Mitochondrial fusion protein 2 electrochemical immunosensor and application thereof in cardiotoxicity detection

Abstract

The invention discloses a mitochondrial fusion protein 2 electrochemical immunosensor and application thereof in cardiotoxicity detection, and a preparation method of the mitochondrial fusion protein 2 electrochemical immunosensor comprises the following steps: performing ultrasonic dispersion on a composite material M-NTO-MWCNT by using a solvent to obtain an M-NTO-MWCNT dispersion liquid; the pretreated GCE electrode is modified with the M-NTO-MWCNT dispersion liquid, and an M-NTO-MWCNT coated GCE electrode is obtained after air drying; a mitochondrial fusion protein 2 antibody solution is dropwise added to the surface of the M-NTO-MWCNT (at) GCE electrode, incubation is carried out, and an Mfn2-Ab (at) M-NTO-MWCNT (at) GCE electrode is obtained; and placing in a confining liquid to seal active sites which are not loaded with the mitochondrial fusion protein 2 antibody, thereby obtaining the mitochondrial fusion protein 2 antibody. The electrochemical immunosensor is wide in monitoring range, low in detection limit and high in sensitivity.

Description

Technical Field

[0001] The present application relates to the technical field of drug development and clinical drug safety monitoring, and in particular to a mitochondrial fusion protein 2 electrochemical immunosensor and its application in cardiac toxicity detection. Background Art

[0002] Drug-induced cardiotoxicity (DIC) is a significant challenge faced during new drug development and clinical use. Characterized by multiple sources, high prevalence, insidiousness, susceptibility to specific populations, and diverse clinical manifestations, it has become a critical issue urgently requiring resolution in the field of drug safety. To date, evaluation techniques such as electrocardiograms, echocardiography, biomarkers, cardiac magnetic resonance imaging, and endocardial biopsy, as well as animal experiments and alternative technologies, have provided important technical support for DIC assessment. However, these techniques still present challenges such as radiation risks, demanding operator requirements, time-consuming procedures, and difficulties in portability and widespread adoption.

[0003] Mitochondrial fusion protein 2 (Mfn2), a transmembrane motor protein located in the outer mitochondrial membrane, is primarily involved in regulating mitochondrial fusion, endoplasmic reticulum stress, mitophagy, cellular energy metabolism, and apoptosis. Existing studies have shown that Mfn2 may serve as a potential biomarker for early warning assessment of drug-induced cardiotoxicity.

[0004] Currently, methods for Mfn2 detection primarily include enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and immunofluorescence. However, these methods require expensive equipment and experienced laboratory technicians, are cumbersome and time-consuming, and are difficult to popularize. Consequently, they are unable to provide ultra-early, highly sensitive early warning of cardiotoxicity. Therefore, developing new technologies for highly sensitive and rapid Mfn2 detection is crucial for the prevention and treatment of drug-induced cardiotoxicity. Summary of the Invention

[0005] Objective: To overcome the deficiencies in the prior art, the present invention provides a mitochondrial fusion protein 2 electrochemical immunosensor and its application in the detection of cardiotoxicity. The electrochemical immunosensor is simple to operate and has sensitive detection. After incubating mitochondrial fusion protein 2 antibodies on the electrode surface, it can achieve specific detection of mitochondrial fusion protein 2, a biomarker of drug-induced cardiotoxicity.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a method for preparing a mitochondrial fusion protein 2 electrochemical immunosensor, comprising: A composite material M-NTO-MWCNT composed of sodium titanate nanorods and multi-walled carbon nanotubes was prepared by oxidative alkalization of MXene, and an M-NTO-MWCNT dispersion was obtained after ultrasonic dispersion in a solvent. The M-NTO-MWCNT dispersion droplets were applied on the pretreated GCE electrode and dried to obtain the M-NTO-MWCNT@GCE electrode; The mitochondrial fusion protein 2 antibody solution was dropped onto the surface of the M-NTO-MWCNT@GCE electrode and incubated to obtain the Mfn2-Ab@M-NTO-MWCNT@GCE electrode; The active sites of the unloaded mitochondrial fusion protein 2 antibody are blocked in a blocking solution to obtain the product.

[0008] Based on electrochemical immunosensing technology, this invention uses a composite material (M-NTO-MWCNT) composed of sodium titanate nanorods and multi-walled carbon nanotubes (MWCNTs) prepared by oxidation and alkalization of MXene as the sensing substrate. This new electrochemical immunosensor based on the highly sensitive detection of Mfn2 is used for early warning and assessment of drug-induced cardiotoxicity. M-NTO, with its unique nanorod structure, abundant active sites, and high specific surface area, significantly enhances sensor sensitivity and provides a large number of antibody attachment sites. MWCNTs, with their outstanding conductivity and network structure, further improve electron transfer efficiency and enhance sensor selectivity.

[0009] In some embodiments, the GCE electrode pretreatment method includes: polishing the GCE electrode on suede sprinkled with 0.3 μm and 0.05 μm aluminum oxide polishing powder for 5-10 minutes, ultrasonically cleaning with methanol and water for 10-15 minutes, and drying.

[0010] In some embodiments, the solvent in the M-NTO-MWCNT dispersion is deionized water, and the concentration of M-NTO-MWCNT in the M-NTO-MWCNT dispersion is 2-3 mg / mL.

[0011] In some embodiments, the incubation condition of the mitofusin 2 antibody solution is 20-25° C. for 4-5 h.

[0012] In some embodiments, the method further comprises: adding a mitochondrial fusion protein 2 antibody solution dropwise onto the surface of the M-NTO-MWCNT@GCE electrode, and rinsing with PBST after incubation; PBST is a phosphate buffer containing 0.05-0.10 v / v% Tween-80, pH=7.0-7.2.

