Myoglobin electrochemical immunosensor and application thereof

Abstract

The electrochemical immunosensor is characterized in that a biotinylated monoclonal antibody HRP-Strept-Biotin-Ab-Mb of myoglobin Mb labeled by horse radish peroxidase avidin is used as a signal probe, a Fe3O4-MWCNTs-COOH / GCE electrode of Mb loaded in a myoglobin standard solution or a solution to be detected is used as a basic electrode, and the biotinylated monoclonal antibody HRP-Strept-Biotin-Ab-Mb labeled by horse radish peroxidase avidin is used as the signal probe. And constructing to obtain the HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE electrochemical immunosensor. The electrochemical immunosensor constructed by the invention can realize high-sensitivity and rapid detection of Mb, shows better analysis performance, and has lower detection limit and wider detection range; and the method has good specificity, reproducibility and stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical biosensors, and particularly relates to a myoglobin electrochemical immunosensor and its application. Background Art

[0002] Acute Myocardial Infarction (AMI) has become one of the main causes of death from non-communicable diseases due to its rapid onset, high rescue difficulty, and high fatality rate. Currently, early clinical diagnosis of AMI mainly relies on methods such as electrocardiogram and echocardiogram, but these methods have problems such as low sensitivity and high missed diagnosis rate, imposing a heavy burden on society and families. Therefore, it is particularly important to develop rapid early warning technologies for AMI with high sensitivity and high specificity. Myoglobin (Mb), as an early warning marker for AMI, its rapid and accurate detection is of great significance for timely initiating treatment. However, existing detection technologies such as fluorescence analysis, electrochemiluminescence, surface plasmon resonance, and enzyme-linked immunosorbent assay have deficiencies such as complex operation, high cost, and long detection time.

[0003] Therefore, it is of great significance to develop an electrochemical immunosensor for sensitive, efficient, and instant detection of Mb. Summary of the Invention

[0004] Objective: To solve the deficiencies of the existing technology, the present invention provides a myoglobin electrochemical immunosensor and its application. By optimizing the electrode material and signal amplification technology, high-sensitivity and rapid detection of Mb are achieved, providing a reliable technical means for early warning of AMI. This application exhibits high selectivity, good stability, and repeatability, and has extremely high sensitivity for the detection of trace Mb in complex biological samples.

[0005] Technical Solution: To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, a myoglobin electrochemical immunosensor is provided, using the biotinylated monoclonal antibody HRP-Strept-Biotin-Ab-Mb of myoglobin Mb conjugated with horseradish peroxidase-labeled avidin as a signal probe, and the Fe3O4-MWCNTs-COOH / GCE electrode loaded with Mb in a myoglobin standard solution or a test solution as a basic electrode, to construct an HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE electrochemical immunosensor;

[0007] The preparation method of the Fe3O4-MWCNTs-COOH / GCE electrode includes:

[0008] S1. Mix Fe3O4 NPs and carboxylated multi-walled carbon nanotubes MWCNTs-COOH, and ultrasonically disperse them with a solvent to obtain a dispersion of Fe3O4-MWCNTs-COOH magnetic composite material;

[0009] S2. Modify the dispersion of Fe3O4-MWCNTs-COOH magnetic composite material on the GCE electrode and dry it to obtain the Fe3O4-MWCNTs-COOH / GCE electrode;

[0010] The preparation method of the signal probe HRP-Strept-Biotin-Ab-Mb includes: mixing HRP-Strept and Biotin-Ab-Mb and incubating them to obtain it.

[0011] In some embodiments, in S1, the solvent is selected from at least one of methanol, ethanol, and isopropanol.

[0012] In some embodiments, in S1, the mass ratio of Fe3O4 NPs to carboxylated multi-walled carbon nanotubes MWCNTs-COOH is 1:1 to 1:3, preferably 1:2.

[0013] In some embodiments, in S2, take 1-11 μL of the dispersion of Fe3O4-MWCNTs-COOH magnetic composite material and modify it on the GCE electrode, preferably 7 μL.

[0014] In some embodiments, the preparation method of the Fe3O4 NPs includes: dissolving ferric chloride in ethylene glycol, adding sodium acetate and ethylenediamine, and reacting in a reaction kettle to obtain Fe3O4 nanoparticles Fe3O4 NPs.

[0015] In the second aspect, an application of the described electrochemical immunosensor in myoglobin detection is provided.

[0016] Furthermore, the myoglobin detection specifically includes:

[0017] S31: Place the Fe3O4-MWCNTs-COOH / GCE electrode in myoglobin standard solutions with different concentrations, incubate, rinse, and dry to obtain the Mb / Fe3O4-MWCNTs-COOH / GCE electrode, and then combine the Mb / Fe3O4-MWCNTs-COOH / GCE electrode with the signal probe HRP-Strept-Biotin-Ab-Mb to obtain the processed electrochemical immunosensor;

[0018] S32: Use the processed electrochemical immunosensor as the working electrode, perform differential pulse voltammetry scanning using a three-electrode system, record the peak current-Mb concentration relationship curve, establish a linear relationship between the peak current and the logarithm of the myoglobin Mb concentration, and obtain a linear regression equation;

[0019] S33: Re-take the Fe3O4-MWCNTs-COOH / GCE electrode, place it in the solution to be measured for incubation, then rinse and dry it to obtain the Mb / Fe3O4-MWCNTs-COOH / GCE electrode. Then, combine the Mb / Fe3O4-MWCNTs-COOH / GCE electrode with the signal probe HRP-Strept-Biotin-Ab-Mb. Use the processed electrochemical immunosensor as the working electrode, and perform differential pulse voltammetry scanning using a three-electrode system. According to the measured peak current and in combination with the linear regression equation, obtain the concentration of myoglobin Mb in the solution to be measured.

