Hemoglobin beta subunit electrochemical immunosensor and application thereof in cardiotoxicity detection

Abstract

The invention discloses a hemoglobin beta subunit electrochemical immunosensor and application thereof in cardiotoxicity detection. A preparation method of the electrochemical immunosensor comprises the following steps: dissolving MWCNTs (multi-walled carbon nanotubes) in N 'N-dimethylformamide to prepare a suspension; dripping the suspension on the surface of the pretreated glassy carbon electrode to obtain an MWCNTs / GCE electrode; the MWCNTs / GCE electrode is sequentially placed in an acetic acid buffer solution of o-phenylenediamine and an H2SO4 solution for cyclic voltammetry scanning, and a PoPD-MWCNTs / GCE electrode is obtained; the PoPD-MWCNTs / GCE electrode is sequentially placed in a dopamine solution and an H2SO4 solution for cyclic voltammetry scanning, and a PDA-PoPD-MWCNTs / GCE electrode is obtained; and incubating the antibody solution on the PDA-PoPD-MWCNTs / GCE electrode, thereby obtaining the product. The electrochemical immunosensor has relatively high sensitivity and specificity, and is simple and convenient to operate and rapid in detection.

Description

Technical Field

[0001] This application relates to the technical fields of environmental monitoring and quality safety monitoring of medicinal plants, and particularly relates to a hemoglobin β-subunit electrochemical immunosensor and its application in cardiac toxicity detection. Background Art

[0002] Aconitine (AC), as a highly toxic diester alkaloid, often causes poisoning or even death due to improper processing or overdose. A large number of clinical cases of aconitine poisoning show that highly individualized toxic reactions are the main characteristics of aconitine-induced cardiac toxicity. Due to the individualized reaction sensitivity, the toxic dose of aconitine is unpredictable. Therefore, the large individual response differences lead to the fact that aconitine drugs may cause unpredictable and inevitable cardiac toxicity, which is the main obstacle to their clinical safe application.

[0003] Hemoglobin β-subunit (HB-β) is a blood toxicity response protein with 89% homology to humans and does not exist in free form under normal circumstances. Some studies have shown that aconitine-induced cardiac toxicity can trigger the high expression of HB-β in the human body. Therefore, HB-β can be used as a specific biomarker for cardiac toxicity caused by aconitine drugs. Achieving highly sensitive and rapid determination of HB-β is crucial for the prevention and treatment of individualized cardiac toxicity of aconitine drugs.

[0004] Currently, the detection methods of HB-β mainly include liquid chromatography-high resolution mass spectrometry (LC-HR-MS), microfluidic capillary electrophoresis-mass spectrometry (CE-MS), enzyme-linked immunosorbent assay (ELISA), etc. However, these methods require experienced laboratory technicians and expensive equipment, and the preparation work required before testing is complex and time-consuming, with a narrow detection range and a high detection limit. Summary of the Invention

[0005] Objective: To overcome the deficiencies in the prior art, the present invention provides a hemoglobin β-subunit electrochemical immunosensor and its application in cardiac toxicity detection. The electrochemical immunosensor is easy to operate and highly sensitive. After incubating the HB-β antibody on the electrode surface, it can achieve specific detection of the cardiac toxicity biomarker hemoglobin β-subunit.

[0006] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] In the first aspect, the present invention provides a preparation method of an electrochemical immunosensor, including: Dissolve MWCNTs in N,N-dimethylformamide and sonicate to obtain a uniformly dispersed MWCNTs suspension; Drop the MWCNTs suspension on the surface of the pretreated glassy carbon electrode, and dry it to obtain the MWCNTs / GCE electrode; Place the MWCNTs / GCE electrode in an acetic acid buffer solution of o-phenylenediamine, scan it by cyclic voltammetry, take it out, wash it, and then place it in an H2SO4 solution, and scan it by cyclic voltammetry again. After taking it out and washing it, obtain the PoPD-MWCNTs / GCE electrode; Place the PoPD-MWCNTs / GCE electrode in a dopamine solution, scan it by cyclic voltammetry, take it out, wash it, and then place it in an H2SO4 solution, and scan it by cyclic voltammetry again. After taking it out and washing it, obtain the PDA-PoPD-MWCNTs / GCE electrode; Drop the antibody solution on the PDA-PoPD-MWCNTs / GCE electrode, incubate it, rinse it, and dry it to obtain the product.

[0008] In some embodiments, the pretreatment method of the glassy carbon electrode includes: polishing the glassy carbon electrode on suede sprinkled with 0.3 μm and 0.05 μm alumina polishing powder for 5-10 min, ultrasonically cleaning it with methanol and water for 10-15 min, and drying it.

[0009] In some embodiments, the cyclic voltammetry scan includes: Place the MWCNTs / GCE electrode in an acetic acid buffer solution of 0.2-0.3 mg / mL o-phenylenediamine with pH = 4.5-5, and perform cyclic voltammetry scan for 8-10 cycles within the potential range of -0.2-1.0 V; then place the electrode in a 0.5-1 mol / L H2SO4 solution, and perform cyclic voltammetry scan for 30-40 cycles within the potential range of -0.2-0.8 V; Place the PoPD-MWCNTs / GCE electrode in a 0.3-0.4 mg / mL dopamine solution, and perform cyclic voltammetry scan for 8-10 cycles within the potential range of -0.2-1.0 V; then place the electrode in a 0.5-1 mol / L H2SO4 solution, and perform cyclic voltammetry scan for 30-40 cycles within the potential range of -0.2-0.8 V.

