Electrochemical immunosensor for detecting anti-mutant citrullinated waveform protein antibody

Abstract

The invention discloses an electrochemical immunosensor for detecting an anti-mutant citrullinated waveform protein antibody. The electrochemical immunosensor comprises: (1) an electrode, the surface of which is modified with an anti-mutant citrullinated waveform protein antigen; (2) a signal amplification probe, wherein the signal amplification probe is composed of a cobalt-doped COF (at) ZIF-8 core-shell type hybrid material and a carboxylated multi-walled carbon nanotube; and (3) hydrogen peroxide and o-phenylenediamine. The electrochemical immunosensor provided by the invention shows good linear response to Anti-MCV, and shows good recovery rate, selectivity and reproducibility in an actual sample test. Compared with the existing detection technology, the electrochemical immunosensor provided by the invention can realize rapid and high-sensitivity detection of Anti-MCV.

Description

Technical Field

[0001] The present invention belongs to the field of immunosensors, and particularly relates to an electrochemical immunosensor for detecting anti-mutated citrullinated vimentin antibody. Background Art

[0002] Rheumatoid arthritis (RA), as a systemic autoimmune disease, seriously threatens the working ability and independent living ability of patients. However, the early clinical diagnosis of RA faces multiple challenges, mainly due to the non-specificity of its clinical manifestations, the uncertainty of autoantibody test results, and the diversity of diagnostic criteria. To address this challenge and improve the accuracy of diagnosis, it is crucial to identify reliable biomarkers. There are many biomarkers for RA, mainly including rheumatoid factor (RF), anti-perinuclear factor (APF), anti-keratin (AKA), anti-cyclic citrullinated peptide antibody (Anti-CCP), RA33, anti-Sa, glucose-6-phosphate isomerase, etc. Among them, Anti-CCP is currently the most widely used.

[0003] Compared with Anti-CCP, anti-mutated citrullinated vimentin antibody (Anti-MCV) is a new biomarker, which shows significant specificity (90%-96%) and sensitivity (75%-85%) in the diagnosis of RA. Therefore, developing a rapid and highly sensitive detection and analysis method for Anti-MCV is of great significance for improving the accuracy and efficiency of RA diagnosis. However, the currently commonly used Anti-MCV detection methods, such as enzyme-linked immunosorbent assay (ELISA), have certain limitations in terms of sensitivity, operational convenience, and detection timeliness, which limit their wide application in clinical practice. Summary of the Invention

[0004] Aiming at the problems existing in the current detection of anti-mutated citrullinated vimentin antibody, the present invention provides an electrochemical immunosensor capable of rapidly and highly sensitively detecting anti-mutated citrullinated vimentin antibody.

[0005] To achieve the above object, the present invention adopts the following technical scheme: An electrochemical immunosensor for detecting anti-mutated citrullinated vimentin antibody, comprising: (1) An electrode, the surface of which is modified with anti-mutated citrullinated vimentin antigen; (2) A signal amplification probe, which is composed of cobalt-doped COF@ZIF-8 core-shell hybrid material and carboxylated multi-walled carbon nanotubes; (3) Hydrogen peroxide and o-phenylenediamine.

[0006] Preferably, the surface of the electrode is further modified with polyaniline and gold nanoparticles.

[0007] Preferably, the electrode is prepared by a method comprising the following steps: (a) Subjecting the electrode to cyclic voltammetry scanning in a solution containing aniline to obtain a polyaniline-modified electrode; (b) Subjecting the polyaniline-modified electrode to cyclic voltammetry scanning in a solution containing chloroauric acid to obtain a polyaniline / gold nanoparticle-modified electrode; (c) Incubating the polyaniline / gold nanoparticle-modified electrode with a solution containing an anti-mutated citrullinated vimentin antigen, and then blocking with bovine serum albumin.

[0008] More preferably, in step (a), the concentration of aniline is 0.3 mol / L, the potential range of cyclic voltammetry scanning is -0.4 V to 1.0 V, the scanning rate is 50 mV / s, and the number of scanning cycles is 6 to 15.

[0009] More preferably, in step (b), the concentration of chloroauric acid is 1 mg / mL, the potential range of cyclic voltammetry scanning is -0.2 V to 1.2 V, the scanning rate is 100 mV / s, and the number of scanning cycles is 10 to 20.

[0010] More preferably, in step (c), the incubation time is 8 to 14 hours.