[0020] In some embodiments, in S31 and S33, the incubation time is 1 to 3 hours, preferably 2 hours.

[0021] In some embodiments, in S31 and S33, after obtaining the Mb / Fe3O4-MWCNTs-COOH / GCE electrode, it further includes: placing the Mb / Fe3O4-MWCNTs-COOH / GCE electrode in a blocking solution to block the active sites on the electrode surface that are not loaded with Mb, wherein the blocking solution contains bovine serum albumin.

[0022] In some embodiments, during the differential pulse voltammetry scanning process, the electrochemical immunosensor is placed in a reaction bottom solution to achieve the quantitative detection of Mb, wherein the reaction bottom solution is a PBS solution containing o-phenylenediamine and H2O2, and the molar ratio of o-phenylenediamine to H2O2 in the reaction bottom solution is 1:2.

[0023] Beneficial effects: An electrochemical immuno-sensor for myoglobin and its application provided by the present invention uses horseradish peroxidase-labeled avidin (HRP-Strept) to specifically recognize the biotinylated monoclonal antibody HRP-Strept-Biotin-Ab-Mb of AMI biomarker - myoglobin Mb as a signal probe, and Fe3O4-MWCNTs-COOH / GCE loaded with Mb as a base electrode, and successfully constructs an HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE electrochemical immuno-sensor for precise and rapid detection of myoglobin Mb. Since the composite material prepared by combining Fe3O4 magnetic nanoparticles with MWCNTs-COOH has excellent electrochemical properties, good stability and excellent biocompatibility and other characteristics, it significantly improves the response signal of the sensor. And the HRP-Strept labeled antibody probe further amplifies the detection signal, providing conditions for highly sensitive and precise detection of myoglobin Mb. Under the optimized experimental conditions (the mixing mass ratio of Fe3O4 and MWCNTs-COOH is 1:2, the modification amount of the magnetic composite material is 7 μL, and the Mb incubation time is 2 h), the sensor shows a good linear relationship in the range of 0.0195 ng·mL -1 to 640 ng·mL -1 , and the detection limit (S / N = 3) is as low as 0.1007 ng·mL -1 . The constructed electrochemical immuno-sensor is used to analyze trace Mb in complex biological samples, and this method shows high selectivity, good stability and repeatability, providing a convenient and efficient new method for the detection of AMI-related biomarkers, and also providing new strategies and scientific and technological support for the early prevention, clinical diagnosis and disease typing evaluation of AMI. Description of the Drawings

[0024] Figure 1 : Among them, (A) FT-IR spectra of MWCNTs-COOH and Fe3O4-MWCNTs; (B) Raman spectra of MWCNTs-COOH and Fe3O4-MWCNTs-COOH; (C) Scanning electron micrographs of MWCNTs-COOH, (D) Fe3O4-MWCNTs-COOH; (E) Energy dispersive spectrum of Fe3O4-MWCNTs-COOH; (F) C, (G) O, (H) EDS elemental stratification images of Fe.

[0025] Figure 2: CV curves (A), EIS curves (B), chrono-Coulomb curves (C), and Q-t1 / 2 curves (D) of GCE (a), Fe3O4NPs / GCE (b), MWCNTs-COOH / GCE (c) and Fe3O4-MWCNTs-COOH / GCE (d); (E) CV measurement of magnetic composite materials at different scanning rates; (F) The relationship between the logarithm of peak current and the logarithm of scanning rate; (G) The relationship between the peak potential of magnetic composite materials and the logarithm of scanning rate.

[0026] Figure 3 :Among them, (A) CV peak current relationship curve of different mixing mass ratios of Fe3O4 and MWCNTs-COOH; (B) peak current relationship curve of different modification amounts of Fe3O4-MWCNTs-COOH; (C) peak current relationship curve of different incubation times of Mb.

[0027] Figure 4 :(A) ELISA analysis of absorbance values corresponding to different concentrations of Mb;(B) HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE electrochemical immunosensor analysis of different concentrations of Mb (640ng·mL -1 ~ 0ng·mL -1 ) DPV response diagram; (C) DPV peak current (Ip) and Mb concentration curve; (D) DPV peak current and LgC Mb Linear relationship diagram between .

[0028] Figure 5 :Schematic diagram of the specificity, reproducibility and stability test results of electrochemical immunosensor; (A) investigation of the specificity of electrochemical immunosensor; (B) investigation of the reproducibility of electrochemical immunosensor (between batches); (C) investigation of the reproducibility of electrochemical immunosensor (within batch); (D) investigation of the long-term stability of electrochemical immunosensor. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the examples. The following examples are only used to more clearly illustrate the performance of the present invention, and are not limited to the following examples.

