An electrochemical immunosensing interface for aquaporin 4 antibody and its construction method

Abstract

This invention discloses an electrochemical immunosensing interface for aquaporin 4 (AQP4) antibodies and its construction method. The technical solution uses a screen-printed carbon electrode (SPCE) as a substrate, immobilizing Ni²⁺ onto the working electrode surface via potentiostatic adsorption. AQP4 is then directionally immobilized using the chelation effect of Ni²⁺ with a histidine tag. After blocking with bovine serum albumin, an immunosensing interface is constructed for the electrochemical detection of AQP4 antibodies (Anti-AQP4). This interface construction method is simple and rapid, provides uniform and stable Ni²⁺ immobilization, high efficiency in the directional binding of AQP4, and exhibits good selectivity and anti-interference capabilities, offering a new approach for the detection of antibodies related to autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical immunosensors, specifically relating to an electrochemical immunosensing interface based on Ni²⁺ chelation of aquaporin 4 antibody and its construction method. Background Technology

[0002] The diagnosis of autoimmune diseases such as neuromyelitis optica spectrum disorder (NMOSD) relies on the detection of specific antibodies, among which aquaporin 4 antibody (Anti-AQP4) is an important serological marker for NMOSD. Currently, the main methods for clinical detection of Anti-AQP4 include enzyme-linked immunosorbent assay (ELISA) and cell immunofluorescence assay (CBA), but these methods have drawbacks such as cumbersome operation, long processing time, and expensive equipment. Electrochemical immunosensors, with their advantages of high sensitivity, rapid response, and low cost, show broad application prospects in the field of biomarker detection.

[0003] The core of electrochemical immunosensors lies in the construction of the immunosensing interface, and the key lies in the immobilization method and efficiency of the biorecognition element. Traditional physical adsorption methods have drawbacks such as weak binding force, easy desorption, and random orientation; covalent cross-linking methods may damage protein active sites.

[0004] Metal chelate affinity techniques utilize the specific coordination binding of transition metal ions to histidine tags to achieve targeted protein immobilization, preserving the protein's native conformation and biological activity. Nickel ions (Ni²⁺) are the most commonly used metal ion in metal chelate affinity techniques. Protein immobilization is achieved by forming coordination bonds with the imidazole groups of histidine residues. The basic principle is as follows: the transition metal Ni... 2+ A relatively stable metal chelate is formed by electronegative elements (O, N) on the electron donor ligand, specifically the carboxyl or amino group, which can interact with metal ions. Typically, a Ni... 2+ There are six coordination sites, which are immobilized on the adsorbent by chelates containing 3, 4, or 5 electron donor groups (coordination sites), while the remaining unoccupied sites are exposed in solution and can specifically bind to proteins with histidine, cysteine, and tryptophan side chains or histidine tags, while other impurity proteins cannot bind or can only bind weakly. Meanwhile, purified proteins in existing immunoassay kits often have a small amount of His tag transcribed at the amino acid terminus, with a small molecular weight (approximately 0.84 KD), which does not affect the function of the target protein. Therefore, theoretically, based on the excellent binding properties of Ni²⁺ to histidine, histidine-labeled target antigens or antibodies could be immobilized on the electrochemical immunosensing interface. However, research has found that this technology has not yet been applied to the construction of electrochemical immunosensing interfaces. Summary of the Invention The purpose of this invention is to overcome the shortcomings of the existing technology and provide an electrochemical immunosensing interface based on Ni²⁺ chelation of aquaporin 4 antibody and its construction method.

[0005] To achieve the above objectives, the technical solution of the present invention is to provide a method for constructing an electrochemical immunosensing interface for aquaporin 4 antibody. Using a screen-printed carbon electrode (SPCE) as a substrate, Ni²⁺ is rapidly immobilized onto the surface of the working electrode by a constant potential adsorption method. Aquaporin 4 (AQP4) is oriented and immobilized by the chelation effect of Ni²⁺ and histidine tags. After being blocked with bovine serum albumin, an immunosensing interface is constructed.

[0006] Further settings include the following steps: (1) SPCE pretreatment: After washing with ultrapure water and drying with nitrogen, the SPCE was activated in 0.1 mol / L PBS buffer solution by cyclic voltammetry. The scanning range was -0.6 V to 1.6 V, the number of scans was 40, and the scan rate was 200 mV / s. (2) Ni²⁺ potentiostatic adsorption: The activated SPCE was placed in a 0.1 mol / L NiCl2 aqueous solution and adsorbed for 100 s under a constant potential of -0.6 V using the chronoamperometry method. The SPCE was then rinsed with ultrapure water and air-dried at room temperature. (3) Directional fixation of AQP4: 10 μg / mL AQP4 solution was added dropwise to the Ni-modified interface, incubated overnight at 4°C, washed with PBS and dried with nitrogen; (4) Blocking treatment: 1% BSA solution was added to the interface and reacted at room temperature for 40 min. The interface was washed with PBST and PBS and dried with nitrogen to obtain the immunosensing interface.