[0011] Preferably, the monomers of the COF are 2,4,6-trihydroxy-1,3,5-benzenetricarboxaldehyde and 2,2'-bipyridine-5,5'-dicarboxylic acid.

[0012] More preferably, the mass ratio of 2,4,6-trihydroxy-1,3,5-benzenetricarboxaldehyde to 2,2'-bipyridine-5,5'-dicarboxylic acid is 1:1.3 to 1:1.4.

[0013] Preferably, the signal amplification probe is prepared by a method comprising the following steps: First, mixing and stirring ZIF-8 and the monomers of COF in a solvent for a certain period of time to obtain COF@ZIF-8; Then, mixing and stirring COF@ZIF-8 and cobalt acetate in a solvent for a certain period of time to obtain a cobalt-doped COF@ZIF-8 core-shell hybrid material; Then, mixing the cobalt-doped COF@ZIF-8 core-shell hybrid material and carboxylated multi-walled carbon nanotubes to obtain the signal amplification probe.

[0014] More preferably, the mass ratio of ZIF-8 to the monomers of COF is 1:1.

[0015] More preferably, the time for mixing and stirring ZIF-8 and the monomers of COF in a solvent is 20 to 30 hours.

[0016] More preferably, the mass ratio of the COF@ZIF-8 to cobalt acetate is 5:2.

[0017] More preferably, the time for mixing and stirring the reaction of the COF@ZIF-8 and cobalt acetate in the solvent is 8 to 20 hours.

[0018] More preferably, the mass ratio of the cobalt-doped COF@ZIF-8 core-shell hybrid material to the carboxylated multi-walled carbon nanotubes is 1:1.

[0019] Preferably, the core of the COF@ZIF-8 core-shell hybrid material is COF and the shell is ZIF-8.

[0020] Preferably, the molar ratio of hydrogen peroxide to o-phenylenediamine is 2:1.

[0021] Preferably, the carboxyl content of the carboxylated multi-walled carbon nanotubes is 1 to 10 wt%.

[0022] More preferably, the carboxyl content of the carboxylated multi-walled carbon nanotubes is 3 to 5 wt%.

[0023] A method for detecting anti-mutated citrullinated vimentin antibody using the above electrochemical immunosensor, which method is for non-disease diagnosis or treatment purposes, includes: (1) After mixing and incubating the signal amplification probe with the sample solution to be tested for a certain time, resuspend it with a pH buffer solution; (2) Place the electrode in the resuspended solution obtained in step (1) and incubate for a certain time; (3) Place the electrode incubated in step (2) in a pH buffer solution containing hydrogen peroxide and o-phenylenediamine, detect the current response by differential pulse voltammetry, and then obtain the concentration of anti-mutated citrullinated vimentin antibody in the sample solution to be tested according to the linear relationship between the concentration of anti-mutated citrullinated vimentin antibody and the current response value.

[0024] Preferably, the incubation time in step (1) is 10 to 15 hours respectively.

[0025] Preferably, after the incubation in step (1), the signal amplification probe is blocked with bovine serum albumin.

[0026] Preferably, the incubation time in step (2) is 1 to 2 hours respectively.

[0027] Preferably, the pH value of the pH buffer solution in steps (1) and (3) is 7 to 8.

[0028] Preferably, the concentration of hydrogen peroxide in the pH buffer solution in step (3) is 2 to 6 mmol / L, and the concentration of o-phenylenediamine is 1 to 3 mmol / L. Description of the Drawings

[0029] Figure 1 It is a schematic diagram of the construction and working principle of the electrochemical immunosensor of the present invention.

[0030] Figure 2 They are the morphology diagrams of different probe materials.

[0031] Figure 3 They are the X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) spectra of different probe materials.

[0032] Figure 4 They are the differential pulse voltammetry (DPV) curves and standard curves of the electrochemical immunosensor at different concentrations of Anti-MCV.

[0033] Figure 5 It is the calibration curve of the peak current detected by DPV and the concentration of Anti-MCV.

[0034] Figure 6 It is the selectivity of the electrochemical immunosensor of the present invention.

[0035] Figure 7 It is the repeatability and stability of the electrochemical immunosensor of the present invention.

[0036] Figure 8 They are the cyclic voltammetry (CV) curves of the glassy carbon electrode (GCE) after modification with polyaniline and gold nanoparticles.

[0037] Figure 9 They are the CV curves of the GCE modified with different probe materials.