[0030] Experimental materials and equipment

[0031] Carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) (Jiangsu Xianfeng Nano Materials Technology Co., Ltd), ferric chloride (FeCl3•6H2O) (Shanghai Macklin Biochemical Technology Co., Ltd), ethylene glycol (CH2OH)2 (Shanghai Titan Technology Co., Ltd), anhydrous sodium acetate (C2H3NaO2) (Sigma-Aldrich Shanghai Co., Ltd), ethylenediamine (C2H 10 N2O) (Tianjin Kemiou Chemical Reagent Co., Ltd), glutaraldehyde (C5H8O2) (Shanghai Zhongqin Chemical Reagent Co., Ltd), bovine serum albumin (BSA, >98.0) (Solarbio), ELISA kit (Wuhan Elabscience Biotechnology Co., Ltd, Lot: FU00FZF03639), Antigen-Mb (Wuhan Elabscience Biotechnology Co., Ltd), horseradish peroxidase-labeled avidin HRP-Strept (Wuhan Elabscience Biotechnology Co., Ltd), biotin-labeled myoglobin monoclonal antibody Biotin-Ab-Mb (Wuhan Elabscience Biotechnology Co., Ltd), proline (Shanghai Zhongqin Chemical Reagent Co., Ltd), methionine (Shanghai Zhongqin Chemical Reagent Co., Ltd), ascorbic acid (Xi'an Tianmao Chemical Industry Co., Ltd), glucose (Tianjin Damao Chemical Reagent Factory), glycine (Tianjin Fuchen Chemical Reagent Factory).

[0032] CHI 760E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd); all electrochemical experiments were carried out at 25 °C using a traditional three-electrode system, including a working electrode of a glassy carbon electrode or a modified glassy carbon electrode (GCE, diameter 3 mm), a 232-type saturated calomel electrode and a platinum wire electrode (≥99.99%) as the reference electrode and the auxiliary electrode; SB-5200DTD ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd); LC-HK-100 reactor (Shanghai Lichen Instrument Technology Co., Ltd); DHG-9030 electrothermal blast drying oven (Shanghai Yiheng Technology Co., Ltd); IFS66v / S Fourier transform infrared spectrometer (Llantrisant, UK); LabRAM HR Evolution confocal Raman microscope (HORIBA JobinYvon S.A.A., France); JSM-6701F cold field emission scanning electron microscope (JEOL, Japan);

[0033] Example 1: A preparation method of a myoglobin electrochemical immunosensor, comprising:

[0034] 1. Preparation of Fe3O4 nanoparticles: Fe3O4 magnetic nanoparticles (Fe3O4 NPs) were synthesized by a one-step thermal synthesis method.

[0035] First, 2 g of anhydrous ferric chloride (FeCl3) was dissolved in 40 ml of ethylene glycol ((CH2OH)2) to obtain a yellow clear solution. Subsequently, 6 g of anhydrous sodium acetate (NaAc) and 20 ml of ethylenediamine (C2H8N2) were added to this solution, and the mixture was vigorously stirred for 30 min. Next, the mixture was poured into a reaction kettle and continuously heated at 200 °C for 8 h. During the entire reaction process, the heating and cooling rates did not need to be considered. After the reaction was completed, the reaction kettle was cooled to room temperature. Subsequently, the precipitate was washed repeatedly with a large amount of ultrapure water and separated by magnetic separation. When the supernatant was neutral, the washing was stopped, and then the precipitate was dried in an oven at 50 °C to obtain Fe3O4 NPs for standby.

[0036] 2. Preparation of Fe3O4-MWCNTs-COOH magnetic composite

[0037] The Fe3O4-MWCNTs-COOH magnetic composite was prepared by ultrasonic synthesis. 1.5 mg of the prepared Fe3O4 NPs and 2.0 mg of MWCNTs-COOH were weighed and mixed, and ultrasonically dispersed in 3 ml of methanol solution for 1 h to obtain a dispersion of the Fe3O4-MWCNTs-COOH magnetic composite, which was stored for standby at 4 °C.

[0038] 3. Preparation of Fe3O4-MWCNTs-COOH / GCE electrode

[0039] Treatment of the electrode: Before modifying the surface of the GCE electrode, the surface of the electrode was polished with alumina powder on suede, ultrasonically cleaned with methanol and deionized water to remove the residual alumina powder, and then dried under an infrared lamp.

[0040] 7 μL of the dispersion of the Fe3O4-MWCNTs-COOH magnetic composite was dropped onto the surface of the GCE electrode and dried under an infrared lamp. The electrode surface was rinsed with 0.1 mol·L -1 PBST buffer solution (pH 7.0 phosphate buffer containing 0.05% Tween-80) to remove physically adsorbed Fe3O4-MWCNTs-COOH, and the Fe3O4-MWCNTs-COOH modified GCE electrode (Fe3O4-MWCNTs-COOH / GCE) was prepared.

[0041] 4. Preparation of Mb / Fe3O4-MWCNTs-COOH / GCE electrode

[0042] Then, the Fe3O4-MWCNTs-COOH / GCE electrode was placed in a standard solution of Mb or a solution to be tested and incubated overnight at 4 °C. Through the amide bond formed between the carboxyl groups (-COOH) on the surface of the magnetic composite material and the amino groups (-NH2) on Mb, the myoglobin Mb molecules were firmly immobilized on the electrode surface. Then, the modified electrode was rinsed with 0.1 mol·L -1 PBST to remove physically adsorbed Mb, obtaining Mb / Fe3O4-MWCNTs-COOH / GCE. Finally, the GCE modified with Mb / Fe3O4-MWCNTs-COOH was placed in a blocking solution (0.1 mol·L of 0.05% Tween-80 and 2% BSA -1 PBS (pH 7.0)) to block the active sites not loaded with Mb. The prepared modified electrode was stored at 4 °C for later use.

