Highly stable carbene-alkynyl probe molecule self-assembled interface, preparation method and application thereof

Abstract

The application discloses a kind of high-stable carbene-alkynyl probe molecule self-assembly interface and preparation method thereof, and systematically proposes the stability change of highly ordered monomolecular self-assembly interface under different experimental environments.The construction method includes: design and synthesis have with carbene-alkynyl as bonding assembly group probe molecule (CB-C≡C);Second, CB-C≡C molecule is self-assembled on gold nano surface by gold-carbene bond, gold-alkynyl bond, forms gold-carbene-alkynyl trident assembly sensing interface.And by electrode surface in situ combination ferrocene (Fc) electrochemical response group, generate gold-carbene-alkynyl-Fc molecular self-assembly interface, and systematically evaluate in different determination environments containing high concentration glutathione (5-10mMGSH), 25-50 DEG C, pH2-10 Interface high stability.The application utilizes this new CB-C≡C self-assembly molecular probe can realize high-stable determination under different determination environments for up to 90 days.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical analysis and detection technology, and relates to a highly stable carbene-alkynyl probe molecule self-assembly interface, its preparation method and application. Background Technology

[0002] Reasonable and effective interface control is the core of sensor construction. Forming a uniform and highly ordered monolayer through molecular self-assembly at the interface is one of the most widely used methods for sensor construction. Among these, the gold-thiol (Au-S) based molecular self-assembly is the most classic interface assembly model. However, due to the relatively weak Au-S bond (~45 kcal / mol), the resulting film is stable under ultra-high vacuum conditions without light, but easily oxidized and degraded in air. Furthermore, it is easily substituted in environments rich in thiol groups such as glutathione (GSH), leading to instability in interface properties. To address this issue, previous studies have developed Au-Se and gold-alkynyl molecular self-assembly methods, which can effectively resist interference from high concentrations of GSH, thus enabling long-term analysis in thiol-rich environments. However, besides the potential interfacial instability caused by high concentrations of sulfides, the Au-S bond is also susceptible to temperature and pH effects. Chemical bond breakage easily occurs under relatively high temperatures and acidic or alkaline conditions, severely limiting the practical application of Au-S-based sensors. Therefore, developing novel molecular self-assembly methods to achieve highly stable measurements in very low-temperature systems and under different acid-base conditions remains a key scientific problem that urgently needs to be solved. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention discloses a highly stable carbene-alkynyl probe molecule self-assembly interface and its preparation method, and systematically studies the stability changes of the highly ordered single-molecule self-assembly interface under different experimental environments. The construction method includes: designing and synthesizing a probe molecule (CB-C≡C) with carbene-alkynyl as the bonding assembly group; secondly, self-assembling the CB-C≡C molecule on a gold nanoparticle surface via gold-carbene and gold-alkynyl bonds to form a gold-carbene-alkynyl tridentate assembly sensing interface. Furthermore, by in-situ binding of ferrocene (Fc) electrochemical responsive groups to the electrode surface, a gold-carbene-alkynyl-Fc molecule self-assembly interface is generated, and the high stability of the interface is systematically evaluated in different measurement environments containing high concentrations of glutathione (5-10 mM GSH), 25℃-50℃, and pH 2-10. This invention utilizes this novel CB-C≡C self-assembled molecular probe to achieve highly stable measurements for up to 90 days under different measurement environments.

[0004] The carbene-acetylene probe molecule self-assembly interface described in this invention has the advantage of excellent high stability under high concentrations of GSH, different temperatures, and different pH values, and is resistant to high temperature, acid and alkali, and thiol attack.