[0038] Figure 10 They are the electrochemical impedance spectroscopy (EIS) curves of the GCE modified with different probe materials.

[0039] Figure 11 They are the ultraviolet-visible absorption spectra (UV-vis) of different probe materials in the TMB + H2O2 system. Detailed Description of the Invention

[0040] The technical solutions of the present invention will be further described in detail below in conjunction with the embodiments.

[0041] Materials and Instruments All electrochemical experiments were carried out on an AUTOLAB PGSTAT302N (Metrohm China Co., Ltd.) electrochemical workstation, using a traditional three - electrode system. The screen - printed carbon electrode (SPCE) was used as the base electrode of the electrochemical sensor. In the SPCE, the working electrode was a carbon electrode, the counter electrode was also a carbon electrode, the reference electrode was an Ag / AgCl electrode, and the substrate material was polyethylene terephthalate (PET). The glassy carbon electrode (GCE) was polished to a mirror surface with 0.05 μm Al2O3, rinsed with ultrapure water, and dried with nitrogen. Scanning electron microscope (SEM) images were obtained by a JSM - 6701F cold field emission scanning electron microscope (JEOL Ltd., Japan). Transmission electron microscope (TEM) images were collected by a JEM - 2100 high - resolution transmission electron microscope (JEOL Ltd., Japan). X - ray photoelectron spectroscopy (XPS) analysis was performed using a Kratos AXISUltra DLD type X - ray photoelectron spectrometer (Kratos Analytical Ltd., UK). X - ray diffraction (XRD) patterns were recorded by an X - ray diffractometer (Rigaku Corporation, Japan). Fourier transform infrared (FTIR) spectra were measured by an FTIR - 650 type Fourier transform infrared spectrometer (Tianjin Gangdong Technology Development Co., Ltd.).

[0042] Carboxylated multi - walled carbon nanotubes (-COOH content 3.86 wt%) were provided by Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences. Anti - mutant citrullinated vimentin antibody and antigen were provided by Shanghai Kexing Trading Co., Ltd. Bovine serum albumin (BSA) was provided by Shanghai Senbeijia Biotechnology Co., Ltd. 0.01 M phosphate - buffered saline (PBS) was prepared with NaH2PO4, Na2HPO4, and KCl as supporting electrolytes.

[0043] The metal - organic framework material (MOF) was ZIF - 8, which could be commercially available or self - made. In this invention, it was synthesized from 2 - methylimidazole and zinc sulfate heptahydrate as raw materials by a traditional method. The specific process was as follows: ZnSO4·7H2O (300 mg, 1.01 mmol) was dissolved in 15 mL of methanol. Then, 2 - methylimidazole (660 mg, 8.04 mmol) dissolved in 15 mL of methanol was added to the above solution, and the mixture was stirred at room temperature for 6 h. The obtained product was centrifuged and washed three times with methanol, and then dried in vacuo to obtain ZIF - 8.

[0044] Example 1 COF represents covalent organic framework material.

[0045] The construction process of the electrochemical immunosensor of this invention is as Figure 1 shown below: 1. Preparation of signal - amplified probe material Dissolve 257.32 mg of 2,4,6-trihydroxy-1,3,5-benzenetricarbaldehyde (TP) in 30 mL of tetrahydrofuran (THF), dissolve 345.56 mg of 2,2'-bipyridine-5,5'-dicarboxylic acid (BPY) in 30 mL of THF, and disperse 600 mg of ZIF-8 in 340 mL of THF. Then, add the above TP solution and BPY solution to the ZIF-8 dispersion, sonicate for 30 minutes, and stir at 25 °C for 24 h to obtain COF@MOF.

[0046] Disperse 0.5 g of COF@MOF in 500 mL of ethanol (EtOH), then add 500 ml of cobalt acetate (200 mg) solution, and stir at room temperature for 12 h. Wash the product with EtOH and THF, and then dry it under vacuum to obtain Co-COF@MOF. Redisperse it with water to prepare an aqueous dispersion with a concentration of 1 mg / mL for standby.

[0047] Mix the aqueous dispersion of carboxylated multi-walled carbon nanotubes (NTs) with a concentration of 1 mg / mL and the aqueous dispersion of Co-COF@MOF with a concentration of 1 mg / mL at a volume ratio of 1:1, and stir at room temperature for 24 h to obtain a Co-COF@MOF-NTs solution with a concentration of 1 mg / mL for standby.