[0007] The present invention also provides an electrochemical detection method for aquaporin 4 antibody, wherein the Anti-AQP4 solution to be tested is incubated with an immunosensing interface at 37°C for 60 min, and 0.2 M NaOH (containing 5 mM glucose) is used as the detection solution. The current response is measured at a potential of 0.55 V using the chronoamperometry method to realize the detection of Anti-AQP4.

[0008] The sensing interface construction process and fixation effect provided by the present invention were characterized and confirmed using techniques such as SEM, XPS and electrochemical impedance spectroscopy.

[0009] The innovative mechanism and advantages of this invention are as follows: For the first time, free Ni²⁺ is used as the constructing material for the immunosensing interface. Ni²⁺ is immobilized on the SPCE surface through a combination of electrostatic adsorption and potentiostatic deposition. Then, the specific chelation between Ni²⁺ and the 6His tag achieves the directional immobilization of AQP4. This method avoids the use of traditional chelating ligands, simplifies the interface construction steps, and reduces costs. The coordination binding of Ni²⁺ to the histidine tag is highly specific, which helps maintain the native conformation and biological activity of AQP4. The directionally immobilized AQP4 can effectively recognize and capture Anti-AQP4, forming an immune complex. Signal detection is achieved by detecting changes in the catalytic current of Ni²⁺ on glucose.

[0010] The immunosensing interface constructed in this invention has a simple and rapid preparation process, mild and controllable conditions, uniform and stable Ni²⁺ fixation, high AQP4 binding efficiency, good selectivity and anti-interference ability, and provides a new method for the detection of antibodies related to autoimmune diseases. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0012] Figure 1 Schematic diagram of sensor interface construction Figure 2 SEM scans of Ni / SPCE and AQP4 / Ni / SPCE; Figure 3 : Percentage of Ni and Cl atomic contents in different interfaces; Figure 4 Cyclic voltammetric scans at different interfaces; Figure 5 Electrochemical impedance spectroscopy at different interfaces; Figure 6 SPCE activation scan cycle optimization diagram; Figure 7 Optimized adsorption potential diagram for Ni²⁺ electrodeposition; Figure 8 Optimization diagram of Ni²⁺ electrodeposition adsorption time; Figure 9 AQP4 concentration optimization diagram; Figure 10 Optimization diagram of standard product incubation time; Figure 11: Current response diagram of the sensor to different interfering substances. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0014] Preparation Examples 1. Reagent preparation (1) 0.1 mol / L NiCl2 aqueous solution: Weigh 2.3769 g NiCl2·6H2O, dissolve in ultrapure water and make up to 100 mL, store at 4℃ protected from light, and equilibrate at room temperature for 20-30 min before use; (2) 10 µg / mL AQP4 working solution: Take 1 µL of AQP4 stock solution (0.7 mg / mL), dilute with ultrapure water to 70 µL, and use immediately.

[0015] (3) 1% BSA solution: Weigh 50 mg BSA, dissolve in 1 mL 0.01 mol / L PBS, vortex to mix, let stand at 4℃ for 12 h, and store in aliquots at -20℃; (4) Detection solution: 0.2 M NaOH (containing 5 mM glucose), i.e., take 1 mL of 0.2 M NaOH and mix it with 5 µL of 1 M glucose, and prepare it fresh for use; 2. Construction of Immune Sensing Interface (1) SPCE pretreatment: Remove the SPCE protective layer, rinse with ultrapure water, and dry with nitrogen; insert the adapter, cover the electrode with 50 µL of 0.1 mol / L PBS, and activate by CV scan (-0.6 V to 1.6 V, 40 cycles, 200 mV / s); rinse with ultrapure water and dry with nitrogen; (2) Ni²⁺ potentiostatic adsorption: 50 µL of 0.1 mol / L NiCl2 was used to cover the electrode, and adsorption was performed by chronoamperometry (-0.6V, 100 s); the electrode was rinsed with ultrapure water and dried at room temperature to obtain Ni / SPCE.

[0016] (3) AQP4 fixation: Add 2.5 µL of 10 µg / mL AQP4 to the working area and incubate overnight at 4℃; wash with PBS and dry with nitrogen to obtain AQP4 / Ni / SPCE.

[0017] (4) BSA blocking: Add 3 µL of 1% BSA to the working area and incubate at room temperature for 40 min; wash with PBST and PBS, dry with nitrogen, and obtain BSA / AQP4 / Ni / SPCE, which is stored at 4℃.

[0018] 3. Electrochemical detection Add 2.5 µL of the Anti-AQP4 solution to the interface and incubate at 37 °C for 60 min; wash with PBST and PBS. Cover the electrode with 50 µL of the detection solution and measure the current using chronoamperometry (0.55 V, 120 s), recording the current value after 100 s.

