from white to transparent brown within 5 min and finally becomes opaque black. After the reduction reaction, in the present invention, it is preferably to cool the obtained reduction reaction solution to room temperature, followed by solid-liquid separation, washing, and drying in sequence. In the present invention, the solid-liquid separation is preferably centrifugation, the washing is preferably washing with deionized water, and the number of washing times is preferably three; the drying is preferably vacuum drying, the drying temperature is preferably 40 °C, and the time is preferably overnight.

[0048] 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 diameter is preferably 1.89 nm, and the pore volume is preferably 0.50 cm 3 g -1 .

[0049] In the present invention, the preparation method of the ZIF-67@PDA@PtCu nanozyme includes the following steps:

[0050] Mix the ZIF-67 solution, the buffer solution, and the hydrochloric acid dopamine solution for the first assembly to obtain ZIF-67@PDA;

[0051] Mix the alcohol solution of ZIF-67@PDA with the alcohol solution of PtCu alloy nanoflowers for the second assembly to obtain ZIF-67@PDA@PtCu nanozyme.

[0052] In the present invention, a ZIF-67 solution, a buffer solution, and a dopamine hydrochloride solution are mixed for the first assembly to obtain ZIF-67@PDA. In the present invention, the solvent of the ZIF-67 solution is preferably a mixed solution of ethanol and water, and the volume ratio of 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 .

[0053] In the present invention, the buffer solution is preferably a Tris-HCl buffer solution, the concentration of the Tris-HCl buffer solution 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 .

[0054] In the present invention, the dosage ratio of the ZIF-67, the buffer solution, and the dopamine hydrochloride solution is preferably 100 mg: 5 mL: 20 μL.

[0055] In the present invention, the first assembly is preferably carried out under stirring conditions, the temperature of the first assembly is preferably room temperature, and the time is preferably 6 h. 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.

[0056] After obtaining the ZIF-67@PDA, the present invention mixes the alcohol solution of the ZIF-67@PDA with the alcohol solution of PtCu alloy nanoflowers for the second assembly to obtain 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 10-20 mg mL -1 . In the present invention, the volume ratio of the alcohol solution of the ZIF-67@PDA to the alcohol solution of the PtCu alloy nanoflowers is preferably 1-2: 1-2.

[0057] In the present invention, the second assembly is preferably carried out under stirring conditions, the temperature of the second assembly is preferably room temperature, and the time is preferably 8 h. 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.

[0058] The present invention provides the application of the above ZIF-67@PDA@PtCu nanozyme in the field of electrochemical biosensing.

[0059] The present invention provides an AQP4-IgG detection probe, which includes the above ZIF-67@PDA@PtCu nanozyme and a secondary antibody chemically conjugated 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 linked to the amino group in the ZIF-67@PDA@PtCu nanozyme, and the amino group is derived from polydopamine.

[0060] In the present invention, the preparation method of the AQP4-IgG detection probe preferably includes the following steps:

[0061] Mix the ZIF-67@PDA@PtCu nanozyme with a buffer solution, add EDC·HCl and the secondary antibody, and carry out a coupling reaction;

[0062] After the coupling reaction, add a BSA solution to block the unreacted sites.

[0063] 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 mixture is preferably 0.5~3 mg·mL -1 , more preferably 2mg·mL -1 .

[0064] 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 2.5 h.

[0065] In the present invention, the mass concentration of the BSA solution is preferably 10%; the time for blocking the unreacted sites is preferably 2 h.

[0066] The present invention provides an electrochemical immunosensor based on the ZIF-67@PDA@PtCu nanozyme, which includes an electrochemical immunosensor matrix and the above AQP4-IgG detection probe.

[0067] In the present invention, the electrochemical immunosensor matrix includes a substrate electrode, a graphene oxide-polydopamine composite attached to the surface of the substrate electrode, a nano-gold film deposited on the surfaces of the substrate electrode and the graphene oxide-polydopamine composite, AQP4 modified on the surface of the nano-gold film, and bovine serum albumin modified on the surface of the nano-gold film for blocking unbound sites.