[0043] 5. Preparation of the signal probe HRP-Strept-Biotin-Ab-Mb

[0044] It was obtained by mixing and incubating 100 µL of HRP-Strept and 100 µL of effective Biotin-Ab-Mb.

[0045] 6. Preparation of the electrochemical immunosensor

[0046] The Mb / Fe3O4-MWCNTs-COOH / GCE electrode was immersed in a solution containing the signal probe HRP-Strept-Biotin-Ab-Mb to form the HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE electrochemical immunosensor.

[0047] After the Fe3O4-MWCNTs-COOH / GCE electrode was prepared in this application, it was characterized by infrared (FT-IR) spectroscopy, Raman spectroscopy, scanning electron microscopy (SEM), energy-dispersive spectroscopy, and elemental stratification (EDS) images. The results are as Figure 1 shown Figure 1 Figure A shows the FT-IR spectra of MWCNTs and Fe3O4-MWCNTs. In the infrared spectrum of MWCNTs-COOH, the peak at 3675 cm -1 is broad and scattered, and this absorption peak corresponds to the stretching vibration peak of O-H of -COOH. The absorption peak at 1766 cm -1 is generated by the C=O stretching vibration at -COOH on MWCNTs-COOH. The absorption peak of the Fe3O4-MWCNTs composite material at 567 cm -1The peak at [specific position] corresponds to the Fe-O group, which is consistent with the characteristic absorption peak of magnetic particles. In addition, due to the stretching vibration of the C-N bond, a characteristic absorption peak appears at 1128 cm -1 at [specific position]. The characteristic absorption peak at 3185 cm -1 is attributed to the stretching vibration of the O-H bond.

[0048] Figure 1 As shown in B of , the intensity ratio of the D band to the G band (I D / I G ) of MWCNTs-COOH is 0.82, which indicates that in the structure of MWCNTs-COOH, the atomic arrangement in some regions deviates from the ideal graphite structure, generating defect sites. When MWCNTs-COOH is combined with Fe3O4 NPs to form the Fe3O4-MWCNTs-COOH magnetic composite material, I D / I G slightly rises to 0.84, which means that the structural disorder of the composite material is enhanced relative to pure multi-walled carbon nanotubes. This may be attributed to the loading of Fe3O4 NPs on the surface of MWCNTs-COOH, resulting in changes in the surface atomic arrangement, generating more defects and disordered regions. This change in structure will further affect the electrochemical performance of the magnetic composite material, providing more active sites for subsequent interaction with the target analyte.

[0049] The morphology and microstructure of MWCNTs-COOH and the Fe3O4-MWCNTs-COOH magnetic composite material were characterized by scanning electron microscope (SEM). As Figure 1 shown in C of , MWCNTs-COOH presents its iconic tubular structure, and the tube wall is smooth and continuous, without significant defects or impurities attached. When the Fe3O4-MWCNTs-COOH magnetic composite material is formed by combining Fe3O4 NPs with MWCNTs-COOH, Figure 1 it can be clearly seen from D of that the surface of the composite material is slightly rougher than that of pure MWCNTs-COOH. This roughness may be caused by the attachment of Fe3O4 NPs, but it does not affect the overall structure of MWCNTs. In addition, the SEM image also shows that the overall morphology of the composite material is stable and does not change significantly due to the addition of Fe3O4 NPs, maintaining the original tubular structure of MWCNTs.

[0050] The results of energy dispersive spectrum (EDS) analysis show (as Figure 1In (E), the Fe3O4-MWCNTs-COOH magnetic composite material is mainly composed of three elements: carbon (C), iron (Fe), and oxygen (O). This result strongly confirms the successful combination of Fe3O4 NPs and MWCNTs-COOH in the composite material. Figure 1 In (F - H), the distribution of elements such as C, O, and Fe in the Fe3O4-MWCNTs-COOH magnetic composite material can be clearly observed. Among them, the strong signal of carbon element originates from multi-walled carbon nanotubes. As the main structure of the composite material, the presence of carbon element not only provides excellent electrical conductivity but also provides a stable carrier for the attachment of Fe3O4 NPs. The co-occurrence of Fe and O is direct evidence of the existence of Fe3O4 NPs. As an important magnetic material, the introduction of Fe3O4 NPs endows the composite material with remarkable magnetic properties, which is crucial for the application of the composite material in fields such as electrochemical immunosensors.

[0051] Electrochemical measurements and reaction processes

[0052] 1. Electrochemical characterization of the working electrode

[0053] Electrochemical measurements include cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), chronocoulometry (CC), and differential pulse voltammetry (DPV). The GCE, Fe3O4 NPs / GCE, MWCNTs-COOH / GCE, and Fe3O4-MWCNTs-COOH / GCE were characterized and compared.

[0054] CV parameter settings: The scanning voltage range is from -0.4 V to 0.8 V, and the scanning rate is fixed at 60 mV s -1 .

[0055] EIS parameter settings: The frequency range is from 1 Hz to 106 Hz, the amplitude of the sine wave signal is set to 5 mV, and the measurement is carried out at a voltage of 0.22 V. All of the above are tested in an electrolyte solution containing 4 mmol·L -1 [Fe(CN)6] 3- / 4- and 0.01 mol·L -1 KCl.