[0013] In some embodiments, the blocking solution is PBST containing 0.05-0.10 v / v% Tween-80 and 2-3 wt% BSA, pH=7.0-7.2.

[0014] In some embodiments, the method for blocking the active site of the unloaded mitochondrial fusion protein 2 antibody comprises: placing the Mfn2-Ab@M-NTO-MWCNT@GCE electrode in a blocking solution at 20-25° C. for 50-60 minutes, removing it and washing it to obtain a BSA@Mfn2-Ab@M-NTO-MWCNT@GCE electrode.

[0015] In a second aspect, the present invention provides a mitochondrial fusion protein 2 electrochemical immunosensor, which is prepared by the method described in the first aspect.

[0016] In a third aspect, the present invention provides use of the mitochondrial fusion protein 2 electrochemical immunosensor as described in the first aspect in the detection of drug-induced cardiotoxicity.

[0017] Mitochondrial fusion protein 2 is a biomarker for drug-induced cardiotoxicity, and the application method includes: Blood samples were collected and centrifuged, and the supernatant was collected. Mfn2-Ag solutions with different standard concentrations were added to the supernatant; The mitochondrial fusion protein 2 electrochemical immunosensor was used as the working electrode, combined with a reference electrode and an auxiliary electrode in a three-electrode system, and the supernatant of the blood sample added with different standard concentrations of Mfn2-Ag solution was dripped on the surface of the working electrode, and differential pulse voltammetry scanning was performed to record the DPV response value; A linear relationship between the DPV response value and the logarithm of the Mfn2 concentration was established, and a linear regression equation was obtained; The sample solution to be tested is dropped onto the surface of the working electrode, differential pulse voltammetry scanning is performed, and the DPV response value is recorded. The concentration of Mfn2 in the sample is calculated based on the DPV response value of the sample combined with the linear regression equation.

[0018] Beneficial effects: The mitochondrial fusion protein 2 electrochemical immunosensor provided by the present invention uses M-NTO-MWCNT composite material as the sensing substrate, and constructs an electrochemical immunosensor based on high-sensitive trace detection of Mfn2 to provide a technology for early warning and evaluation of drug-induced cardiotoxicity, which can achieve specific detection of mitochondrial fusion protein 2 (Mfn2), a potential biomarker of drug-induced cardiotoxicity.

[0019] In this sensor, M-NTO, leveraging its unique nanorod structure, abundant active sites, and high specific surface area, significantly enhances sensor sensitivity and provides a large number of antibody immobilization sites. MWCNT, leveraging its outstanding conductivity and network structure, further improves electron transfer efficiency and enhances sensor selectivity. After incubation with mitochondrial fusion protein 2 antibodies, this electrochemical immunosensor combines specific immune responses with electrochemical sensing technology. Through the specific recognition and binding of antigen and antibody, it correlates antigen concentration with electrical signal intensity, enabling the detection of mitochondrial fusion protein 2, a biomarker for drug-induced cardiotoxicity.

[0020] The mitochondrial fusion protein 2 electrochemical immunosensor provided by the present invention can achieve rapid on-site testing without the need for large-scale sample preparation or complex equipment. It has the advantages of simple operation, short detection time and good stability. It not only provides a new rapid and portable detection solution for early warning of drug-induced cardiotoxicity, but also provides technical support and scientific reference for the safety assessment of new drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the scanning electron microscope, Raman spectroscopy and infrared spectroscopy characterization results of MWCNT, MXene, M-NTO and M-NTO-MWCNT in the embodiment of the present invention. Figure 1 A in the middle is a scanning electron microscope image of MWCNT; Figure 1 B is a scanning electron microscope image of MXene; Figure 1 C in the middle is a scanning electron microscope image of M-NTO; Figure 1 D in the middle is the scanning electron microscope image of M-NTO-MWCNT; Figure 1 Figure E in the middle is the Raman spectrum image of MWCNT, MXene and M-NTO-MWCNT; Figure 1 (F) is the infrared spectrum image of MWCNT, MXene and M-NTO-MWCNT.

[0022] Figure 2 Schematic diagram of electrochemical characterization results of cyclic voltammetry, electrochemical impedance spectroscopy and chronocoulometry of GCE electrode, M-NTO@GCE electrode, MWCNT@GCE electrode, M-NTO-MWCNT@GCE (CS) electrode and M-NTO-MWCNT@GCE (DI) electrode in the embodiment of the present invention. Figure 2 Figure A is the cyclic voltammetry characterization results of GCE electrode, M-NTO@GCE electrode, MWCNT@GCE electrode, M-NTO-MWCNT@GCE (CS) electrode and M-NTO-MWCNT@GCE (DI) electrode; Figure 2Figure B shows the electrochemical impedance spectroscopy of the GCE electrode, M-NTO@GCE electrode, MWCNT@GCE electrode, M-NTO-MWCNT@GCE (CS) electrode, and M-NTO-MWCNT@GCE (DI) electrode. Figure 2 Middle C is the chrono-coulometry plots of GCE electrode, M-NTO@GCE electrode, M-NTO-MWCNT@GCE (DI) electrode, MWCNT@GCE electrode, and M-NTO-MWCNT@GCE (CS) electrode; Figure 2 Figure D is the Qt 1 / 2 curve of GCE electrode, M-NTO@GCE electrode, M-NTO-MWCNT@GCE (DI) electrode, MWCNT@GCE electrode and M-NTO-MWCNT@GCE (CS) electrode.

[0023] Figure 3 This is a schematic diagram showing the effects of different experimental conditions on the preparation of the immunosensor according to the embodiment of the present invention. Figure 3 A in the middle is the CV curve of M-NTO-MWCNT@GCE electrode at different scan rates; Figure 3 B in the figure is the linear relationship between the logarithm of the redox peak current of the M-NTO-MWCNT@GCE electrode and the logarithm of the scan rate; Figure 3 C in the figure is the logarithmic linear relationship between the redox peak potential of the M-NTO-MWCNT@GCE electrode and the scan rate.