[0068] In the present invention, the graphene oxide-poly dopamine composite includes graphene oxide and poly dopamine coated on the surface of the graphene oxide. In the present invention, the preparation method of the graphene oxide-poly dopamine composite (GO@PDA) preferably includes the following steps:

[0069] Ultrasonically mix the graphene oxide with the solvent, and sequentially add dopamine hydrochloride and Tris-HCl buffer solution for a loading reaction.

[0070] 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 dosage ratio of the graphene oxide to the solvent is preferably 80 mg:140 mL. In the present invention, the time for ultrasonic mixing is preferably 5 min.

[0071] In the present invention, the mass ratio of the graphene oxide to the dopamine hydrochloride is preferably 80:1-2. In the present invention, the concentration of the Tris-HCl buffer solution is preferably 10 mM, and the pH value is preferably 8.5.

[0072] 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 washing is preferably ethanol and deionized water; the drying temperature is preferably 50 °C.

[0073] In the present invention, the preparation method of the electrochemical immunosensor matrix includes the following steps:

[0074] Load the graphene oxide-poly dopamine composite solution onto the surface of the substrate electrode, and obtain a substrate electrode modified with the graphene oxide-poly dopamine composite after drying;

[0075] Electrodeposit a nano-gold film on the surface of the substrate electrode modified with the graphene oxide-poly dopamine composite to obtain a substrate electrode deposited with the nano-gold film;

[0076] Load the AQP4 solution onto the surface of the substrate electrode deposited with the nano-gold film for incubation to obtain a substrate electrode modified with AQP4;

[0077] Load the bovine serum albumin solution onto the surface of the substrate electrode modified with AQP4 for incubation to obtain the electrochemical immunosensor matrix.

[0078] In the present invention, a graphene oxide-poly dopamine composite solution is loaded onto the surface of a substrate electrode, and after drying, a substrate electrode modified with the graphene oxide-poly dopamine composite is obtained. In the present invention, the substrate electrode is preferably a glassy carbon electrode (GCE). Before use, the present invention preferably pre-treats the substrate electrode, and the pre-treatment preferably includes: polishing, cleaning, and drying the substrate electrode in sequence. In the present invention, the polishing agent used for polishing is preferably Al2O3 slurry, and the polishing time is preferably 8 min.

[0079] In the present invention, the concentration of the graphene oxide-poly dopamine composite solution is preferably 1-6 mg ml -1 , and the dosage is preferably 10 μL; the drying time is preferably 37 °C.

[0080] In the present invention, a nano-gold film is electrodeposited on the surface of the substrate electrode modified with the graphene oxide-poly dopamine composite to obtain a substrate electrode deposited with the nano-gold film. In the present invention, the electrodeposition is preferably carried out in a HAuCl4 solution, and the mass concentration of the HAuCl4 solution is preferably 1.0%. In the present invention, the potential of the electrodeposition is preferably -0.2 V, and the time is preferably 30 s. After the electrodeposition, the present invention preferably rinses the electrode with deionized water and air-dries it at room temperature.

[0081] In the present invention, an AQP4 solution is loaded onto the surface of the substrate electrode deposited with the nano-gold film and incubated 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 , and the loading amount is preferably 10 μL. In the present invention, the incubation temperature is preferably 37 °C, and the time is preferably 1 h.

[0082] In the present invention, 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. In the present invention, the concentration of the bovine serum albumin solution is preferably 1 wt%, and the loading amount is preferably 10 μL. In the present invention, the incubation temperature is preferably 37 °C, and the time is preferably 1 h. After the incubation, the present invention preferably rinses the electrode with a 10 mM, pH 7.4 PBS buffer solution to remove any unbound proteins.

[0083] 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 binds to AQP4-IgG (antibody) and anti-AQP4-IgG antibody in the test sample to construct a sandwich-type electrochemical immunosensor.