[0048] From Figure 2 it can be seen that the SEM image of ZIF-8 ( Figure 2 a) shows spherical particles with a hollow structure, and its transmission electron microscope image ( Figure 2 b) shows that its outer shell is darker in color and the inside is bright, which confirms its hollow structure. The SEM image of COF@MOF ( Figure 2 c) and TEM image ( Figure 2 d) show a uniform particle distribution and a clear core-shell boundary, and the outer shell is darker in color. After further doping with Co, the morphology of Co-COF@MOF ( Figure 2 e-f) is more aggregated and the core-shell interface is blurred, which may be related to the increase in surface energy and boundary effect caused by the introduction of Co, and may affect the electron transfer process, thus affecting the electrochemical performance.

[0049] XPS analysis reveals the contents and chemical states of Co and Zn in Co-COF@MOF-NTs. The XPS full spectrum confirms the presence of C 1s, N1s, O 1s, Co 2p and Zn 2p ( Figure 3a). Specifically, in the surface atomic composition of Co-COF@MOF-NTs, carbon (C) accounts for 75.4 at.%, nitrogen (N) accounts for 5.8 at.%, oxygen (O) accounts for 14.9 at.%, and it contains trace amounts of Co at 1.2 at.% and Zn at 2.7 at.% ( Figure 3 b, c).

[0050] The FT-IR of ZIF-8, COF@MOF, and Co-COF@MOF composites is as Figure 3 shown in d. For ZIF-8, the absorption peak at 1600 cm⁻¹ comes from the vibration of the C=N bond in the imidazole ring. The peak at 420 cm⁻¹ confirms the Zn-N coordination. In COF@MOF, a characteristic peak exists at 1600 cm⁻¹, corresponding to the C=N (imine) bond derived from BPY and TP, indicating that the COF has been formed and the MOF and COF have been successfully combined. The stretching vibration of C=C in the benzene ring results in a peak at 1450 cm⁻¹, and due to symmetry, the bending vibration of C-H in the benzene ring appears near 760 cm⁻¹. In addition, after doping Co atoms, new peak positions appear at 690 cm⁻¹ (Co-O) and 1300 cm⁻¹ (Co-N) in the Co-COF@MOF sample, indicating that Co atoms have been successfully incorporated into the COF@MOF structure.

[0051] As Figure 3 shown in e, the structural characteristics and crystal conformations of Co-COF@MOF and Co-COF@MOF-NTs were observed using XRD. Introducing Co clusters into COF@MOF mainly produced new diffraction peaks at lower 2θ angles, reflecting the influence of metal centers on the crystal structure. The interaction between carboxylated carbon nanotubes and Co-COF@MOF changed the overall crystal structure and packing mode. The carboxylated multi-walled carbon nanotubes (NTs) are characterized by a peak at 26.4°, corresponding to the (002) crystal plane. These carbon nanotubes may be inserted into the voids of Co-COF@MOF or interact with its surface, thus changing the pore structure or layer spacing and causing a shift in the diffraction peaks.

[0052] 2. Modification of the electrode First, the SPCE was placed in a solution containing 0.3 M aniline and 0.5 M sulfuric acid, and 6 cyclic voltammetry scans were performed at a scanning rate of 50 mV / s in the potential range from -0.4 V to 1.0 V to deposit a layer of polyaniline film (PANI) on the surface of the SPCE, obtaining PANI / SPCE.

[0053] Subsequently, the PANI / SPCE was placed in a solution containing 1 mg / mL chloroauric acid and 0.5 M sulfuric acid, and 10 cyclic voltammetry scans were performed at a scanning rate of 100 mV / s within the potential range of -0.2 V to 1.2 V, thereby depositing a layer of gold nanoparticles (AuNPs) on the electrode surface to obtain PANI / AuNPs / SPCE.

[0054] Next, 80 μL of anti-mutated citrullinated vimentin antigen (MCV) solution was carefully dropped onto the surface of PANI / AuNPs / SPCE, incubated at 4 °C for 11 hours, then 50 μL of 1% BSA solution was added and incubated at 37 °C for 1.5 h. After that, the electrode was rinsed with PBS to obtain MCV / PANI / AuNPs / SPCE.