[0024] Figure 4 Schematic diagram of the analytical performance results of ELISA and mitochondrial fusion protein 2 electrochemical immunosensor for detecting Mfn2 in an embodiment of the present invention. In FIG4 , A is the absorbance value corresponding to different concentrations of Mfn2 analyzed by the ELISA method; Figure 4 Middle B is the DPV response result of mitochondrial fusion protein 2 immunosensor to different concentrations of Mfn2; Figure 4 Middle C is a graph showing the relationship between the different concentrations of Mfn2 detected by the mitochondrial fusion protein 2 immunosensor and the corresponding DPV response values; Figure 4 Middle D is the linear relationship between the DPV response value of the mitochondrial fusion protein 2 immunosensor and the Mfn2 concentration.

[0025] Figure 5 Schematic diagram of the reproducibility, stability and selectivity test results of mitochondrial fusion protein 2 in the embodiment of the present invention. Figure 5 Middle A is a broken line diagram of the reproducible detection results of mitochondrial fusion protein 2 electrochemical immunosensor; Figure 5 Middle B is a bar graph showing the reproducible detection results of mitochondrial fusion protein 2 electrochemical immunosensor; Figure 5Middle C is a schematic diagram of the stability test results of the mitochondrial fusion protein 2 electrochemical immunosensor; Figure 5 Middle D is a bar graph showing the stability test results of the mitochondrial fusion protein 2 electrochemical immunosensor; Figure 5 Middle E is a broken line diagram of the selective detection results of mitochondrial fusion protein 2 electrochemical immunosensor; Figure 5 Middle F is a bar graph showing the selective detection results of the mitochondrial fusion protein 2 electrochemical immunosensor. DETAILED DESCRIPTION

[0026] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention, its application, or use.

[0027] Unless otherwise specifically stated, the relative arrangement of the parts and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention. At the same time, it should be understood that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to actual proportional relationships. The techniques, methods and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the techniques, methods and equipment should be considered as part of the authorization specification. In all examples shown and discussed here, any specific values ​​should be interpreted as being merely exemplary, not as limitations. Therefore, other examples of the exemplary embodiments may also include different values. It should be noted that similar numbers and letters represent similar items in the following figures, and therefore, once an item is defined in one figure, it does not need to be further discussed in subsequent figures.

[0028] The present invention will be further described below with reference to the examples.

[0029] In the following examples, the experimental materials used were: multi-walled carbon nanotubes (MWCNTs, ≥95% deacetylation, San Chemical Technology (Shanghai) Co., Ltd.); MXene (Ti3C2, Suzhou Beike Nanotechnology Co., Ltd.); HCl (Henan Dongke Chemical Products Sales Co., Ltd.); NaOH (Tianjin Baishi Chemical Co., Ltd.); H2O2 (Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.); potassium ferrocyanide (K3[Fe(CN)6], Yantai Shuangshuang Chemical Co., Ltd.); potassium ferrocyanide (K4[Fe(CN)6]·3H2O, Tianjin Baishi Chemical Co., Ltd.); KCl (Tianjin Guangfu Technology Development Co., Ltd.); glucose (Tianjin Damao Chemical Reagent Factory); glycine (Shanghai MacLean Biochemical Technology Co., Ltd.); L-methionine (Shanghai Zhongqin Chemical Reagent Co., Ltd.); proline (Shanghai MacLean Biochemical Technology Co., Ltd.); bovine serum albumin (BSA, Solarbio); ELISA kit (Shanghai ELISA Biotechnology Co., Ltd.); biotinylated Mfn2 antibody (Shanghai ELISA Biotechnology Co., Ltd.); Mfn2 standard (Shanghai ELISA Biotechnology Co., Ltd.); Mfn2-Ag (Shanghai ELISA Biotechnology Co., Ltd.).

[0030] In the following examples, the experimental instruments used were: FA2204B electronic analytical balance, HG4-21K desktop high-speed centrifuge, DHG-9030 electric blast drying oven (Huapu Biotechnology Co., Ltd.); 100 mL hydrothermal synthesis reactor (Haoxing Biotechnology Co., Ltd.); P916 desktop push-button pH meter (Shanghai Youke Instrument Co., Ltd.); CJJ78-1 magnetic stirrer (Changzhou Jintan Dadi Automation Instrument Factory); SB-5200DTD ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.); JSM-6701F cold field emission scanning electron microscope (Japan Electron Optics Co., Ltd.); LabRAM HR Evolution microconfocal Raman spectrometer (HORIBA Jobin Yvon SAA, France); IFS66v / S infrared spectrometer (Llantrisant, UK); SpectraMax M3 microplate reader (MeiGu Molecular Instrument (Shanghai) Co., Ltd.); and CHI 660E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.). All electrochemical experiments were performed at 25 °C using a conventional three-electrode system, which included a glassy carbon electrode or modified glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode, a saturated calomel electrode (type 232), and a platinum wire electrode (≥99.99%) as the reference electrode and auxiliary electrode.

[0031] Example 1:

[0032] This embodiment provides a method for preparing a mitochondrial fusion protein 2 electrochemical immunosensor, comprising: 1. Preparation of M-NTO-MWCNT nanocomposites First, M-NTO composite powder was prepared. Ti3AlC2 was then liquid-phase exfoliated using a mixture of LiF and HCl. The mixture in situ generated HF, which selectively etched the Al layer of the original MAX phase to produce MXene.