[0019] Performance Test Examples 1. Morphology and elemental characterization SEM and EDS analyses showed that ( Figure 2 , Figure 3 The AQP4 / Ni / SPCE interface exhibited a silver ear-like structure, and the contents of Ni and Cl elements were significantly lower than those in Ni / SPCE, confirming that AQP4 was successfully immobilized.

[0020] 2. Characterization of electrochemical behavior CV and EIS characterization show that ( Figure 4 , Figure 5 As the modification steps proceed, the oxidation peak current decreases step by step, while the electron transfer impedance increases step by step, indicating that the interface has been successfully constructed.

[0021] 3. Condition Optimization The optimal number of activation cycles for SPCE is 40 cycles. Figure 6 The optimal deposition potential for Ni²⁺ is -0.6 V. Figure 7 The optimal deposition time is 100 s. Figure 8 The optimal concentration of AQP4 is 10 μg / mL. Figure 9 The optimal incubation time for Anti-AQP4 is 60 min. Figure 10 ).

[0022] 4. Selective Testing Using BSA, ATVY, Piezo1, and NLRP3 as interfering substances (concentration ratio 10:1), the sensor showed significant differences in its response to Anti-AQP4, indicating good selectivity. Figure 11 ).

[0023] The above description is merely a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. Equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for constructing an electrochemical immunosensing interface for aquaporin 4 antibody, characterized in that, Includes the following steps: (1) Pretreatment of screen-printed carbon electrode (SPCE): After cleaning with ultrapure water and drying with nitrogen, the SPCE was activated in 0.1 mol / L PBS buffer solution by cyclic voltammetry. The scanning range was -0.6 V to 1.6 V, the number of scans was 40, and the scan rate was 200 mV / s. (2) Ni²⁺ constant potential adsorption: The activated SPCE was placed in a 0.1 mol / L NiCl2 aqueous solution and adsorbed for 100 s under a constant potential of -0.6 V using the chronoamperometry method, so that Ni²⁺ was electrostatically adsorbed and fixed on the surface of the SPCE working electrode. After rinsing with ultrapure water and drying at room temperature, the Ni / SPCE interface was obtained. (3) Directional fixation of AQP4: 10 μg / mL aquaporin 4 (AQP4) solution was added to the working electrode region of the Ni / SPCE interface and incubated overnight at 4°C to allow AQP4 to be directionally fixed by chelating with Ni²⁺ through its histidine tag. After washing with PBS and drying with nitrogen, the AQP4 / Ni / SPCE interface was obtained. (4) Blocking treatment: 1% bovine serum albumin (BSA) solution was added dropwise to the AQP4 / Ni / SPCE interface and reacted at room temperature for 40 min to block the unbound Ni²⁺ active sites. After washing with PBST and PBS and drying with nitrogen, the BSA / AQP4 / Ni / SPCE immunosensing interface was obtained.

2. The construction method according to claim 1, characterized in that: The pH of the PBS buffer solution in step (1) is 7.

4. The purpose of the activation treatment is to clean the oxides on the electrode surface and carboxylate the graphite sheet.

3. The construction method according to claim 1, characterized in that: The NiCl2 aqueous solution mentioned in step (2) needs to be equilibrated at room temperature for 20-30 min before use. The constant potential adsorption parameters are initial voltage -0.6 V, standing time 2 s, and running time 100 s.

4. The construction method according to claim 1, characterized in that: The AQP4 mentioned in step (3) is a recombinant protein with 6 histidine tags (6His) at the N-terminus, and the AQP4 solution is added in a volume of 2.5 µL.

5. The construction method according to claim 1, characterized in that: In step (4), the amount of BSA solution added is 3 µL, and the PBST washing solution is a PBS solution containing 0.05% Tween-20.

6. An electrochemical immunosensing interface for aquaporin 4 antibody, characterized in that: The electrode is prepared by the construction method according to any one of claims 1-5, comprising, from bottom to top, a screen-printed carbon electrode substrate, a Ni²⁺ modification layer, an AQP4 recognition layer, and a BSA sealing layer.

7. The electrochemical immunosensing interface according to claim 6, characterized in that: The Ni²⁺ modified layer binds to the carboxyl groups on the SPCE surface via electrostatic adsorption, and the AQP4 recognition layer is oriented and immobilized by the chelation of Ni²⁺ with histidine tags.

8. An electrochemical detection method for aquaporin 4 antibody, characterized in that: The electrochemical immunosensing interface according to claim 6 or 7 includes the following steps: (1) Immunological reaction: The standard solution of Anti-AQP4 to be tested was added to the immunosensing interface and incubated at 37°C for 60 min to allow AQP4 to bind with Anti-AQP4. Unbound substances were removed by washing with PBST and PBS. (2) Electrochemical detection: The detection solution was covered on the electrode surface, and a voltage of 0.55 V was applied by chronoamperometry. The standing time was 2 s and the running time was 120 s. The current-time response curve was recorded, and the quantitative detection of Anti-AQP4 was achieved by the change in current value at 100 s. The detection solution is a mixture of 0.2 M NaOH and 5 mM glucose.

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