[0055] 3. Detection of Anti-MCV 10 µL of Anti-MCV (Ab) solution was thoroughly mixed with 1 mL of Co-COF@MOF-NTs solution with a concentration of 1 mg / mL, and stirred at a constant temperature of 4 °C for 12 h. Subsequently, 100 μL of 1% bovine serum albumin (BSA) solution was added to the mixture, and stirring was continued at 4 °C for 1 h to block the excess binding sites on the material surface. After that, the obtained Ab-Co-COF@MOF-NTs was rinsed with PBS and dispersed in 1 mL of phosphate buffer solution (pH 7.4), and stored at 4 °C to maintain its stability for subsequent use.

[0056] The MCV / PANI / AuNPs / SPCE was placed in the above-mentioned Ab-Co-COF@MOF-NTs dispersion, incubated at 37 °C for 2 h, and then the electrode was rinsed with PBS.

[0057] The differential pulse voltammetry (DPV) method was used to detect the current response of the electrode in a 0.01 M phosphate buffer solution (PBS, pH 7.4) containing 2 mM o-phenylenediamine (oPD) and 4 mM H2O2. The parameters of the DPV test were: initial potential -0.3 V, final potential 1.4 V, pulse height 50 mV, step height 4 mV, and pulse width 0.05 s. The results are as Figure 4 shown.

[0058] It can be seen from Figure 4 that the peak current gradually increases with the increase in the concentration of Anti-MCV, and the calibration curve ( Figure 5) showed that in the range of Anti-MCV concentration from 0.25 to 36 ng / mL, there was a good linear relationship between the increase in peak current and the concentration of Anti-MCV. The calibration curve showed a strong linear correlation, and its regression equation was as follows: I (μA) = 3.52569×C Anti-MCV + 58.74512 (R² = 0.99936). The limit of detection (LOD) calculated based on the signal-to-noise ratio (S / N = 3) was 0.20 ng / mL.

[0059] 4. Selectivity, repeatability, and stability To evaluate the specificity of the electrochemical immunosensor, six interfering substances commonly found in the sera of RA patients were used. These interfering substances included tumor necrosis factor-alpha (TNF-α), rheumatoid factor (RF), antinuclear antibody (ANA), anti-cyclic citrullinated peptide antibody (Anti-CCP), interleukin-6 (IL-6), and interleukin-β (IL-β), and the DPV method was used for detection. The results are shown in Figure 6 . The concentration of each interfering substance was set to half of the maximum detectable concentration specified by the ELISA kit. Their electrochemical responses were extremely low, close to those of the blank samples, and significantly lower than that of 4 ng / mL Anti-MCV. In addition, the peak current of the mixed solution of the interfering substances and Anti-MCV was similar to that of the pure Anti-MCV solution. These results indicate that the electrochemical immunosensor of the present invention has excellent selectivity.

[0060] The repeatability and stability of the electrochemical immunosensor of the present invention were evaluated using an Anti-MCV solution with a concentration of 4 ng / mL. The results are shown in Figure 7 . As Figure 7 shown in a, in the tests of five parallel electrochemical immunosensors, it was observed that the fluctuation of their net charge values was very small, and the relative standard deviation (RSD) was 3.82% (n = 3), indicating that the electrochemical immunosensor has good repeatability. As Figure 7 shown in b, the net charge of the immunosensor only decreased by 13.90% on the 7th day, demonstrating its good long-term stability.

[0061] 5. Analysis of Anti-MCV in the spiked recovery experiment of rat serum The accuracy and precision of the electrochemical immunosensor of the present invention were evaluated by performing a standard addition recovery experiment in rat serum samples at different dilution ratios. Different concentrations of Anti-MCV (1 ng / mL, 6 ng / mL, and 12 ng / mL) were added to rat serum diluted to 10% with PBS (pH 7.4) buffer, and the net charge signal of the electrochemical immunosensor was recorded. As shown in Table 1, the recovery rate of this electrochemical immunosensor was between 98.32% - 108.34%, and the relative standard deviation (RSD) was in the range of 6.47% - 14.35%, indicating that this immunosensor has excellent accuracy and precision in detecting Anti-MCV in actual biological samples.

[0062] Table 1 Results of the standard addition recovery experiment

[0063] Example 2 First, the GCE was placed in a solution containing 0.3 M aniline and 0.5 M sulfuric acid, and 6 cyclic voltammetry scans were performed at a scanning rate of 50 mV / s in the potential range of -0.4 V to 1.0 V, thereby depositing a layer of polyaniline film (PANI) on the surface of the SPCE to obtain PANI / GCE.