[0084] The present invention provides the application of the above-mentioned electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme in the preparation of a detection kit for neuromyelitis optica.

[0085] The present invention provides a method for detecting AQP4-IgG for non-diagnostic purposes, comprising the following steps:

[0086] Loading a sample to be tested onto the surface of the electrochemical immunosensor matrix, performing a first incubation to obtain a first incubation product;

[0087] Loading an AQP4-IgG detection probe solution onto the surface of the first incubation product, performing a second incubation to obtain a working electrode;

[0088] Placing the working electrode in a buffer solution containing H2O2, performing differential pulse voltammetry testing to obtain a peak current signal, and obtaining the content of AQP4-IgG in the sample to be tested according to the peak current signal and a predetermined standard curve;

[0089] The standard curve is a linear relationship curve between the logarithmic concentration of AQP4-IgG and the peak current signal;

[0090] 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.

[0091] In the present invention, a sample to be tested is loaded onto the surface of the electrochemical immunosensor matrix, and a first incubation is performed to obtain 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 1 h. After the first incubation, the present invention preferably rinses the electrode with a 10 mM, pH 7.4 PBS buffer solution to remove any unbound proteins.

[0092] After obtaining the first incubation product, an AQP4-IgG detection probe solution is loaded onto the surface of the first incubation product, and a second incubation is performed to obtain a working electrode. In the present invention, the concentration of the AQP4-IgG detection probe solution is preferably 0.5-3 mg mL -1 , and 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 1 h. After the second incubation, the present invention preferably rinses the electrode with a 10 mM, pH 7.4 PBS buffer solution to remove any unbound proteins. In the present invention, the working electrode is preferably stored at 4 °C for subsequent analysis.

[0093] After obtaining the electrode to be tested, the present invention places the electrode to be tested in a buffer solution containing H2O2 for differential pulse voltammetry testing to obtain the peak value of the current signal. In the present invention, the buffer solution containing H2O2 is preferably a 10 mM, pH 7.4 PBS buffer solution containing H2O2, and the concentration of H2O2 is preferably 5 mM.

[0094] In the present invention, the scanning potential range of the differential pulse voltammetry testing 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.

[0095] After obtaining the current peak value, the present invention obtains the content of AQP4-IgG in the sample to be tested according to the peak value of the current signal 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. In the present invention, the method for obtaining the standard curve preferably includes the following steps:

[0096] Provide AQP4-IgG standard solutions with known gradient concentrations;

[0097] Using the AQP4-IgG standard solutions with known gradient concentrations as the samples to be tested, load them onto the surface of the electrochemical immunosensor matrix, sequentially load the AQP4-IgG detection probe solution and perform differential pulse voltammetry testing in the above manner to obtain the peak values of the current signals corresponding to different concentrations of AQP4-IgG, and draw a standard curve according to the logarithmic concentration of AQP4-IgG and the peak value of the current signal.

[0098] In the present invention, the source of AQP4-IgG is commercially available.

[0099] 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 detection limit (LOD) is calculated as 0.05 U mL -1 .

[0100] In the present invention, the linear detection range of AQP4-IgG is preferably 0.1 U mL -1 ~ 80 U mL -1 .

[0101] The following is a detailed description of a ZIF-67@PDA@PtCu nanozyme and its electrochemical immunosensor, and a method for detecting AQP4-IgG provided by the present invention in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0102] Example 1 Preparation of ZIF-67@PDA@PtCu nanozyme, which is carried out by the following steps:

[0103] (1) Synthesis of PtCu NPs

[0104] Mix 38.19 mg of K2PtCl4, 12.37 mg of CuCl2 and 640 mg of CTAC with 40 mL of deionized water in a flask to form a flocculent mixture, and ultrasonicate it for 10 minutes at 100 W. While performing the ultrasonication, add 3 mL (17.6 mg mL -1 ) of freshly prepared ascorbic acid aqueous solution to the mixture. The resulting homogeneous solution is vigorously stirred in an oil bath at 90 °C for 5 min, and then cooled to room temperature. During the reaction, the appearance of the solution changes from white to transparent brown within 5 minutes and finally to opaque black. The obtained product is centrifuged, washed three times with deionized water, and dried overnight in vacuo at 40 °C to obtain PtCu NPs.