[0056] DPV parameter settings: The potential range is from 0.4 V to -0.4 V, the resting time is 2 s, the voltage amplitude is 0.05 V, the pulse period is 0.5 s, and the sampling width is 0.0167 s.

[0057] CC parameter settings: Initial potential = 0 V, termination potential = 0.3 V, pulse width = 0.25 s, standing time = 2 s, sampling interval = 2.5×10 -4s. In 0.1 mol·L -1 KCl and 1 mM Fe(CN)6 3- / 4- The electroactive surface areas of GCE, Fe3O4 NPs / GCE, MWCNTs-COOH / GCE, and Fe3O4-MWCNTs-COOH / GCE at their optimal modification amounts were studied in the probe solution.

[0058] Subsequently, electrochemical characterization was carried out to characterize the preparation process and performance of the electrodes through electrochemical testing methods. The results are as Figure 2 shown, as Figure 2 shown in A, the current intensity increases with the increase of the electrode surface modification material. A weak background current and small redox peaks can be observed on the bare electrode (GCE) (curve a). Compared with GCE, the redox peak currents of magnetic Fe3O4 NPs (curve b) and carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) (curve c) have obvious improvements. However, the magnetic Fe3O4 NPs are not firmly bound to the GCE surface, resulting in a lower peak current. Due to its large specific surface area and good conductivity, MWCNTs-COOH promotes the progress of the redox reaction, and obvious oxidation and reduction peaks appear, indicating that multi-walled carbon nanotubes can effectively enhance the electrochemical reaction activity of the electrode. Combining magnetic Fe3O4 NPs and MWCNTs-COOH to form the magnetic composite material Fe3O4-MWCNTs-COOH (curve d), it can be seen from the figure that its current response further increases. This may be due to the introduction of magnetism, which enhances the adsorption ability of the electrode to electroactive substances, and its oxidation peak current is as high as 1.82×10 -4 A, and the reduction peak current is also significantly higher than that of the MWCNTs-COOH modified electrode.

[0059] To further prove the excellent conductivity of the magnetic composite material Fe3O4-MWCNTs-COOH, electrochemical impedance spectroscopy (EIS) was used to characterize GCE (curve a, Ret = 724.9 Ω), Fe3O4 NPs (curve b, Ret = 618.1 Ω), MWCNTs-COOH (curve c, Ret = 410.6 Ω), and Fe3O4-MWCNTs-COOH (curve d, Ret = 348.73 Ω) ( Figure 2In B). Among them, the electron transfer resistance (Ret) reflects the ease of the electron transfer process at the interface between the electrode and the electrolyte. According to the measured Ret of different modified electrodes, it can be seen that after the magnetic composite material Fe3O4-MWCNTs-COOH (curve d, Ret = 348.73 Ω) modifies the GCE (curve a, Ret = 724.9 Ω), the Ret value decreases significantly, indicating that Fe3O4 nanoparticles are attached to MWCNTs-COOH, promoting the transfer of electrons through the solution-electrode interface. This result is consistent with the characterization results of the CV technique, indicating that the electrochemical immunosensor in this application is successfully constructed.

[0060] As Figure 2 shown in C and D in, Q and t 1 / 2 show a good linear relationship. According to the Anson equation:

[0061]

[0062] A is the surface area of the working electrode, c is the concentration of the probe solution substance, F is the Faraday electrolysis constant, D is the diffusion coefficient, Q dl is the double-layer charge, Q ads is the Faraday charge, and n is the number of electron transfers.

[0063] According to the above formula, the electrochemically active surface area of Fe3O4-MWCNTs-COOH / GCE is calculated to be 0.739 cm 2 , which is much higher than that of the bare GCE (0.044 cm 2 ), Fe3O4 NPs / GCE (0.050 cm 2 ), and MWCNTs-COOH / GCE (0.257 cm 2 ). This data confirms that Fe3O4-MWCNTs-COOH / GCE has excellent electron migration ability and can achieve good current response.

[0064] As shown in E in Figure 2, the peak current (I p ) of the CV response of Fe3O4-MWCNTs-COOH / GCE at different scan rates increases with the increase of the scan rate, and the peak potential also gradually shifts. With the increase of the scan rate, the relevant peak current increases and tends to a linear equation. As Figure 2 shown in F, it can be seen that the logarithm of the redox peak current has a good linear relationship with the logarithm of the scan rate within a certain range, that is, LgI pa = 0.5476 Lgν (mV s -1 ) + 0.0746 (R² = 0.9998) and, LgIpc = 0.5795 Lgν (mV s -1 ) + 0.013(R² = 0.9996)。The experimental results show that the reaction on the electrode surface in this application is controlled by both adsorption and diffusion. In addition, as Figure 2 shown in G of pa , the oxidation peak potential (E pc ) and the reduction peak potential (E pa ) of Fe3O4-MWCNTs-COOH / GCE show a good linear relationship with the logarithm of the scan rate, specifically: E pa (V) = 0.1532logν (v s -1 ) + 0.0619 R 2 = 0.9941 and E pc (V) = -6.9693logν(v s -1 ) + 1.9974 R 2 = 0.9926. According to the Laviron equation:

[0065]

[0066]

[0067]

[0068] R is the gas constant (8.314 J mol -1 K -1 ), T is the room temperature (298.15 K), F is the Faraday electrolysis constant, is the difference between the oxidation peak potential and the reduction peak potential. From the above formula, the electron transfer coefficient α can be calculated as 0.5200, the electron transfer number n is 0.8046 ≈ 1, and the electrode transfer rate Ks is 0.107 V s -1 . This result is consistent with the number of electron transfers in the redox reaction in the electrolyte, indicating that there is a rapid electron transfer process of the magnetic electroactive substance Fe3O4-MWCNTs-COOH / GCE on the electrode surface.