[0033] Specifically, 100 mg of MXene powder was dispersed in 5 mL of deionized water and sonicated at 300 W for 20 minutes to form a homogeneous suspension. 0.5 mL of glacial acetic acid was then added to adjust the pH to 6-7. 0.3 mL of 2 M NaOH solution was then added dropwise to raise the pH to 10, and the mixture was magnetically stirred for 15 minutes to dissolve. 0.34 mL of 30% H₂O₂ solution (corresponding to an initial molar ratio of NaOH:H₂O₂ ≈ 1:1) was then slowly added. The mixture was transferred to a 10 mL polytetrafluoroethylene-lined autoclave and hydrothermally reacted at 140°C for 12 hours. After cooling, the mixture was neutralized with 1 mol / L hydrochloric acid until neutral, centrifuged at 5000 rpm for 10 minutes, and the precipitate was collected. The precipitate was washed three times with deionized water and ethanol, followed by drying under vacuum at 60°C for 8 hours to obtain the M-NTO composite powder.

[0034] Next, prepare the M-NTO-CNT material. 50 mg of the M-NTO composite powder was dispersed in 50 mL of deionized water to form a 1 mg / mL M-NTO dispersion. The dispersion was then ultrasonicated in an ice bath for 30 minutes at a power of 150 W.

[0035] MWCNTs were added to a three-necked flask, followed by a 3:1 volume ratio of concentrated H₂SO₄ and concentrated HNO₃ solution. The mixture was ultrasonically dispersed for 30 minutes, then refluxed under condensation and reacted with magnetic stirring at 60°C for 3 hours. The acid-treated MWCNTs were poured into distilled water and washed repeatedly until the filtrate was colorless and transparent with a neutral pH. The acid-treated MWCNTs were filtered through filter paper and dried in a vacuum oven at 60°C for 12 hours. The acid-treated MWCNTs were then ground using an agate mortar to obtain acidified MWCNT powder.

[0036] Disperse 50 mg of acidified MWCNT powder in 50 mL of deionized water, add 2 mg of sodium dodecyl sulfate (SDS), and sonicate for 30 minutes. The total volume of the mixture was adjusted to 100 mL and magnetically stirred at 500 rpm for 2 hours. Ultrasonicate in an ice bath for 10 minutes at 100 W to avoid local overheating. Dry in a vacuum oven at 60°C for 12 hours to obtain the M-NTO-MWCNT composite powder.

[0037] 2. Preparation of modified electrodes (1) Pretreatment of glassy carbon electrode (GCE): The GCE was polished on suede sprinkled with 0.3 μm and 0.05 μm aluminum oxide polishing powder for 5 min, respectively. The electrode was then ultrasonically cleaned with methanol and water for 10 min, respectively, and dried in air.

[0038] (2) Preparation of modified electrodes: 20 mg of M-NTO-MWCNT powder was dispersed in 10 mL of deionized water and ultrasonicated for 10 min to obtain 2 mg mL -1 20 μL of M-NTO-MWCNT dispersion was applied to the surface of the GCE working electrode and allowed to dry naturally to prepare the M-NTO-MWCNT@GCE electrode.

[0039] 3. Preparation of electrochemical immunosensor 40 μL of 100× diluted biotinylated Mfn2 antibody (Mfn2-Ab) was added dropwise to the surface of the M-NTO-MWCNT@GCE working electrode and incubated at 20°C for 4 h. -1 The electrode was washed with PBST (phosphate buffer containing 0.05 v / v% Tween-80, pH = 7.0) to remove the physically adsorbed Mfn2-Ab, thus obtaining the Mfn2-Ab@M-NTO-MWCNT@GCE electrode. The electrode was placed in a blocking solution (0.1 mol L containing 0.05 v / v% Tween-80 and 2% BSA) at 20 °C. -1 The active sites were blocked by incubating in PBST (pH = 7.0) for 50 min to obtain the BSA@Mfn2-Ab@M-NTO-MWCNT@GCE electrode. This electrochemical immunosensor for Mfn2 detection was constructed and stored at 4 °C for future use.

[0040] After constructing the BSA@Mfn2-Ab@M-NTO-MWCNT@GCE electrode in this example, it was characterized by Raman spectroscopy, infrared spectroscopy, and SEM electron microscopy. Figure 1 As shown, Figure 1 As can be seen in Figure A, the multi-walled carbon nanotubes have a uniform nanotube structure and agglomerate when dispersed in deionized water; Figure 1 As can be seen in Figure B, MXene (Ti3C2) usually presents an accordion-like layer stacking. Due to air oxidation, the material has layer fragmentation, holes or surface loosening; Figure 1 Middle C is a scanning electron micrograph of M-NTO, which was synthesized by a hydrothermal method and exhibits a sea urchin-like morphology with an open macroporous structure and a large specific surface area. Figure 1Figure D is the microscopic morphology of the composite material M-NTO-MWCNT. It can be seen that MWCNT can be evenly dispersed in the macroporous structure of M-NTO, which proves that M-NTO can be used to load and disperse MWCNT. At the same time, the pores of the composite are large and present a fiber network structure, which proves the successful preparation of the M-NTO-MWCNT composite material.

[0041] Figure 1 E in the figure is the Raman spectrum image of MWCNT, MXene and M-NTO-MWCNT. It can be seen from the figure that MXene, MWCNT and M-NTO-MWCNT electrode materials are all at 1350 cm -1 、1580 cm -1 Characteristic peaks appear on the left and right, corresponding to the D and G characteristic peaks of graphene-like and carbon nanotube materials. The D band originates from sp 3 A1g vibration of hybrid carbon or defects, G band corresponds to sp 2 The ratio of the D peak intensity to the G peak intensity of the E2g in-plane stretching vibration of hybrid carbon reflects the degree of defects. A high ratio (>0.2) indicates structural disorder or oxidation damage. As can be seen from the figure, the defects of MWCNT and MXene are very small, indicating excellent material properties. 285 cm -1 and 375 cm -1 The characteristic peaks at are attributed to the characteristic metal-carbon / nitrogen stretching vibration bands in MXene-based materials. Raman spectroscopy reveals that M-NTO-MWCNT possesses distinct bands from both MWCNT and MXene. The increased intensity of the MWCNT RBM peak indicates that its role as a conductive backbone effectively enhances the composite's electron transport efficiency. The broadening of the MXene interlayer vibration peak reflects that the MWCNT mitigates the volume expansion of the M-NTO. This spectrum also demonstrates the successful preparation of the M-NTO-MWCNT composite.