[0010] In some embodiments, the scan rate of the cyclic voltammetry is 100 mV / s.

[0011] Placing the electrode in a 0.5-1 M H2SO4 solution and performing cyclic voltammetry scan for 30-40 cycles can clean and activate the electrode surface to obtain a stable polymer layer.

[0012] In some embodiments, the method for incubating the antibody solution includes: dropping the antibody solution onto the PDA-PoPD-MWCNTs / GCE electrode, incubating at 4-5 °C for 1-1.5 hours, taking it out and washing with Tris-HCl buffer solution with pH = 7.4-7.5, placing it in the blocking solution for 50-60 min, and then taking it out and rinsing with PBS solution and Tris-HCl solution with pH = 7.2-7.3.

[0013] In some embodiments, the blocking solution is 0.1-0.2 mol L-1 PBS containing 3%-5% BSA, with pH = 7.4-7.5.

[0014] In a second aspect, the present invention provides an electrochemical immunosensor prepared by the method described in the first aspect.

[0015] In a third aspect, the present invention provides an application of the electrochemical immunosensor described in the second aspect in cardiac toxicity detection. The biomarker for cardiac toxicity is HB-β. The antibody solution incubated on the PDA-PoPD-MWCNTs / GCE electrode is HB-β antibody solution. After incubating the antibody solution, the HB-β-Ab / PDA-PoPD-MWCNTs electrode is obtained.

[0016] The application method includes: Taking a blood sample, centrifuging after coagulation, and collecting the supernatant; Dropping the supernatant onto the surface of the HB-β-Ab / PDA-PoPD-MWCNTs electrode and incubating; Combining a reference electrode and an auxiliary electrode in a three-electrode system, using differential pulse voltammetry to measure the DPV peak current of different concentrations of HB-β standard products, fitting a linear regression equation, and calculating the concentration of HB-β in the sample according to the DPV peak current of the sample.

[0017] In some embodiments, the scanning potential range during the measurement by differential pulse voltammetry is -0.2 to 0.3 V.

[0018] In some embodiments, the incubation conditions of the supernatant are incubation at room temperature for 80-90 min.

[0019] The composite material composed of multi-walled carbon nanotubes, poly-o-phenylenediamine and polydopamine contains rich hydrophilic functional groups such as amino and hydroxyl groups. It can directly allow HB-β antibody (HB-β-Ab) to be coupled with dopamine on the PDA-PoPD-MWCNTs electrode under mild conditions and be fixed on the electrode surface to obtain the HB-β-Ab / PDA-PoPD-MWCNTs electrode. The HB-β-Ab on the electrode can specifically bind to the HB-β antigen in the sample to be tested, thereby realizing the specific detection of HB-β.

[0020] Beneficial effects: The electrochemical immunosensor for HB-β detection provided by the present invention can achieve specific detection of the cardiac toxicity biomarker hemoglobin β subunit (HB-β). A poly-o-phenylenediamine-dopamine copolymer film (PDA-PoPD) with good biocompatibility and high conductivity was modified on the MWCNTs electrode by an electrodeposition method. With the abundant hydrophilic functional groups amino and hydroxyl groups on the PDA-PoPD-MWCNTs composite material, the HB-β antibody can be directly linked to the DA molecules in the polymer on the PDA-PoPD-MWCNTs electrode under mild conditions and fixed on the electrode surface to obtain the HB-β-Ab / PDA-PoPD-MWCNTs working electrode. This electrochemical immunosensor relies on the specific binding of the HB-β antigen to HB-β-Ab, thereby achieving specific detection of HB-β. It has high sensitivity, specificity, reproducibility and stability, is easy to operate, has a short detection time and good stability, and can be applied to on-site rapid detection, providing a reference for the evaluation and early warning of individualized cardiac toxicity during the application of aconitine drugs. Description of the Drawings

[0021] Figure 1 These are the infrared spectra, Raman spectra and SEM characterization results of MWCNTs, PoPD-MWCNTs and PDA-PoPD-MWCNTs in the examples of the present invention; among them, Figure 1 A in it is the infrared spectrum diagram of MWCNTs, PoPD-MWCNTs and PDA-PoPD-MWCNTs, Figure 1 B in it is the Raman spectrum diagram of MWCNTs, PoPD-MWCNTs and PDA-PoPD-MWCNTs, Figure 1 C in it is the SEM image of MWCNTs, Figure 1 D in it is the SEM image of PDA-MWCNTs, Figure 1 E in it is the SEM image of PDA-PoPD-MWCNTs;