[0105] (2) Synthesis of ZIF-67

[0106] Dissolve 0.5 mmol of Co(NO3)2·6H2O and 5 mmol of 2-methylimidazole in 10 mL of methanol at 37 °C respectively to obtain a salt solution and a ligand solution. Then add the salt solution to the ligand solution and stir vigorously. After stirring for 12 minutes, let it stand for 20 hours. The obtained solid product is centrifuged and collected, washed 3 times with methanol, and dried in vacuo at 40 °C for 24 hours to obtain ZIF-67.

[0107] (3) Synthesis of ZIF-67@PDA

[0108] Synthesize PDA-modified ZIF-67 by a one-pot assembly method. Specifically, dissolve 100 mg of ZIF-67 in a mixture of 5 mL of ethanol and water (3:4, v / v), and mix the above solution with 5 mL of 10 mM Tris-HCl buffer (pH 8.5) under ultrasonication. Then, add 20 µL of 2 mg mL -1 hydrochloric acid dopamine solution to the mixture and stir continuously for 6 hours. The obtained ZIF-67@PDA is separated by centrifugation at 12,000 rpm for 5 minutes, washed twice with methanol, and then redispersed in 2 mL of methanol.

[0109] (4) Synthesis of ZIF-67@PDA@PtCu

[0110] Add 1 mL of PtCu nanoparticle methanol solution with a concentration of 20 mg mL -1 to 2 mL of a solution with a concentration of 10 mgmL-1 Stirred in the ZIF-67@PDA dispersion for 8 hours. The obtained product was centrifuged, washed three times with methanol, and then redispersed in deionized water for later use.

[0111] Example 2 Construction of the electrochemical immunosensor matrix (BSA / AQP4 / GO@PDA / GCE)

[0112] (1) Construction of GO@PDA

[0113] First, 80 mg of graphene oxide was ultrasonically dispersed in a mixture of 140 mL of ethanol and water (3:4, v / v) for 5 minutes. Then, 2 mg of dopamine hydrochloride was added to the solution, and it was stirred 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 continuously stirred overnight. Subsequently, the obtained sediment was collected by centrifugation and thoroughly washed with ethanol and deionized water until the supernatant was clear. Finally, the black precipitate was dried at 50 °C.

[0114] (2) Synthesis of ZIF-67@PDA@PtCu@Ab probe

[0115] Dissolve 2 mg of ZIF-67@PDA@PtCu nanoparticles in 1 mL of 0.2 M boric acid buffer solution (BBS) (pH 7.4). Then add 400 μg of EDC·HCl and 200 μg of mouse anti-human IgG labeled antibody, and shake well for 2.5 hours. To block any unreacted sites, add 110 μL of 10 wt% BSA solution, and then shake for another 2 hours. Finally, collect the ZIF-67@PDA@PtCu@Ab probe, centrifuge at 12,000 rpm for 10 minutes, and redisperse in 0.5 mL of fresh BBS solution.

[0116] (3) Construction of BSA / AQP4 / GO@PDA / GCE

[0117] First, polish the bare GCE with Al2O3 slurry for 8 minutes until a mirror-like surface is obtained. Then ultrasonically clean it with deionized water for 5 cycles and dry it with nitrogen in preparation for modification. Subsequently, 10 μL of the GO@PDA solution with a concentration of 3 mg mL -1 was loaded onto the cleaned GCE and dried at 37 °C. Electrodeposit AuNPs on the GCE surface by maintaining a constant potential of -0.2 V in 1.0% HAuCl4 solution for 30 seconds to form an AuNPs / GO@PDA film. Then rinse the electrode with deionized water and air-dry it at room temperature. Add 10 μL of AQP4 solution (20 μg mL -1),(Incubate at 37 °C for 1 hour. To block non-specific binding sites, take 10 μL of 1 wt.% BSA solution and incubate at 37 °C for 1 hour. Finally, rinse the obtained electrochemical immunosensor (BSA / AQP4 / GO@PDA / GCE) with 10 mM PBS (pH 7.4) and store it at 4 °C for later use.)