[0069] Optimization of experimental conditions: To obtain the best electrochemical performance, this study systematically optimized the mixing mass ratio of Fe3O4 NPs and MWCNTs-COOH. By changing the mixing ratio of the two, a series of magnetic composites with different mass ratios (Fe3O4:MWCNTs-COOH = 1:1, 1:2, 1:3, 2:1, 3:1) were prepared using methanol as the solvent. After drying under an infrared lamp at 20 - 80 °C, CV measurements were carried out to determine the optimal mixing mass ratio of Fe3O4 NPs and MWCNTs-COOH. In addition, by changing the modification amount of the magnetic composite, electrodes with different modification amounts of 1 μL, 3 μL, 5 μL, 7 μL, 9 μL, and 11 μL were prepared, and the performance of these electrodes in electrochemical tests was evaluated using CV to determine the optimal modification amount of the magnetic composite. Finally, to investigate the effect of Mb incubation time on the response of the immunosensor system, CV measurements were performed on the electrochemical behavior of the interaction between Mb and the Fe3O4-MWCNTs-COOH / GCE electrode at different incubation times (1, 1.5, 2, 2.5, 3 h) to determine the optimal Mb incubation time. All the above CV measurements were carried out at 25 ± 2 °C within a voltage range of -0.4 to 0.8 V and a scan rate of 60 mV s -1 under the condition of.

[0070] As Figure 3 shown in A, when the mixing mass ratio of Fe3O4 NPs and MWCNTs-COOH was set to 1:2, the composite material exhibited the best electrochemical performance. This ratio not only significantly increased the electroactive specific surface area, providing more active sites for electrochemical reactions, but also optimized the conductivity and stability of the material, thus enhancing the overall electrochemical performance. Therefore, the optimal mixing mass ratio of Fe3O4 and MWCNTs-COOH in this application was finally determined to be 1:2. In addition, the modification amount of the electrode modification material has a certain impact on the sensitivity of the sensor. As Figure 3 shown in B, with the increase in the modification amount of 1:2 (m:m) Fe3O4-MWCNTs-COOH on the GCE, the oxidation peak current obtained from the cyclic voltammetric scan of the modified electrode in the probe solution gradually increased. When the modification amount of the composite material reached 7 μL, the electrode exhibited the optimal electrochemical performance. Therefore, the optimal modification amount of Fe3O4-MWCNTs-COOH was determined to be 7 μL. Finally, the length of the Mb incubation time also has a significant impact on the sensor sensitivity, as Figure 3As shown in C, by observing the change in the current response, we found that when the incubation time reached 2 h, the current response tended to be stable, indicating that Mb was stably loaded on the electrode and the insulating effect on the electrode surface had not yet appeared. Therefore, considering both the detection sensitivity and the response time, we selected 2 h as the optimal incubation time for Mb to ensure the effective application of the electrochemical immunosensor in the early warning of acute myocardial infarction.

[0071] Finally, under the optimized conditions (the mixing mass ratio of Fe3O4 and MWCNTs-COOH was 1:2, the modification amount of the magnetic composite was 7 μL, and the Mb incubation time was 2 h), we used the DPV method to evaluate the analytical performance of the electrochemical immunosensor constructed using the HRP-Strept-Biotin-Ab system. To ensure the specific binding between Mb and Biotin-Ab-Mb, we first blocked the remaining active sites on the surface of the Mb / Fe3O4-MWCNTs-COOH / GCE electrode with 2% bovine serum albumin (BSA) to prevent unnecessary interference. Then, we immobilized a limited number of Mb on the electrode surface by binding it with a small amount of biotin antibody. Next, we immobilized HRP on the immunosensor through the specific interaction between the Biotin-Ab-Mb complex and HRP-Strept. To determine the Mb content, we used standard solutions containing different concentrations of Mb (640 ng·mL -1 、320 ng·mL -1 、160 ng·mL -1 、80 ng·mL -1 、40 ng·mL -1 、20 ng·mL -1 、10 ng·mL -1 、5 ng·mL -1 、2.5 ng·mL -1 、1.25 ng·mL -1 、0.625 ng·mL -1 、0.3125 ng·mL -1 、0.156 ng·mL -1 、0.0781 ng·mL -1 、0.039 ng·mL -1 、0.0195 ng·mL -1 、0 ng·mL -1 ) to prepare the electrochemical immunosensor, and the Fe3O4-MWCNTs-COOH / GCE electrode was used with 0.1 mol L -1Rinse with PBST (phosphate buffer solution at pH 7.0 containing 0.05% Tween-80) and dry at room temperature. Then soak it in a solution containing 100 µL Biotin-Ab-Mb and 100 µL effective HRP-Strept to form an HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE electrochemical immunosensor with different HRP contents. Then, place the prepared immunosensor in 10 ml of PBS solution (pH = 7.4) containing 2 mmol L -1 o-phenylenediamine (o-PD) and 4 mmol L -1 H2O2 to achieve the quantitative detection of Mb. As shown in B of Figure 4 , as the concentration of Mb in the test bottom solution decreases, the DPV response of the HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE immunosensor also gradually decreases. This indicates that under this environment, HRP catalyzes the decomposition of H2O2, promoting the conversion of o-phenylenediamine to 2,3-diaminophenothiazine. The electrochemical signal generated during this conversion process, which originates from the reversible reaction of 2,3-diaminophenothiazine, provides the basis for us to detect the content of Mb. This result also further demonstrates that this application has successfully constructed an electrochemical immunosensor capable of rapidly and sensitively determining Mb, and its DPV response value is positively correlated with the Mb concentration in the sample.