[0022] Figure 2 These are the electrochemical characterization results of the glassy carbon electrode, MWCNTs / GCE electrode, PoPD-MWCNTs / GCE electrode and PDA-PoPD-MWCNTs / GCE electrode in the examples of the present invention; among them, Figure 2 A in it is the CV curves of the glassy carbon electrode, MWCNTs / GCE electrode, PoPD-MWCNTs / GCE electrode and PDA-PoPD-MWCNTs / GCE electrode, Figure 2In B, the EIS curves of the glassy carbon electrode, MWCNTs / GCE electrode, PoPD-MWCNTs / GCE electrode, and PDA-PoPD-MWCNTs / GCE electrode are shown. Figure 2 In C, the chronocoulometry curves of the glassy carbon electrode, MWCNTs / GCE electrode, PoPD-MWCNTs / GCE electrode, and PDA-PoPD-MWCNTs / GCE electrode are shown. Figure 2 In D, the linearized plots of the chronocoulometric analysis data of the glassy carbon electrode, MWCNTs / GCE electrode, PoPD-MWCNTs / GCE electrode, and PDA-PoPD-MWCNTs / GCE electrode are shown. Figure 2 In E, the CV curves of the PDA-PoPD-MWCNTs / GCE electrode at different scan rates are shown. Figure 2 In F, the fitting curve of the logarithm of the scan rate and the logarithm of the redox peak current of the PDA-PoPD-MWCNTs / GCE electrode is shown.

[0023] Figure 3 This shows the results of the influence of different experimental conditions on the preparation of the working electrode in the embodiments of the present invention. Among them, Figure 3 In A, the influence of the MWCNTs drop-coating amount on the anodic peak current is shown. Figure 3 In B, the influence of the number of o-PD polymerization cycles on the anodic peak current is shown. Figure 3 In C, the influence of the number of DA polymerization cycles on the anodic peak current is shown. Figure 3 In D, the influence of the pH of the detection system on the peak current is shown. Figure 3 In E, the influence of the HB-β-Ab incubation time on the peak current is shown. Figure 3 In F, the influence of the HB-β incubation time on the peak current is shown.

[0024] Figure 4 This shows the test results of the detection performance of the HB-β-Ab / PDA-PoPD-MWCNTs / GCE electrochemical immunosensor under the optimized experimental conditions in the embodiments of the present invention. Among them, Figure 4 In A, the differential pulse voltammetry (DPV) responses to different concentrations of HB-β are shown. Figure 4 In B, the DPV peak current value I of the HB-β-Ab / PDA-PoPD-MWCNTs / GCE electrochemical immunosensor p and the curve of the HB-β antigen concentration are shown. Figure 4 In C, the calibration curve of the change value ∆I of the DPV peak current of the HB-β-Ab / PDA-PoPD-MWCNTs / GCE electrochemical immunosensor and the logarithm of the HB-β antigen concentration (lgC HB-β ) is shown.

[0025] Figure 5This is the graph showing the reproducibility, stability, and selectivity detection results of the HB-β-Ab / PDA-PoPD-MWCNTs / GCE sensor in the embodiments of the present invention; among them, Figure 5 In A is the graph showing the reproducibility detection results of the electrochemical immunosensor, Figure 5 In B is the graph showing the stability detection results of the electrochemical immunosensor, Figure 5 In C is the graph showing the selectivity detection results of the electrochemical immunosensor. Detailed implementation manners

[0026] Next, the technical solutions in the embodiments will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and in no way limits the present invention and its application or use.

[0027] Unless otherwise specifically stated, the relative arrangements, numerical expressions, and numerical values of the components and steps set forth in these embodiments do not limit the scope of the present invention. At the same time, it should be understood that for the convenience of description, the sizes of the various parts shown in the drawings are not drawn in actual proportional relationships. Technologies, methods, and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the said technologies, methods, and devices should be regarded as part of the authorization specification. In all the examples shown and discussed here, any specific value should be construed as merely exemplary and not as a limitation. Therefore, other examples of the exemplary embodiments may also include different values. It should be noted that: similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0028] The present invention will be further described below in conjunction with embodiments.

[0029] In the following embodiments, the experimental materials used are: multi-walled carbon nanotubes (MWCNTs, Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.), o-phenylenediamine (o-PD, Tianjin Damao Chemical Reagent Factory), dopamine hydrochloride (DA, Shanghai Macklin Biochemical Technology), bovine serum albumin (BSA, purity > 98.0%, Solarbio), human hemoglobin β subunit (HB-β) enzyme-linked immunosorbent assay kit (ELISA, Nanjing Kamillo Bioengineering Co., Ltd., Lot: 241204), HB-β (Nanjing Kamillo Bioengineering Co., Ltd.), biotin-labeled HB-β monoclonal antibody (HB-β-Ab, Nanjing Kamillo Bioengineering Co., Ltd.), methionine (Shanghai Zhongqin Chemical Reagent Co., Ltd.), ascorbic acid (Xi'an Tianmao Chemical Industry Co., Ltd.), glucose (Tianjin Damao Chemical Reagent Factory).

[0030] In the following examples, all electrochemical experiments were carried out at 25 °C using a traditional three-electrode system. The experimental equipment used was as follows: CHI 760E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.); the working electrode was a glassy carbon electrode or a modified glassy carbon electrode (GCE, diameter 3 mm), the reference electrode was a saturated calomel electrode (type 232), and the auxiliary electrode was a platinum wire electrode (≥99.99%); SB-5200DTD ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); IFS66v / S Fourier transform infrared spectrometer (Llantrisant, UK); LabRAM HR Evolution confocal Raman microscope (HORIBA Jobin Yvon S.A.A., France); JSM-6701F cold field emission scanning electron microscope (JEOL, Japan).