[0118] Structural Characterization

[0119] Analyze the structures and compositions of GO@PDA and ZIF-67@PDA@PtCu NPs, and the obtained results are as Figure 1 shown. As Figure 1 shown in A of Figure 1 , the structure of GO@PDA was observed by transmission electron microscopy (TEM), and the results confirmed the successful synthesis of GO@PDA. As Figure 1 shown in B of Figure 1 , 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. As Figure 1 shown in C of Figure 1 , 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 reveal that these PtCu NPs form a nanoflower-like structure (see Figure 1 D and E of Figure 1 ), and PtCu NPs are successfully encapsulated on the surface of ZIF-67. When loaded onto ZIF-67, the PtCu NPs on the ZIF-67 nanocomposite exhibit a typical mesoporous morphology. As -1 shown in F - K of

[0120] , Pt and Cu are mainly distributed on the surface of the nanoparticle structure, while Co, N, C, and O are uniformly distributed inside. The XRD peaks of ZIF-67@PDA@PtCu ( Figure 1 in L) are consistent with those of ZIF-67@PDA, indicating that ZIF-67@PDA@PtCu retains its phase structure. Subsequently, XPS was used to analyze the elemental composition and chemical bonds of the ZIF-67@PDA@PtCu nanocomposite. Figure 1 The XPS spectrum of the nanocomposite shown in M of Figure 1 contains peaks of C 1s, Cu 2p, N 1s, Pt 4f, O 1s, and Co 2p. As -1 shown in N of

[0120] , the FT-IR spectrum shows that compared with ZIF-67@PDA, ZIF-67@PDA@PtCu has a new peak at 912 cm -1 , indicating that PtCu has been effectively loaded on the surface of ZIF-67.

[0120] Example 3 Electrochemical Detection of AQP4-IgG

[0121] (1) Equal aliquots of AQP4-IgG solutions (serum samples) with different concentrations, 8 μL each, were applied to the surface of BSA / AQP4 / GO@PDA / GCE and incubated at 37 °C for 1 hour. Subsequently, 10 μL of the ZIF-67@PDA@PtCu@Ab probe with a concentration of 2.0 mg mL -1 was added to the modified electrode and incubated for another 1 hour 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 measurement was carried out in a 10 mM PBS (pH 7.0) solution containing 5 mM H2O2.

[0122] (2) Optimization of experimental variables of the biosensing strategy

[0123] To obtain the best analytical performance, various experimental parameters were systematically studied (where the concentration of AQP4-IgG was 10 U mL -1 ), and the results are as Figure 2 shown.

[0124] The first step was to optimize the concentration of ZIF-67@PDA@PtCu to enhance the electrocatalytic activity and reduce the cost of the developed electrochemical immunosensor. As shown in A of Figure 2 , the best performance of the ZIF-67@PDA@PtCu nanocomposite was achieved at a concentration of 2.0 mg mL -1 , after which the electrochemical current began to decline. Therefore, 2.0 mg mL -1 of ZIF-67@PDA@PtCu was determined as the ideal concentration for detecting AQP4-IgG. Next, the concentration of GO@PDA was adjusted, considering its significant impact on the charge transfer kinetics at the electrode surface. As shown in B of Figure 2 , the heights of the oxidation and reduction peaks reached the maximum at a GO@PDA concentration of 3 mg mL -1 . Based on these results, 3 mg mL -1 of GO@PDA was selected as the optimal concentration in this study. The pH value may be a key factor affecting the stability of the electrochemical immunosensor because it affects both the biological activities of antigens and antibodies and the electrochemical properties of ZIF-67@PDA@PtCu. Various PBS solutions with pH values between 6.0 and 8.5 were tested. Figure 2In C, it 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. After that, as the pH value further increases, the signal starts to decline. Finally, the incubation time of AQP4-IgG is another important factor affecting the performance of the whole system. As Figure 2 shown in D of, the DPV signal gradually increases with the prolongation of the incubation time and reaches the maximum current response at 60 minutes.