[0072] We evaluated the performance of the immunosensor by analyzing Mb standard solutions with different concentrations under the same conditions. The relationship between the DPV peak current (I p ) and the Mb concentration is shown in C of Figure 4 , and the linear relationship between the Mb concentration and its DPV response is shown in D of Figure 4 . In the concentration range of 0.0195 ~ 640 ng·mL Figure 4 , there is a good linear relationship between the DPV peak current and LgC -1 . The linear equation is: I Mb (μA) = 29.644 LgC p + 104.07, R Mb = 0.9946. The lowest detection limit (LOD) of this immunosensor is 0.1007 ng·mL 2 (S / N = 3). -1 (S / N = 3).

[0073] Traditional enzyme-linked immunosorbent assay (ELISA) was used to analyze Mb standards with different concentrations as a control. Figure 4In it, A represents the absorbance value (OD) corresponding to the analysis of Mb at different concentrations by ELISA method. It can be seen from the experimental results that in the Mb concentration range of 0.31 ng·mL -1 to 20 ng·mL -1 , the OD value of the ELISA method shows a good linear relationship with the Mb concentration. The linear equation is: OD = 0.1234 LgC Mb (ng mL -1 ) + 0.1065, and the linear correlation coefficient is R 2 = 0.9742, and the detection limit is 0.19 ng·mL -1 .

[0074] By comparing two different analytical methods, it is found that the electrochemical immunosensor constructed in this application exhibits better analytical performance, with lower detection limit and wider detection range.

[0075] The specificity of the electrochemical immunosensor is one of the important indicators for evaluating its performance. In this application, 20 ng·mL -1 Mb, 12.5 ng·mL -1 cardiac troponin (cTnI), 4.00 ng·mL -1 brain natriuretic peptide (BNP), and 2.50 μLmL -1 proline (PRO) are selected as potential interfering substances to test the specific recognition ability of the sensor for the target analyte Mb. As Figure 5 shown in A, the concentration of the interfering substance does not affect the determination of the content of Mb, and there is also no significant effect on the DPV current response when Mb and the interfering substance coexist (less than 5%). In summary, the electrochemical immunosensor constructed in this application shows good specificity for Mb, which not only provides an efficient and accurate method for the detection of myoglobin, but also provides new ideas and directions for the application of electrochemical immunosensors in the biomedical field.

[0076] In the performance evaluation of the electrochemical immunosensor, reproducibility is a crucial indicator. To verify the reproducibility of the HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE electrochemical immunosensor, we placed 6 electrodes with the same concentration of Mb (20 ng·mL -1 ) prepared simultaneously into a PBS solution (pH = 7.4) containing 2 mmol L -1 o-PD and 4 mmol L -1 H2O2 and performed 3 DPV scans respectively. As Figure 5As shown in B, the electrochemical signals of the 6 sensors were relatively stable, with an RSD of only 2.13%. In addition, the same electrode was prepared 6 times using the same method and placed in the same test base solution for 3 parallel DPV tests. The results are as Figure 5 shown in C. The DPV signals of the 6 tests were also relatively stable, with an RSD of only 3.53%. In summary, the RSD between batches and within batches was less than 5%, and I p was stable at around 142 μA, indicating that the electrochemical immunosensor constructed in this application had good reproducibility.

[0077] Finally, to investigate the stability of the electrochemical immunosensor, 7 Fe3O4-MWCNTs-COOH / GCE electrodes were prepared in parallel and stored at 4 °C for 0, 5, 10, 15, 20, 25, 30 days and then subjected to DPV tests under a 20 ng·mL -1 Mb standard solution (as shown in D). On the 25th day, compared with the initial current response, the electrochemical response of the electrochemical immunosensor maintained 92.67% of the initial response. From the above results, it can be seen that the HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE electrochemical immunosensor constructed in this application had acceptable stability for Mb content determination. Figure 5 Actual sample collection: In this experiment, the blood of healthy adults (which had passed ethical review) was used as the actual sample for Mb detection. The collected blood samples were placed in EP tubes containing EDTA-Na2 and centrifuged at a speed of 1000×g for 15 min within 30 minutes. Then, the ELISA method and the electrochemical immunosensor constructed in this application were used to detect the Mb content in the supernatant.