[0031] Example 1:

[0032] This example provides a method for preparing an electrochemical immunosensor, including:

[0033] 1. Preparation of MWCNTs suspension 8.0 mg of MWCNTs was dissolved in 2 mL of N,N-dimethylformamide (DMF), and ultrasonicated for 2 h to form a uniformly dispersed black MWCNTs suspension, which was stored at 4 °C for later use.

[0034] 2. Preparation of modified electrode (1) Pretreatment of glassy carbon electrode (GCE): The GCE was polished on suede sprinkled with 0.3 μm and 0.05 μm alumina polishing powder for 5 min, then ultrasonically cleaned with methanol and water for 10 min respectively, and then dried in air; (2) Preparation of modified electrode: 7.5 μL of MWCNTs suspension was dropped onto the surface of GCE and dried under an infrared lamp to obtain the MWCNTs / GCE electrode; The MWCNTs / GCE electrode was placed in an acetic acid buffer solution (ABS, pH = 4.5) containing 0.2 mg / mL o-phenylenediamine (oPD) and cycled 10 times in the potential range of -0.2 to 1.0 V at a scanning rate of 100 mV / s; after the electrode was rinsed with distilled water, cyclic voltammetry scanning was performed 30 times in the potential range of -0.2 to 0.8 V in 0.5 M H2SO4 solution at a scanning rate of 100 mV / s to obtain a stable polymer layer; rinsed with distilled water to obtain the PoPD-MWCNTs / GCE electrode; The PoPD-MWCNTs / GCE electrode was placed in a 0.3 mg / mL dopamine (DA) solution and cyclic voltammetry scanning was performed 8 times in the potential range of -0.2 to 1.0 V at a scanning rate of 100 mV / s. After the electrode was rinsed clean with distilled water, cyclic voltammetry scanning was carried out 30 times in a 0.5 M H2SO4 solution in the potential range of -0.2 to 0.8 V at a scanning rate of 100 mV / s to obtain a stable polymer layer. After rinsing clean with distilled water, the modified electrode - PDA-PoPD-MWCNTs / GCE was obtained.

[0035] 3. Preparation of Electrochemical Immunosensor 8 μL of HB-β antibody solution was dropped onto the modified electrode and incubated at 4 °C for 1 h, and then washed with Tris-HCl with pH = 7.4 to remove the unbound HB-β antibody. After incubation, the electrode was placed in a blocking solution (0.1 mol L -1 PBS containing 3% BSA, pH = 7.4) at 25 °C for 50 min to block the active sites not loaded with HB-β antibody. It was gently rinsed with PBS solution (PH = 7.2) and Tris-HCl solution with pH = 7.4, and dried at room temperature to obtain the HB-β-Ab / PDA-PoPD-MWCNTs / GCE electrode, which was stored at 4 °C for standby.

[0036] Using the HB-β-Ab / PDA-PoPD-MWCNTs / GCE electrode as the working electrode, a saturated calomel electrode (type 232) as the reference electrode, and a platinum wire electrode as the auxiliary electrode, an electrochemical immunosensor was obtained.

[0037] As Figure 1 shown, after the HB-β-Ab / PDA-PoPD-MWCNTs / GCE modified electrode was constructed in this example, infrared spectroscopy, Raman spectroscopy, and SEM electron microscopy characterization were performed on it.

[0038] Figure 1 The curves of A in -1 are the FTIR spectral images of MWCNTs, PoPD-MWCNTs, and PDA-PoPD-MWCNTs respectively. In the spectrum of MWCNTs, the absorption peak at 3442.3 cm -1 belongs to the stretching vibration peak of O-H of -COOH, and the absorption peak at 1637.9 cm -1There are wide spectral bands on both sides, which are caused by N-H stretching vibration, and PDA-PoPD-MWCNTs has stronger absorption near 3400 cm -1 compared with PoPD-MWCNTs; the absorption peaks of both at around 1640 cm -1 are due to the stretching vibration of C=N, and the absorption peak at around 1400 cm -1 is related to the stretching vibration of C=C in the benzene-like ring and quinone ring. This proves the successful polymerization of PDA-PoPD copolymer film on MWCNTs.

[0039] Figure 1 In B of , the Raman spectra of MWCNTs, PoPD-MWCNTs, and PDA-PoPD-MWCNTs are shown. The carbon structure changes of MWCNTs before and after modification can be studied through Raman spectra. From Figure 1 B in , the two different peaks centered at 1343.9 cm -1 and 1590.1 cm -1 are the D peak and G peak respectively, which represent the vibration of sp 3 carbon atoms in the functional group and the in-plane stretching vibration of sp 2 carbon atoms. Comparing the positions of the D peak and G peak, the D peak and G peak of PoPD-MWCNTs and PDA-PoPD-MWCNTs have shifted, indicating an interaction between the PDA-PoPD copolymer and MWCNTs.