[0042] Figure 1 F in the figure is the infrared spectrum image of MWCNT, MXene and M-NTO-MWCNT. It can be seen from the figure that at 3400 cm -1 The OH stretching vibration peak appears near 2900 cm, which is due to the presence of adsorbed water molecules or surface hydroxyl groups. -1 The CH stretching vibration peak appeared near the 1600 cm -1 The C=C stretching vibration peak appears near the carbon nanotube (MWCNT) 2 Hybridized carbon skeleton. In addition, at 1000 cm -1The CO stretching vibration peak appears in the region, which is related to the oxygen-containing functional groups on the surface of carbon nanotubes. The spectrum shows that the infrared spectrum of the M-NTO-MWCNT composite is a superposition of the characteristic peaks of M-NTO and MWCNT, indicating effective interfacial bonding between M-NTO and MWCNT, proving the successful preparation of the composite.

[0043] Example 2:

[0044] This example provides an electrochemical characterization experiment of the mitochondrial fusion protein 2 electrochemical immunosensor as described in Example 1.

[0045] 1. Electrochemical characterization of basic electrodes Electrochemical characterization was carried out on an electrochemical workstation using a three-electrode system with a platinum wire electrode as the counter electrode and a saturated calomel electrode as the reference electrode. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronocoulometry (CC) were used. A 4.0 mM K4[Fe(CN)]6 / K3[Fe(CN)]6 solution containing 0.01 M KCl was used as the electrolyte for electrode characterization.

[0046] For cyclic voltammetry, the scanning potential range was -0.4 to 0.8 V, and the scan rate was 60 mV s -1 .

[0047] For electrochemical impedance spectroscopy, set the frequency range to 1 Hz ~ 10 6 Hz, the amplitude of the sine wave signal is 5mV, and the voltage is 0.22V.

[0048] The electroactive surface areas of five electrodes, namely GCE, M-NTO@GCE, MWCNT@GCE, M-NTO-MWCNT@GCE (DI), and M-NTO-MWCNT@GCE (CS), were investigated by chronocoulometry (CC) in a 1.00 mol KCl electrolyte containing 5.00 mmol K₃[Fe(CN)₆]. The CC parameters were set as follows: initial potential = 0.08 V, end potential = 0 V, pulse width = 0.25 s, and sampling interval = 2.5 × 10 −4 s.

[0049] For the chronocoulometry, the GCE electrode, M-NTO@GCE electrode, M-NTO-MWCNT@GCE (DI) electrode, MWCNT@GCE electrode, and M-NTO-MWCNT@GCE (CS) electrode were tested in a probe solution of 1.0 M KCl containing 5.0 mM K3[Fe(CN)6] at room temperature of 20±2°C in the potential range of 0 ~ 0.08 V. The data were saved, and the electroactive surface area was calculated by Qt curve analysis (fitting the Anson equation).

[0050] 2. Cyclic voltammetry characterization of electrochemical immunosensor The CV method was used in the voltage range of -0.4 ~ 0.8 V with a frequency of 100 mVs -1 The BSA@Mfn2-Ab@M-NTO-MWCNT@GCE electrochemical immunosensor was characterized by CV in 5 mmol / L K4[Fe(CN)]6 / K3[Fe(CN)]6 electrolyte containing 0.01 mmol / L KCl at a scan rate of .

[0051] like Figure 2 As shown in Figure A, the bare GCE (curve a) has almost no conductivity. After the electrode was modified with M-NTO (curve b), the current response was not significant because M-NTO is not electrically active. However, the current response signal of MWCNT@GCE (curve d) increased significantly, and the peak current increased. The current response of M-NTO-MWCNT@GCE (DI) (curve c) was significantly reduced compared with MWCNT@GCE (curve d), which may be due to the poor dispersion of M-NTO-MWCNT in deionized water (DI). Consider using 0.01 M-NTO-MWCNT@GCE (CS) was used as a dispersant; the current response of M-NTO-MWCNT@GCE (CS) (curve e) was significantly improved compared with MWCNT@GCE (curve d) and M-NTO-MWCNT@GCE (DI) (curve c), and the peak current was significantly increased. This is because the dispersion of MWCNT in chitosan (CS) after combining with M-NTO with an open macroporous structure is better than that in deionized water (DI), and the aggregation of MWCNT is greatly reduced. The synthesized M-NTO-MWCNT (CS) combines the advantages of both to achieve the integration of high specific surface area, excellent conductivity and outstanding thermal stability. The dual optimization of its porous interface and conductivity significantly improved the charge transfer efficiency.

[0052] Depend on Figure 2As shown in Figure B, compared with bare GCE (curve a), the charge transfer resistance of M-NTO@GCE (curve b) does not change much, while the charge transfer resistance of MWCNT@GCE (curve c) decreases. However, the charge transfer resistance of M-NTO-MWCNT@GCE (DI) (curve d) increases compared with MWCNT@GCE (curve c, Rct = 267.3 Ω), which is due to poor dispersion. The charge transfer resistance of M-NTO-MWCNT@GCE (CS) (curve e) decreases compared with the other curves, indicating that the electrode modified with the M-NTO-MWCNT@GCE (CS) composite material has a better ability to accelerate electron transfer, which is consistent with the results obtained by CV measurement.