[0064] Subsequently, the PANI / GCE was placed in a solution containing 1 mg / mL chloroauric acid and 0.5 M sulfuric acid, and 10 cyclic voltammetry scans were performed at a scanning rate of 100 mV / s in the potential range of -0.2 V to 1.2 V, thereby depositing a layer of gold nanoparticles (AuNPs) on the electrode surface to obtain PANI / AuNPs / GCE.

[0065] In a 0.1 M KCl solution containing 5 mM [Fe(CN)6]³⁻ / 4 ⁻, cyclic voltammetry (CV) tests were carried out. The reference electrode (RE): silver / silver chloride electrode, the potential range was -0.3 V to 0.6 V, and the scanning rate was 50 mV / s.

[0066] The CV test results are as Figure 8 shown. Compared with the bare GCE, PANI / GCE exhibited a more significant current response due to the electro-deposited polyaniline (PANI) layer improving the conductivity and binding properties. It is worth noting that the current response of the PANI / AuNPs / GCE electrode reached the highest level. On the basis of the PANI-modified GCE, further addition of gold nanoparticles (AuNPs) significantly enhanced its electrochemical performance.

[0067] The CV results show that the electrochemical performance of the electrode can be enhanced by modifying with PANI and then adding AuNPs.

[0068] Example 3 Preparation of COF: 257.32 mg of 2,4,6-trihydroxy-1,3,5-benzenetricarbaldehyde (TP) was dissolved in 30 mL of tetrahydrofuran (THF), and 345.56 mg of 2,2'-bipyridine-5,5'-dicarboxylic acid (BPY) was dissolved in 30 mL of THF. Then, the above TP solution and BPY solution were mixed, sonicated for 30 minutes, and stirred at 25 °C for 24 h to obtain COF.

[0069] The preparation of COF@MOF, Co-COF@MOF, and Co-COF@MOF-NTs was the same as that in Example 1.

[0070] 6 μL suspensions of ZIF-8, COF, COF@MOF, Co-COF@MOF, NTs, and Co-COF@MOF-NTs were respectively dropped onto the surface of GCE and dried at room temperature to obtain ZIF-8 / GCE, COF / GCE, COF@MOF / GCE, Co-COF@MOF / GCE, NTs / GCE, and Co-COF@MOF-NTs / GCE.

[0071] Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) tests were carried out in a 0.1 M KCl solution containing 5 mM [Fe(CN)6]³⁻ / 4 ⁻. The reference electrode (RE): silver / silver chloride electrode. The potential range for CV test was from -0.3 V to 0.6 V, the scanning rate was 50 mV / s, and the frequency range for EIS was from 0.1 Hz to 10 5 kHz.

[0072] As shown in the CV test results, Figure 9 the current response of Co-COF@MOF-NTs / GCE was the most significant, indicating that Co-COF@MOF-NTs had excellent electrocatalytic performance.

[0073] As shown in the EIS test results, Figure 10 at higher frequencies, the diameter of the semicircle was closely related to the electron transfer resistance (Rct). Modifying materials such as ZIF-8 and COF could effectively reduce Rct. Particularly, Co-COF@MOF-NTs / GCE showed the lowest Rct value, which fully demonstrated the excellent charge transfer ability of Co-COF@MOF-NTs.

[0074] Using 3,3',5,5'-tetramethylbenzidine (TMB) as the chromogenic substrate, the peroxidase-like activity of the probe materials in the TMB + H2O2 system was studied, and the results were asFigure 11 As shown, Co-COF@MOF exhibits low peroxidase-like activity, resulting in the lightest solution color. On the contrary, in the system without this material, there is no color change in the solution. On the other hand, the introduction of Co-COF@MOF-NTs significantly enhances the peroxidase-like activity, making the solution show a dark yellow color.

[0075] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An electrochemical immunosensor for detecting anti-mutated citrullinated vimentin antibody, comprising: (1) An electrode, on the surface of which an anti-mutated citrullinated vimentin antigen is modified; (2) A signal amplification probe, which is composed of cobalt-doped COF@ZIF-8 core-shell hybrid material and carboxylated multi-walled carbon nanotubes; (3) Hydrogen peroxide and o-phenylenediamine.

2. The electrochemical immunosensor according to claim 1, wherein: The surface of the electrode is further modified with polyaniline and gold nanoparticles.