[0040] The surface morphology and microstructure of different modified materials are characterized by SEM. Figure 1 C in is the SEM image of MWCNTs. A large number of curved tubular structures can be observed, with a relatively smooth surface and tight winding between the tubes. After electrodepositing o-phenylenediamine (oPD) on MWCNTs / GCE, as can be seen from Figure 1 D in , a layer of PoPD film is formed on the electrode surface, but this polymer film is relatively thin, and the tubular structure of multi-walled carbon nanotubes can still be seen; depositing DA solution on it continuously, as can be seen from Figure 1 E in , the structure of the PDA-PoPD composite film is uniform, indicating that the PDA-PoPD copolymer film prepared under this condition is relatively uniform and has a high degree of polymerization. This indicates the successful preparation of the PDA-PoPD-MWCNTs / GCE electrode.

[0041] Example 2:

[0042] This example provides an electrochemical characterization test of the electrochemical immunosensor as described in Example 1.

[0043] Electrochemical characterization was carried out on an electrochemical workstation. Using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronocoulometry (CC), at room temperature of 20 ± 2 °C, a 2.0 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.1 M KCl was used as the electrolyte for electrode characterization.

[0044] For cyclic voltammetry (CV), the potential scan range was -0.4 to 0.8 V, and the scan rate was 100 mV s -1 , and CV tests were performed on the modified electrodes MWCNTs / GCE, PoPD-MWCNTs / GCE, and PDA-PoPD-MWCNTs / GCE respectively, and the current responses were recorded.

[0045] Electrochemical impedance spectroscopy (EIS) was used to study the electron transfer efficiency between the electrode and the redox pair [Fe(CN)6] 3- / 4- The frequency range was 0.1 Hz to 100 kHz, the amplitude was 100 mV. EIS tests were performed on the modified electrodes MWCNTs / GCE, PoPD-MWCNTs / GCE, and PDA-PoPD-MWCNTs / GCE respectively, and the data was fitted with an equivalent circuit to record the charge transfer resistance (Rct) value.

[0046] Chronocoulometry (CC) was carried out at room temperature of 20 ± 2 °C in a probe solution of 1.0 M KCl containing 5.0 mM K3[Fe(CN)6]. The modified electrodes MWCNTs / GCE, PoPD-MWCNTs / GCE, and PDA-PoPD-MWCNTs / GCE were tested in the potential range of 0 to 0.08 V, the data was saved, and the electroactive surface area was calculated by Q-t curve analysis (fitting the Anson equation).

[0047] As Figure 2 shown, in this example, the preparation and performance of the electrode were characterized by electrochemical testing methods.

[0048] The electrochemical properties of different materials on the electrode surface were tested by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results are shown in Figure 2 A and B. The cyclic voltammograms of different modified electrodes are shown in Figure 2As shown in Figure A, the bare GCE (curve a) has almost no conductivity; after the electrode is modified with MWCNTs (curve b), a significant and sharp increase in the peak current is observed, indicating that MWCNTs have good conductivity and promote electron transfer. Compared with MWCNTs / GCE, the peak current of PoPD-MWCNTs / GCE (curve c) increases, with Ip approximately 156.0 μA; after dopamine is polymerized onto the surface of this modified electrode, the current of PDA-PoPD-MWCNTs / GCE (curve d) increases again, greatly accelerating electron transfer, indicating that the PDA-PoPD-MWCNTs composite film plays an important role in the 3- / 4- redox reaction of [Fe(CN)6]. As Figure 2 shown in Figure B, the test results of EIS correspond to the CV results.

[0049] As Figure 2 shown in Figures C and D, the chronocoulometric curves of GCE, MWCNTs / GCE, PoPD-MWCNTs / GCE, and PDA-PoPD-MWCNTs / GCE show a good linear relationship between Q and t 1 / 2 . According to the Anson equation:

[0050] where A is the surface area of the working electrode, c is the concentration of the probe solution, 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. It can be calculated that the electroactive specific surface areas of GCE, MWCNTs / GCE, PoPD-MWCNTs / GCE, and PDA-PoPD-MWCNTs / GCE are 0.483 cm 2 , 0.865 cm 2 , 1.296 cm 2 , 1.461 cm 2 , respectively. The above results indicate that the PDA-PoPD-MWCNTs composite material and the modified electrode are successfully prepared.

[0051] As Figure 2 shown in Figures E and F, the redox peak current (Ip) increases continuously with the increase of the scan rate (υ) in the range of 10 mV s -1 ~200 mV s -1 , and there is a good linear relationship between Ip and υ. The corresponding linear regression equation is lgI pa = 0.615321 lgυ (mV s -1 ) + 0.89815 (R² = 0.9991), and lgIpc = 0.56464 lgυ (mV s -1 ) +1.00221(R² = 0.9994)。The result shows that the electrochemical redox reaction on PDA-PoPD-MWCNTs / GCE is controlled by both adsorption and diffusion.

[0052] Example 3:

[0053] Based on Example 1, this example provides a method for optimizing experimental conditions and the optimized experimental conditions.