[0053] Figure 2 C in the figure is the chrono-Coulomb curves of GCE electrode, M-NTO@GCE electrode, M-NTO-MWCNT@GCE (DI) electrode, MWCNT@GCE electrode and M-NTO-MWCNT@GCE (CS) electrode, as shown in Figure 2. Figure 2 As shown in D, Q and t 1 / 2 There is a good linear relationship. The following relationship is obtained from the Anson equation:

[0054] Q M-NTO-MWCNT@GCE(CS) = 2.0359t 1 / 2 - 0.0333 ( R 2 = 0.9241); Q MWCNT@GCE = 1.3005t 1 / 2 - 0.1262 ( R 2 = 0.9477); Q M-NTO-MWCNT@GCE(DI) = 0.7479t 1 / 2 - 0.1592 ( R 2 = 0.9805); Q M-NTO@GCE = 0.3652t 1 / 2 - 0.0462 ( R 2 = 0.9808); Q GCE = 0.3535t 1 / 2 - 0.0423 ( R 2 = 0.9821).

[0055] Where A is the surface area of ​​the working electrode, c is the concentration of the electrolyte, F is the Faraday constant, and D is the diffusion coefficient (the diffusion coefficient of 5.00 mmol K3[Fe(CN)6] in 1.00 mol KCl solution is 1.39 × 10 -4 cm 2 s -1 ), Q dl is the double layer charge, Q ads is the Faraday charge, n is the number of electron transfers, and the effective surface area of ​​the electrode is calculated based on the slope.

[0056] Therefore, the electroactive effective specific surface areas of the GCE electrode, M-NTO@GCE electrode, M-NTO-MWCNT@GCE (DI) electrode, MWCNT@GCE electrode, and M-NTO-MWCNT@GCE (CS) electrode are 0.137 cm 2 , 0.142 cm 2 、0.291 cm 2 , 0.507 cm 2 , 0.793 cm 2 .

[0057] In order to further characterize the electrochemical performance of the M-NTO-MWCNT@GCE electrode, its electrochemical performance at different scan rates was investigated. Figure 3 As shown, Figure 3 A is the CV curve of M-NTO-MWCNT@GCE electrode at different scan rates. When the scan rate is from 20 mV s -1 Increased to 240 mV s -1 When , the reduction peak moves toward the negative potential direction, and the oxidation peak moves toward the positive potential direction, which is mainly caused by the internal resistance of the electrode; Figure 3 B is the peak oxidation current of the M-NTO-MWCNT@GCE electrode (I pa ) and the reduction peak current (I pc ) has a good linear relationship with the logarithm of the scan rate, and the linear relationships are: Log I pa (μA) = 0.66536 Lgν(mV s -1 ) – 4.80719, R 2 = 0.99876; Log I pc (μA) = 0.64062 Lgν(mV s -1 ) – 4.77214, R 2 = 0.99882.

[0058] The results show that the electrochemical reaction on the electrode surface of the prepared sensor is controlled by adsorption-diffusion mixing.

[0059] As shown in Figure C, the oxidation peak potential (Epa) and reduction peak potential (Epc) of the M-NTO-MWCNT@GCE electrode show a good linear relationship with the logarithm of the scan rate, and their linear relationships are: Epa (V) = -0.23393Lgν (mV s -1 )-0.31431, R 2 = 0.98143; Epc (V) = 0.44303 Lgν (mV s -1 )+0.29029,R 2 = 0.99307.

[0060] According to the Laviron equation:

[0061] Where R is the gas constant (8.314 J mol -1 K -1 ), T is room temperature (298.15 K), F is the Faraday electrolysis constant (96485.33 C mol -1 ), ∆E p is the difference between the oxidation peak potential and the reduction peak potential. From formula (1), formula (2) and formula (3), it can be calculated that the electron transfer coefficient α is 0.35, the electron transfer number n is 0.386 ≈ 1, and the electrode transfer rate K s 0.101 V s -1 The results are similar to those of Fe(CN)6 3- / 4- Fe in electrolyte Ⅱ (CN)6 4- -e - →Fe Ш (CN)6 3- The electron transfer numbers of the oxidation reactions are consistent, indicating that the electroactive substances have a fast electron transfer process on the surface of the M-NTO-MWCNT@GCE electrode.

[0062] Example 3:

[0063] This embodiment provides an optimization method for experimental conditions and optimized experimental conditions based on the first embodiment.

[0064] The optimization of experimental conditions includes: MWCNT concentration screening, M-NTO-MWCNT composite material concentration screening, M-NTO-MWCNT modification amount screening, Mfn2-Ab incubation time screening, and Mfn2-Ag incubation time screening.

[0065] MWCNT concentration screening: GCE was modified with MWCNTs at different concentrations (0.5 M, 1 M, 2 M, 3 M, and 4 M). Results showed that the peak current of the electrochemical response reached its maximum when the MWCNT concentration reached 2 M. As the modification concentration increased, MWCNTs accumulated on the working electrode surface, hindering electron transfer and resulting in a decrease in the peak current response signal. Therefore, a 2 M MWCNT concentration was selected for this experiment.

[0066] M-NTO-MWCNT concentration screening: GCE was modified with M-NTO-MWCNT at various concentrations (1 M, 1.5 M, 2 M, 2.5 M, and 3 M). Results showed that the peak current of the electrochemical response reached its maximum when the M-NTO-MWCNT concentration reached 2 M. Further increases in the modification amount decreased the peak response signal. This is because high concentrations cause the M-NTO-MWCNT composite to accumulate on the working electrode surface, hindering electron transfer. Therefore, a 2 M concentration of M-NTO-MWCNT was selected for this experiment.

[0067] Screening of M-NTO-MWCNT modification amounts: GCE was modified with different M-NTO-MWCNT modification amounts (10 μL, 12 μL, 14 μL, 16 μL, 18 μL, and 20 μL). Results showed that the peak electrochemical response current reached its maximum when the modification amount reached 14 μL. Further increases in the modification amount decreased the peak response signal. This is because excessive modification causes the M-NTO-MWCNT composite to accumulate on the working electrode surface, hindering electron transfer. Therefore, a modification amount of 14 μL was selected for this experiment.