[0078]

[0079] ​This application aims to evaluate the practical application effects of the ELISA method and the self-constructed electrochemical immunosensor in detecting Mb in human serum. For this purpose, we collected human serum samples and detected them using the above two methods respectively. At the same time, in order to compare the accuracy of the two methods, we used the standard addition method for three parallel tests. As shown in Table 1, the RSD of the ELISA method in three independent experiments was between 4.08% and 5.64%, and the recovery rate fluctuated between 94.2% and 104.15%. While the electrochemical immunosensor we constructed showed an RSD between 3.86% and 5.03% in three independent experiments, and the recovery rate was between 98.32% and 105.30%, showing good precision and accuracy and there was no significant difference in the results obtained by the two measurement methods. In summary, the HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE electrochemical immunosensor constructed in this application shows significant application potential in the quantitative detection of Mb in human serum and is consistent with the results of the ELISA method, with good accuracy.

[0080] Table 1: Detection of the electrochemical immunosensor in actual serum samples

[0081]

[0082] a The average value of three consecutive measurements.

[0083] The present invention has been disclosed in preferred embodiments, but it is not intended to limit the present invention. Any technical solutions obtained by using equivalent replacements or equivalent transformations fall within the protection scope of the present invention.

Claims

1. An electrochemical immunoassay sensor for myoglobin, characterized in that Using the biotinylated monoclonal antibody HRP-Strept-Biotin-Ab-Mb of myoglobin Mb labeled with horseradish peroxidase avidin as a signal probe and the Fe3O4-MWCNTs-COOH / GCE electrode loaded with Mb in the myoglobin standard solution or the solution to be tested as the basic electrode, an HRP-Strept-Biotin-Ab-Mb / Mb / Fe3O4-MWCNTs-COOH / GCE electrochemical immunosensor was constructed; Among them, the preparation method of the Fe3O4-MWCNTs-COOH / GCE electrode includes: S1. Mix Fe3O4 NPs and carboxylated multi-walled carbon nanotubes MWCNTs-COOH, and ultrasonically disperse them with a solvent to obtain a dispersion of Fe3O4-MWCNTs-COOH magnetic composite material; S2. Modify the dispersion of Fe3O4-MWCNTs-COOH magnetic composite material on the GCE electrode and dry it to obtain the Fe3O4-MWCNTs-COOH / GCE electrode; The preparation method of the signal probe HRP-Strept-Biotin-Ab-Mb includes: mixing HRP-Strept and Biotin-Ab-Mb and incubating them to obtain it.

2. The myoglobin electrochemical immunosensor according to claim 1, wherein In S1, the solvent is selected from at least one of methanol, ethanol, and isopropanol.

3. The myoglobin electrochemical immunosensor according to claim 1, characterized in that, In S1, the mass ratio of Fe3O4 NPs to carboxylated multi-walled carbon nanotubes MWCNTs-COOH is 1:1 to 1:3, preferably 1:

2.

4. The myoglobin electrochemical immunosensor according to claim 1, wherein In S2, 1-11 mL of the dispersion of Fe3O4-MWCNTs-COOH magnetic composite material is taken to modify the GCE electrode, preferably 7 mL.

5. The myoglobin electrochemical immunosensor according to claim 1, wherein The preparation method of the Fe3O4 NPs includes: dissolving ferric chloride in ethylene glycol, adding sodium acetate and ethylenediamine, and reacting in a reaction kettle to obtain Fe3O4 nanoparticles Fe3O4 NPs.

6. The application of the electrochemical immunosensor according to any one of claims 1-5 in the detection of myoglobin.

7. The application according to claim 6, characterized in that, Including: S31: Place the Fe3O4-MWCNTs-COOH / GCE electrode in myoglobin standard solutions with different concentrations, incubate, rinse, and dry to obtain the Mb / Fe3O4-MWCNTs-COOH / GCE electrode, and then combine the Mb / Fe3O4-MWCNTs-COOH / GCE electrode with the signal probe HRP-Strept-Biotin-Ab-Mb to obtain the processed electrochemical immunosensor; S32: Using the processed electrochemical immunosensor as the working electrode, perform differential pulse voltammetry scanning using a three-electrode system, record the peak current-Mb concentration relationship curve, establish a linear relationship between the peak current and the logarithm of the myoglobin Mb concentration, and obtain a linear regression equation; S33: Re-take the Fe3O4-MWCNTs-COOH / GCE electrode, place it in the solution to be measured, incubate, rinse, and dry it to obtain the Mb / Fe3O4-MWCNTs-COOH / GCE electrode. Then, combine the Mb / Fe3O4-MWCNTs-COOH / GCE electrode with the signal probe HRP-Strept-Biotin-Ab-Mb. Use the processed electrochemical immunosensor as the working electrode and perform differential pulse voltammetry scanning using a three-electrode system. According to the measured peak current and in combination with the linear regression equation, obtain the concentration of myoglobin Mb in the solution to be measured.

8. The application according to claim 7, wherein Including: In S31 and S33, the incubation time is 1 to 3 hours, preferably 2 hours.

9. The application according to claim 7, characterized in that, In S31 and S33, after obtaining the Mb / Fe3O4-MWCNTs-COOH / GCE electrode, it further includes: placing the Mb / Fe3O4-MWCNTs-COOH / GCE electrode in a blocking solution to block the active sites on the electrode surface that are not loaded with Mb, where the blocking solution contains bovine serum albumin.

10. The application according to claim 7, characterized in that, During the differential pulse voltammetry scanning process, the electrochemical immunosensor is placed in the reaction bottom solution to achieve the quantitative detection of Mb, where the reaction bottom solution is a PBS solution containing o-phenylenediamine and H2O2, and the molar ratio of o-phenylenediamine to H2O2 in the reaction bottom solution is 1:2.

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