[0054] To improve the sensitivity and selectivity of the immunosensor, factors such as the modification amount of MWCNTs, the number of electropolymerization cycles of PoPD and PDA, the pH value of the detection system, the incubation time of HB-β antibody, and the incubation time of antigen were evaluated.

[0055] 4 mg mL -1 MWCNTs suspension (2.5 μL, 5.0 μL, 7.5 μL, 10.0 μL, 12.5 μL) was dropped onto the surface of the GCE electrode, and CV method was used to detect MWCNTs / GCE in the 2.0 mM K3[Fe(CN)6] / K4[Fe(CN)6] probe solution containing 0.1 M KCl, and the corresponding measured current was recorded to draw a trend line to determine the optimal modification amount.

[0056] As Figure 3 shown in A, during the process of the content of MWCNTs suspension (4 mg / mL) modified on the electrode changing from 2.5 to 15.0 μL, the oxidation peak current of MWCNTs reaches the maximum value when the content is 7.5 μL, which indicates that when the content of the modification material reaches a certain value, the thickness of the film on the electrode surface will hinder electron transfer, thus affecting the oxidation process of MWCNTs on the electrode. Therefore, 7.5 μL is selected as the optimal modification amount of MWCNTs.

[0057] PoPD was electropolymerized on MWCNTs / GCE, and different numbers of electropolymerization cycles (5, 10, 15, 20, 25 cycles) were optimized. CV method was used to detect the response current of PoPD-MWCNTs / GCE, and the optimal number of polymerization cycles was obtained.

[0058] As Figure 3 shown in B, with the increase of the number of PoPD polymerization cycles, the oxidation peak current of the modified electrode gradually increases. When the polymerization is 10 cycles, the peak current reaches the maximum value. After that, with the increase of the number of polymerization cycles, the response current gradually decreases, indicating that the current response of the polymerization film is the best when PoPD is polymerized for 10 cycles. Therefore, 10 cycles are selected as the optimal number of polymerization cycles of the PoPD polymerization film.

[0059] Electropolymerize PDA on PoPD-MWCNTs / GCE, optimize different numbers of electropolymerization cycles (2, 4, 6, 8, 10 cycles), detect the response current of PDA-PoPD-MWCNTs / GCE by CV method, and obtain the optimal number of polymerization cycles.

[0060] As Figure 3 shown in C, the oxidation peak current of PDA-PoPD-MWCNTs / GCE reaches the maximum at 8 cycles of electropolymerization. Insufficient or excessive number of electropolymerization cycles will lead to a decrease in the response current. Therefore, in order to achieve the best detection effect of the modified electrode, 8 cycles are selected as the optimal number of PDA electropolymerization cycles.

[0061] After preparing the modified electrode, prepare electrolyte solutions with different pH values (pH = 6.8, 7.0, 7.2, 7.4, 7.6, 7.8) (0.1 M PBS solution containing 2.0 mM [Fe(CN 6) 3- / 4- ), and use the CV method to determine the magnitude of their respective response currents and obtain the optimal pH value of the detection system.

[0062] As Figure 3 shown in D, when the pH is between 6.8 and 7.0, the peak current increases with the increase of pH value and reaches the maximum at pH = 7.0. When the pH is between 7.0 and 7.8, the peak current decreases with the increase of pH value. It can be seen that the immunosensor performs best when the pH of the detection system is 7.0, which may be because some groups in the antibody lose their activity under weak acid or weak base conditions. In addition, this indicates that the pH value of the human physiological system, that is, pH neutral, is most conducive to the immune reaction. Therefore, an electrolyte solution with a pH of 7.0 is used during the preparation process.

[0063] The experiment also studied the influence of antibody incubation time on the detection ability of the immunosensor. Drop 10.0 μL of HB-β antibody (HB-β-Ab) on PDA-PoPD-MWCNTs / GCE and incubate for 30 min, 60 min, 90 min, 120 min, 150 min respectively. Then rinse the electrode with Tris-HCl with a pH of 7.4 to remove physically adsorbed HB-β-Ab, and use the CV method to detect the current response of HB-β-Ab / PDA-PoPD-MWCNTs / GCE and obtain the optimal incubation time.

[0064] As Figure 3 shown in E, with the increase of antibody incubation time, the oxidation peak current (Ip) gradually decreases, and Ip remains basically unchanged after 60 min, indicating that the number of antibodies immobilized on the electrode surface is close to saturation. Therefore, 60 min is selected as the optimal antibody incubation time for the immunosensor.​

[0065] Finally, HB-β (50 μg / mL) was incubated on HB-β-Ab / PDA-PoPD-MWCNTs / GCE for 5 min, 20 min, 40 min, 60 min, 80 min and 100 min, and its response current was measured by CV method to obtain the optimal incubation time.

[0066] As Figure 3 shown in F, after the incubation time reached 80 min, the change of Ip tended to be stable, with only a slight decrease. Considering the time cost of the experiment comprehensively, 80 min was selected as the optimal incubation time of HB-β.

[0067] Under the optimized experimental conditions, gradient concentration samples were detected. As Figure 4 shown, in the concentration range of 1.0 - 4.0×10 5 ng mL -1 , there was a good linear relationship between the concentration of HB-β and the current response ( Figure 4 shown in C), and the linear equation was ΔI = 9.91928 lgCHB-β + 8.26964 (R 2 = 0.99225), and the detection limit was 0.3002 ng mL -1 (S / N = 3).