3. The electrochemical immunosensor according to claim 2, characterized in that: The electrode is prepared by a method comprising the following steps: (a) Subjecting the electrode to cyclic voltammetry scanning in a solution containing aniline to obtain a polyaniline-modified electrode; (b) Subjecting the polyaniline-modified electrode to cyclic voltammetry scanning in a solution containing chloroauric acid to obtain a polyaniline / gold nanoparticle-modified electrode; (c) Incubating the polyaniline / gold nanoparticle-modified electrode with a solution containing an anti-mutated citrullinated vimentin antigen, and then blocking it with bovine serum albumin.

4. The electrochemical immunosensor according to claim 3, wherein: In step (a), the concentration of aniline is 0.3 mol / L, the potential range of cyclic voltammetry scanning is -0.4 V to 1.0 V, the scanning rate is 50 mV / s, and the number of scanning cycles is 6 - 15; In step (b), the concentration of chloroauric acid is 1 mg / mL, the potential range of cyclic voltammetry scanning is -0.2 V to 1.2 V, the scanning rate is 100 mV / s, and the number of scanning cycles is 10 - 20; In step (c), the incubation time is 8 - 14 hours.

5. The electrochemical immunosensor according to claim 1, characterized in that: The monomer of the COF is 2,4,6-trihydroxy-1,3,5-benzenetricarboxaldehyde and 2,2'-bipyridine-5,5'-dicarboxylic acid.

6. The electrochemical immunosensor according to claim 5, characterized in that: The mass ratio of 2,4,6-trihydroxy-1,3,5-benzenetricarboxaldehyde to 2,2'-bipyridine-5,5'-dicarboxylic acid is 1:1.3 - 1:1.

4.

7. The electrochemical immunosensor according to claim 1 or 5, characterized in that: The signal amplification probe is prepared by a method comprising the following steps: First, mixing and stirring the monomers of ZIF-8 and COF in a solvent for a certain time to obtain COF@ZIF-8; Then, mixing and stirring COF@ZIF-8 and cobalt acetate in a solvent for a certain time to obtain a cobalt-doped COF@ZIF-8 core-shell hybrid material; Then, mixing the cobalt-doped COF@ZIF-8 core-shell hybrid material and carboxylated multi-walled carbon nanotubes to obtain the said signal amplification probe.

8. The electrochemical immunosensor according to claim 7, wherein: The mass ratio of ZIF-8 to the monomer of COF is 1:1; The mass ratio of COF@ZIF-8 to cobalt acetate is 5:2; The mass ratio of the cobalt-doped COF@ZIF-8 core-shell hybrid material to the carboxylated multi-walled carbon nanotubes is 1:1; The mixing and stirring reaction time of ZIF-8 and the monomers of COF in a solvent is 20 - 30 hours; The mixing and stirring reaction time of COF@ZIF-8 and cobalt acetate in a solvent is 8 - 20 hours.

9. The electrochemical immunosensor according to claim 1, characterized in that: The core of the COF@ZIF-8 core-shell hybrid material is COF, and the shell is ZIF-8; The molar ratio of hydrogen peroxide to o-phenylenediamine is 2:1; The carboxyl content of the carboxylated multi-walled carbon nanotubes is 1-10 wt%; Preferably, the carboxyl content of the carboxylated multi-walled carbon nanotubes is 3-5 wt%.

10. A method for detecting anti-mutated citrullinated vimentin antibody using the electrochemical immunosensor according to claim 1, which is for non-diagnostic or therapeutic purposes and includes: (1) After mixing the signal amplification probe with the sample solution to be tested and incubating for a certain time, resuspend it with a pH buffer; (2) Place the electrode in the resuspended solution obtained in step (1) and incubate for a certain time; (3) Place the electrode incubated in step (2) in a pH buffer containing hydrogen peroxide and o-phenylenediamine, detect the current response by differential pulse voltammetry, and then obtain the concentration of anti-mutated citrullinated vimentin antibody in the sample solution to be tested according to the linear relationship between the concentration of anti-mutated citrullinated vimentin antibody and the current response value; Preferably, the incubation time in step (1) is 10-15 hours respectively; Preferably, after the incubation in step (1), the signal amplification probe is blocked with bovine serum albumin; Preferably, the incubation time in step (2) is 1-2 hours respectively; Preferably, the pH value of the pH buffer in steps (1) and (3) is 7-8; Preferably, the concentration of hydrogen peroxide in the pH buffer in step (3) is 2-6 mmol / L, and the concentration of o-phenylenediamine is 1-3 mmol / L.

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