[0068] Screening of Mfn2-Ab incubation time: Mfn2-Ab at a 100× concentration was incubated on the M-NTO-MWCNT@GCE electrode for different times (10 min, 20 min, 30 min, 40 min, and 50 min) at room temperature (20°C). The incubation time of Mfn2-Ab significantly affects the electron transfer rate of the electrochemical immunosensor. Too short an incubation time prevents the Mfn2-Ab from being fully and effectively immobilized on the M-NTO-MWCNT@GCE surface, while too long an incubation time results in an excessively high protein content on the modified electrode surface, rendering it insulating. Results showed that the current response peak signal remained unchanged at a 40-min incubation time, indicating that the Mfn2-Ab was stably immobilized on the electrode. Therefore, 40 min was selected as the optimal incubation time for the Mfn2-Ab.

[0069] Mfn2-Ag incubation time screening: at room temperature (20°C), the concentration was 240 ng mL -1 Mfn2-Ag was incubated on a BSA@Mfn2-Ab@M-NTO-MWCNT@GCE electrode for different time periods (20 min, 30 min, 40 min, 50 min, and 60 min). When the Mfn2-Ag incubation time was less than 50 min, the antigen-antibody binding was incomplete. As the Mfn2-Ag incubation time increased, the peak current of the electrochemical reaction no longer significantly changed. Therefore, 50 min was selected as the optimal incubation time for Mfn2-Ag.

[0070] Example 4:

[0071] In this example, under the experimental conditions optimized in Example 3, different concentrations of Mfn2 standard (240 ng mL -1 , 120 ng mL -1 , 60 ng mL -1 , 30 ng mL -1 , 15 ng mL -1 , 7.5 ng mL -1 , 3.75 ng mL -1 , 1.875 ngmL -1 , 0.9375 ng mL -1 ) were used to verify the analytical performance of the constructed electrochemical immunosensor for detecting Mfn2 and compared with the ELISA method.

[0072] Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of 0.9375-240 ng mL -1 The absorbance (OD) value corresponding to Mfn2 within the concentration range is as follows: Figure 4 As shown in A, the OD value of the ELISA method showed a good linear relationship with the Mfn2 concentration, and the linear regression equation was OD = 0.01001C Mfn2 + 0.12309(R 2 = 0.98421), and the detection limit was 36.8901 ng mL -1 (S / N= 3); such as Figure 4 As shown in Figure B, the BSA@Mfn2-Ab@M-NTO-MWCNT@GCE electrochemical immunosensor was -1 , 120 ng mL -1 , 60 ng mL -1 , 30 ng mL -1 , 15 ng mL -1 , 7.5 ng mL-1 , 3.75 ng mL -1 , 1.875 ngmL -1 , 0.9375 ng mL -1 Mfn2 effectively recognizes the reaction within a range of concentrations; e.g. Figure 4 As shown in C and D, the DPV response value decreases with the increase of Mfn2 concentration and shows a good linear relationship. The linear equation is Ip = 1.06382-1.72862LgC Mfn2 (R 2 = 0.98767), and the detection limit was 1.846 ng mL -1 (S / N = 3).

[0073] Therefore, the BSA@Mfn2-Ab@M-NTO-MWCNT@GCE electrochemical immunosensor exhibited excellent analytical performance in detecting Mfn2 and could be used as an effective platform for analyzing Mfn2 in real samples.

[0074] Embodiment 5:

[0075] This example provides methods and results for testing the specificity, repeatability, and stability of the electrochemical immunosensor described in Example 1.

[0076] At 1.875 ng mL -1 Interfering substances were added to the Mfn2 solution in sequence: 1.875 ng mL -1 Glucose (Glu), L-methionine (Met), glycine (Glycine, Gly), ascorbic acid (ASA), horseradish peroxidase (HRP), to test the specificity of the prepared immunosensor, that is, its anti-interference ability. Figure 5 As shown in Figures A and B, the DPV response signals of the sensors incubated with Mfn2 and interfering substances are similar, indicating that these interfering substances have no effect on the detection of Mfn2. Therefore, the mitochondrial fusion protein 2 electrochemical immunosensor provided by the present invention has good specificity for Mfn2.

[0077] In order to test the reproducibility of the mitochondrial fusion protein 2 electrochemical immunosensor provided by the present invention, five electrodes prepared in the same batch were used to perform immunosensor tests. Figure 5 As shown in Figures C and D, the electrochemical signals of the five sensors are relatively stable, with a relative standard deviation (RSD) of only 1.33%, indicating that the mitochondrial fusion protein 2 electrochemical immunosensor provided by the present invention has good reproducibility.

[0078] In order to test the stability of the electrochemical immunosensor for mitochondrial fusion protein 2 provided by the present invention, three BSA@Mfn2-Ab@M-NTO-MWCNT@GCE electrodes were sealed and stored in a refrigerator at 4°C for 0 to 18 days and the electrodes were tested by DPV. Each time, a fixed concentration (1.875 ng mL -1 ) to perform electrochemical detection on Mfn2 and record the electrochemical response value. Figure 5 As shown in Figures E and F, after the prepared immunosensor was stored for 18 days, the electrochemical immunosensor maintained 98% of its function compared with the initial current response, indicating that the mitochondrial fusion protein 2 electrochemical immunosensor provided by the present invention has good stability in detecting Mfn2.

[0079] Example 6:

[0080] To verify the feasibility of the mitochondrial fusion protein 2 electrochemical immunosensor provided by the present invention in actual sample detection, this example provides the practical application method and effect of the electrochemical immunosensor described in Example 1.