[0068] Example 4:

[0069] This example provides a method and results for performance analysis of the electrochemical immunosensor as described in Example 1.

[0070] DPV method was used to analyze the performance of the electrochemical immunosensor. The scanning range was set to -0.2 - 0.3 V. Different concentrations of HB-β (1.0 - 4.0×105 ng mL -1 ) solutions were prepared. 5.0 μL of HB-β solutions with different concentrations were dropped on the surface of the working electrode and incubated at room temperature for 80 min, and the specificity, reproducibility and stability were detected in the three-electrode system. The results are shown in Figure 5 .

[0071] The specificity, reproducibility and stability of HB-β were also detected by DPV method. The method includes: The detection method of reproducibility includes: 5 immunosensors prepared simultaneously were used for DPV detection of HB-β with the same concentration (10 μg mL -1 ) under the same conditions.

[0072] As Figure 5As shown in A, the DPV peak current values of 5 electrodes were recorded after incubating with HB-β, and the calculated relative standard deviation RSD = 0.81%, indicating that the immunosensor has good reproducibility.

[0073] The detection method for stability includes: placing the prepared immunosensor in a refrigerator at 4 °C, and testing the DPV signals of the same HB-β immunosensor at 0 d, 5 d, 10 d, 15 d, and 20 d respectively. The test is carried out in the same test bottom solution (0.1 M PBS solution containing 2.0 mM [Fe(CN6)] 3- / 4- , pH = 7.0).

[0074] As Figure 5 shown in B, after 5 days of stability testing, the response current value is 98.75% of the initial value; after 30 days, the response current value is 96.80% of the initial value. The experimental results prove that the sensor has good stability.

[0075] The detection method for specificity includes: using the immunosensor to detect interferents such as GLU (200 μg mL -1 ), MET (200 μgmL -1 ), VC (200 μg mL -1 ), and BSA (200 μg mL -1 ). Incubate the working electrode with a mixed solution of 50 μg mL -1 HB-β containing GLU, MET, VC, and CRP respectively, and conduct DPV testing.

[0076] As Figure 5 shown in C, the concentrations of the interferents set in this example are much higher than that of HB-β (50 μg mL -1 ). When the electrochemical immunosensor is incubated with a mixed solution of 50 μg mL -1 HB-β containing GLU, MET, VC, and CRP respectively, the response current has no obvious difference from the current detected by incubating only with HB-β. The results prove that the immunosensor has good specificity.

[0077] Example Five:

[0078] This example provides the practical application method and effect of the electrochemical immunosensor as described in Example One.

[0079] The standard addition method was used to determine HB-β in actual serum samples. Blood from healthy adults (ethical approval obtained) was collected as the actual sample for HB-β detection, and the samples were placed in EP tubes without anticoagulants. The samples were left at room temperature for 30 - 60 min to allow blood coagulation, and then the samples were centrifuged at 1000×g for 15 min. The upper layer of liquid was collected. Different concentrations of HB-β (6.25, 12.50, 25.00 μg / mL) were added to the supernatant. Finally, the filtrate containing different concentrations of HB-β was dropped onto the surface of the HB-β-Ab / PDA-PoPD-MWCNTs / GCE working electrode and incubated at room temperature for 80 min, and detection was carried out using DPV in a three-electrode system.

[0080] Meanwhile, under the same experimental conditions, ELISA was used to detect serum samples with different concentrations of HB-β. A total of 3 groups of samples (spiked with 6.25, 12.50, 25.00 μg / mL) were detected. The method included: adding the samples and HB-β standard products with different concentrations (adding standard product diluent to the 0 μg / mL well) to the corresponding wells of the enzyme-linked immunosorbent assay (ELISA) plate (100 μL / well) respectively, sealing the reaction wells with sealing tape, and incubating in an incubator at 37°C for 90 minutes; after washing the plate 2 times, adding the working solution of biotinylated HB-β antibody (100 μL / well) and incubating in an incubator at 37°C for 60 minutes; after washing the plate 3 times, except for the blank well, adding the working solution of enzyme conjugate (100 μL / well). Incubate in an incubator at 37°C in the dark for 30 minutes; after washing the plate 5 times, add the TMB chromogenic working solution (including the blank well) 100 μL / well, incubate in an incubator at 37°C in the dark, and finally add the stop solution (including the blank well) 100 μL / well. After mixing, the OD (450 nm) value can be measured. Using the concentration of the HB-β standard product as the abscissa and the measured OD value as the ordinate, a standard curve was plotted, the concentration of HB-β in the sample was calculated and the data was recorded. Each group of samples was subjected to 3 parallel experiments.

[0081] As shown in Table 1, the serum samples of the same sample were detected by the electrochemical sensor and ELISA respectively. The experimental results showed that the recovery rate of this method for HB-β was between 100.84% and 102.16%, and the relative standard deviation (RSD) of repeated determination of each sample three times was between 0.92% and 1.23%. Using the ELISA method to determine the content of HB-β in serum, the results were consistent. The RSD of its three independent experiments was between 1.11% and 1.75%, and the recovery rate varied within the range of 98.40 - 105.12%. The results showed that the sensor had high sensitivity and could realize the real-time detection of HB-β in actual samples.