[0125] (3)Under the optimized experimental conditions, an electrochemical immunosensor was used to detect AQP4-IgG proteins with different concentrations, and the obtained results are as Figure 3 shown. Figure 3 A in 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). In Figure 3 B, there is an obvious linear relationship (R 2 = 0.978) between the DPV current and the logarithmic concentration of AQP4-IgG. The obtained linear regression equation is I = -27.76 - 21.48 lgC AQP4-IgG , and the limit of detection (LOD) is calculated to be 0.05 U mL -1 , based on three standard deviations of the blank response. Compared with previous studies, this protease-free electrochemical immunosensor of the present invention has a wide linear range and excellent detection limit, thanks to the high catalytic activity of ZIF-67@PDA@PtCu nanozyme towards H2O2 and the excellent conductivity of GO@PDA.

[0126] Example 4 Specificity and Stability Analysis

[0127] The selectivity, reproducibility and stability of the electrochemical immunosensor were evaluated, and the obtained results are as Figure 4 shown.

[0128] To evaluate the specificity of the electrochemical immunosensor of the present invention, several interfering substances including bovine serum albumin (BSA), myelin oligodendrocyte glycoprotein IgG (MOG-IgG), anti-acetylcholine receptor (AChR) antibody, N-methyl-D-aspartic acid receptor (NMDAR) antibody, and a mixture containing AQP4-IgG were used for cross-reactivity tests, and the test conditions refer to Example 3. As Figure 4 shown in A of, the analytes in A are in turn blank (without analyte), sera from MOG, AChR, NMDAR positive patients, mixture (10 U mL -1AQP4-IgG, MOG, AChR, and NMDAR). A significant increase in current was observed in the presence of AQP4-IgG protein, while the signals of other interfering proteins showed no significant change compared to the blank group. These results indicate that the electrochemical immunosensor has high specificity for AQP4-IgG, which may be due to the strong affinity between the antibody and the antigen and the extremely low non-specific adsorption of the nanomaterials.

[0129] In clinical applications, repeatability and stability are crucial for the performance of electrochemical immunosensors. To evaluate these factors, the relative standard deviation (RSD) method was adopted. The reproducibility of electrochemical immunosensors is an important indicator for judging the detection performance. Under the optimal conditions, 10 U mL of AQP4-IgG protein was measured five times continuously. -1 As shown in Figure 4 B, it can be seen that the relative standard deviation (RSD) of the five measurements was less than 5%. In addition, the long-term stability of the electrochemical immunosensor was tested by storing it at 4 °C for 15 days while keeping the concentration of AQP4-IgG (10 U mL -1 ) constant. As shown in Figure 4 C, the long-term stability of the developed electrochemical immunosensor was evaluated with 10 U mL -1 of AQP4-IgG. The EC signal gradually decreased, but still retained 88.1% of the initial current. In summary, the 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.

[0130] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An AQP4-IgG detection probe, characterized in that: It includes a ZIF-67@PDA@PtCu nanozyme, and a secondary antibody chemically bound to the ZIF-67@PDA@PtCu nanozyme; The ZIF-67@PDA@PtCu nanozyme comprises ZIF-67, and polydopamine and PtCu alloy nanoflowers loaded on the surface of the ZIF-67; 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.

2. The AQP4-IgG detection probe 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. 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 1 or 2; 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.

4. The electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme according to claim 3, 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.

5. Use of the electrochemical immunosensor based on ZIF-67@PDA@PtCu nanozyme according to claim 3 or 4 in the preparation of a neuromyelitis optica detection kit.

6. 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 3 or 4.

7. The detection method according to claim 6, 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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