[0081] Mfn2 was measured in real serum samples using the standard addition method. Blood samples were collected from healthy adults (with ethical approval) and collected in EDTA-sodium-containing EP tubes. Within 30 minutes of sample collection, the samples were centrifuged at 1000 × g for 15 minutes, and the supernatant was collected. The supernatant was spiked with various standard concentrations (0 ng mL -1 , 31.25 ng mL -1 , 62.5 ng mL -1 , 125 ng mL -1 Subsequently, serum samples with different concentrations of Mfn2-Ag were added to the BSA@Mfn2-Ab@M-NTO-MWCNT@GCE electrode, and the current response values ​​were measured.

[0082] At the same time, under the same experimental conditions, serum samples with different concentrations of Mfn2 were tested by ELISA. A total of three groups of samples were tested, and three parallel experiments were performed for each group of samples.

[0083] As shown in Table 1, the relative standard deviations (RSDs) of the constructed electrochemical immunosensor and ELISA methods ranged from 1.18% to 3.76% and 0.54% to 3.3%, respectively. The recoveries ranged from 98.3% to 99.4% and 99.5% to 101.3%, respectively. The differences in the results obtained by the two assay methods were not significant. These experimental results demonstrate that the constructed mitofusin 2 electrochemical immunosensor demonstrates accuracy and reliability in detecting Mfn2 and can be used to determine Mfn2 concentrations in real serum samples.

[0084] Table 1 Comparison of electrochemical immunosensor detection results in actual serum samples and ELISA kit test results

[0085] in, a It is the average of three measurements.

[0086] The above is only a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A method for preparing a mitochondrial fusion protein 2 electrochemical immunosensor, characterized in that: include: A composite material M-NTO-MWCNT composed of sodium titanate nanorods and multi-walled carbon nanotubes was prepared by oxidative alkalization of MXene, and an M-NTO-MWCNT dispersion was obtained after ultrasonic dispersion in a solvent. The M-NTO-MWCNT dispersion droplets were applied on the pretreated GCE electrode and dried to obtain the M-NTO-MWCNT@GCE electrode; The mitochondrial fusion protein 2 antibody solution was dropped onto the surface of the M-NTO-MWCNT@GCE electrode and incubated to obtain the Mfn2-Ab@M-NTO-MWCNT@GCE electrode; The active sites of the unloaded mitochondrial fusion protein 2 antibody are blocked in a blocking solution to obtain the product.

2. The method for preparing the mitochondrial fusion protein 2 electrochemical immunosensor according to claim 1, characterized in that: The pretreatment method of the GCE electrode includes: polishing the GCE electrode on suede sprinkled with 0.3 μm and 0.05 μm aluminum oxide polishing powder for 5 to 10 minutes, ultrasonically cleaning the GCE electrode with methanol and water for 10 to 15 minutes, and drying the electrode.

3. The method for preparing the mitochondrial fusion protein 2 electrochemical immunosensor according to claim 2, characterized in that: The solvent in the M-NTO-MWCNT dispersion is deionized water, and the concentration of M-NTO-MWCNT in the M-NTO-MWCNT dispersion is 2-3 mg / mL.

4. The method for preparing the mitochondrial fusion protein 2 electrochemical immunosensor according to claim 1, characterized in that: The incubation condition of the mitochondrial fusion protein 2 antibody solution is 20-25° C. for 4-5 h.

5. The method for preparing the mitochondrial fusion protein 2 electrochemical immunosensor according to claim 1 or 4, characterized in that: Also includes: The mitochondrial fusion protein 2 antibody solution was added dropwise onto the surface of the M-NTO-MWCNT@GCE electrode and then rinsed with PBST after incubation; PBST is a phosphate buffer containing 0.05~0.10 v / v% Tween-80, pH=7.0~7.

2.

6. The method for preparing the mitochondrial fusion protein 2 electrochemical immunosensor according to claim 5, characterized in that: The blocking solution is PBST containing 0.05-0.10 v / v% Tween-80 and 2-3 wt% BSA, with a pH of 7.0-7.

2.

7. The method for preparing the mitochondrial fusion protein 2 electrochemical immunosensor according to claim 6, characterized in that: The method for blocking the active sites of the unloaded mitochondrial fusion protein 2 antibody comprises placing the Mfn2-Ab@M-NTO-MWCNT@GCE electrode in a blocking solution at 20-25° C. for 50-60 minutes, removing the electrode and washing the electrode to obtain the BSA@Mfn2-Ab@M-NTO-MWCNT@GCE electrode.

8. A mitochondrial fusion protein 2 electrochemical immunosensor, characterized in that: The method is prepared by any one of claims 1 to 7. 9 . Use of the mitochondrial fusion protein 2 electrochemical immunosensor according to claim 8 in the detection of drug-induced cardiotoxicity.

10. Use of the mitochondrial fusion protein 2 electrochemical immunosensor according to claim 9 in the detection of drug-induced cardiotoxicity, characterized in that: The mitochondrial fusion protein 2 is a biomarker for drug-induced cardiotoxicity, and the application method includes: Blood samples were collected and centrifuged, and the supernatant was collected. Mfn2-Ag solutions with different standard concentrations were added to the supernatant; The mitochondrial fusion protein 2 electrochemical immunosensor was used as the working electrode, combined with a reference electrode and an auxiliary electrode in a three-electrode system, and the supernatant of the blood sample added with different standard concentrations of Mfn2-Ag solution was dripped on the surface of the working electrode, and differential pulse voltammetry scanning was performed to record the DPV response value; A linear relationship between the DPV response value and the logarithm of the Mfn2 concentration was established, and a linear regression equation was obtained; The sample solution to be tested is dropped onto the surface of the working electrode, differential pulse voltammetry scanning is performed, and the DPV response value is recorded. The concentration of Mfn2 in the sample is calculated based on the DPV response value of the sample combined with the linear regression equation.

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