[0082] Table 1 Comparison of detection results of electrochemical immunosensor in actual serum samples and Elisa kit

[0083] Among them, a is the average value of three measurements.

[0084] The above are only the preferred embodiments of the present application. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present application, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present application.

Claims

1. A preparation method of an electrochemical immunosensor, characterized in that Comprising: Dissolve MWCNTs in N,N-dimethylformamide and ultrasonicate to obtain a uniformly dispersed MWCNTs suspension; Drop the MWCNTs suspension onto the surface of a pretreated glassy carbon electrode and dry to obtain a MWCNTs / GCE electrode; Place the MWCNTs / GCE electrode in an acetic acid buffer solution of o-phenylenediamine and perform cyclic voltammetry scanning. After taking it out and washing, place it in an H2SO4 solution and perform cyclic voltammetry scanning again. After taking it out and washing, obtain a PoPD-MWCNTs / GCE electrode; Place the PoPD-MWCNTs / GCE electrode in a dopamine solution and perform cyclic voltammetry scanning. After taking it out and washing, place it in an H2SO4 solution and perform cyclic voltammetry scanning again. After taking it out and washing, obtain a PDA-PoPD-MWCNTs / GCE electrode; Drop the antibody solution onto the PDA-PoPD-MWCNTs / GCE electrode, incubate, rinse, and air-dry to obtain the product.

2. The preparation method of the electrochemical immunosensor according to claim 1, characterized in that, The pretreatment method of the glassy carbon electrode includes: polishing the glassy carbon electrode on suede sprinkled with 0.3 μm and 0.05 μm alumina polishing powder for 5 - 10 min, ultrasonically cleaning with methanol and water for 10 - 15 min, and drying.

3. The preparation method of the electrochemical immunosensor according to claim 1, characterized in that, The cyclic voltammetry scanning includes: Place the MWCNTs / GCE electrode in an acetic acid buffer solution of 0.2 - 0.3 mg / mL o-phenylenediamine with pH = 4.5 - 5 and perform cyclic voltammetry scanning for 8 - 10 cycles in the potential range of -0.2 - 1.0 V; then place the electrode in a 0.5 - 1 mol / L H2SO4 solution and perform cyclic voltammetry scanning for 30 - 40 cycles in the potential range of -0.2 - 0.8 V; Place the PoPD-MWCNTs / GCE electrode in a 0.3 - 0.4 mg / mL dopamine solution and perform cyclic voltammetry scanning for 8 - 10 cycles in the potential range of -0.2 - 1.0 V; then place the electrode in a 0.5 - 1 mol / L H2SO4 solution and perform cyclic voltammetry scanning for 30 - 40 cycles in the potential range of -0.2 - 0.8 V.

4. The preparation method of the electrochemical immunosensor according to claim 1 or 3, characterized in that, The scanning speed of the cyclic voltammetry is 100 mV / s.

5. The preparation method of the electrochemical immunosensor according to claim 1, characterized in that, The method for incubating the antibody solution includes: dropping the antibody solution onto the PDA-PoPD-MWCNTs / GCE electrode, incubating at 4 - 5 °C for 1 - 1.5 hours, taking it out and washing with a Tris-HCl buffer solution with pH = 7.4 - 7.5, placing it in a blocking solution for 50 - 60 min, and then taking it out and rinsing with a PBS solution and a Tris-HCl solution with pH = 7.2 - 7.

3.

6. The preparation method of the electrochemical immunosensor according to claim 1, characterized in that, The blocking solution is 0.1 - 0.2 mol L-1 PBS containing 3% - 5% BSA, pH = 7.4 - 7.

5.

7. An electrochemical immunosensor, characterized in that, Prepared by the method described in any one of claims 1 - 7.

8. Use of an electrochemical immunosensor as described in claim 7 in cardiac toxicity detection, characterized in that, The biomarker of cardiac toxicity is HB-β, the antibody solution incubated on the PDA-PoPD-MWCNTs / GCE electrode is an HB-β antibody solution, and an HB-β-Ab / PDA-PoPD-MWCNTs electrode is obtained after incubating the antibody solution; The application method includes: Taking a blood sample, centrifuging after coagulation, and collecting the supernatant; Dropping the supernatant onto the surface of the HB-β-Ab / PDA-PoPD-MWCNTs electrode and incubating; Combining a reference electrode and an auxiliary electrode in a three-electrode system, using differential pulse voltammetry to measure the DPV peak current of different concentrations of HB-β standard products, fitting a linear regression equation, and calculating the concentration of HB-β in the sample according to the DPV peak current of the sample.

9. Use of the electrochemical immunosensor according to claim 8 in cardiac toxicity detection, characterized in that, When measuring by the differential pulse voltammetry, the scanning potential range is -0.2~0.3 V.

10. Use of the electrochemical immunosensor according to claim 8 in cardiac toxicity detection, characterized in that, The incubation conditions of the supernatant are incubation at room temperature for 80~90 min.

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