[0005] This invention provides a method for preparing a carbene-alkynyl probe molecule self-assembly interface, the specific steps of which are as follows:

[0006] Step (1): Design and synthesize carbene-alkynyl molecule CB-C≡C;

[0007] Step (2): Electrodeposit gold nanoparticles on a carbon fiber electrode (CFME) as an electrochemical platform substrate;

[0008] Step (3): The carbene-acetylene molecules CB-C≡C obtained in step (1) are self-assembled onto the carbon fiber electrode modified with gold nanoparticles obtained in step (2) through gold-carbene and gold-acetylene bonds. Then, ferrocene (Fc) electrochemical response groups are in situ bound to the electrode surface to generate a CFME / Au / CB-C≡C / Fc self-assembly interface (also known as a carbene-acetylene probe molecule self-assembly interface), forming an electrochemical detection platform.

[0009] In step (1) of this invention, the carbene-alkynyl molecule CB-C≡C has the structure shown in formula (A):

[0010]

[0011] In step (1) of this invention, the preparation method of the carbene-alkynyl molecule CB-C≡C includes the following steps: using 3-nitro-4-aminophenol and N-(2-bromoethyl)phthalimide as raw materials, molecule 1-(2-phthalimide)ethoxy-3-nitro-4-aminophenol is synthesized in a first organic solvent; then, under the action of formic acid, iron, and ammonium chloride, a reaction is carried out to generate 5-(2-phthalimide)ethoxybenzimidazole. Then, 3-bromopropyne and cesium carbonate were added to react and give 1,3-dipropyne-5-(2-phthalimide)ethoxybenzimidazole; hydrazine hydrate was added to further react and synthesize 1,3-dipropyne-5-(2-phthalimide)ethoxybenzimidazole bromide. Bicarbonate ion exchange resin was added to give 1,3-dipropyne-5-(2-amino)ethoxybenzimidazole bicarbonate, which is the carbene-alkynyl molecule CB-C≡C.

[0012] The reaction process is shown in reaction formula (I):

[0013]

[0014] Wherein, the molar ratio of 3-nitro-4-aminophenol to N-(2-bromoethyl)phthalimide is (1-2):(1-2); preferably, it is 1:1;

[0015] Wherein, the molar ratio of 1-(2-phthalimide)ethoxy-3-nitro-4-aminophenol, formic acid, iron and ammonium chloride is (1-2):(1-2):(1-2):(1-2); preferably, it is 1:2:2:2;

[0016] Wherein, the molar ratio of 5-(2-phthalimide)ethoxybenzimidazole, 3-bromopropyne and cesium carbonate is (1-2):(1-2):(1-2); preferably, it is 1:1:2;

[0017] Wherein, the molar ratio of 1,3-dipropynyl-5-(2-phthalimide)ethoxybenzimidazole to hydrazine hydrate is (1-2):(1-2); preferably, it is 1:2;

[0018] The first organic solvent includes one or more of dichloromethane, tetrahydrofuran, N,N-dimethylformamide, ethanol, etc.

[0019] The volume of the first organic solvent used is 20-30 mL;

[0020] In the preparation of the carbene-alkynyl molecule CB-C≡C, the temperature of all reactions is 25-60℃; preferably, it is 60℃.

[0021] In the preparation of the carbene-alkynyl molecule CB-C≡C, the reaction time for all reactions is 12-24 hours; preferably, it is 24 hours.

[0022] In step (2) of this invention, the method for preparing gold nanoparticles electrodeposited on the carbon fiber electrode includes the following steps: using a three-electrode system containing a reference electrode, immersing the carbon fiber electrode in a chloroauric acid solution at room temperature; connecting the electrode using an electrochemical workstation, setting a constant potential for electrodeposition, and obtaining a gold-plated carbon fiber electrode, named CFME / Au;

[0023] In the three-electrode system, the reference electrode is an Ag / AgCl electrode, the auxiliary electrode is a platinum wire electrode, and the working electrode is a carbon fiber electrode.

[0024] The concentration of the chloroauric acid solution is 0.001-0.1M; preferably, it is 0.1M.

[0025] The potential is set to -0.02 to -0.3V; preferably, it is -0.1V.

[0026] The deposition time is 10-120 seconds; preferably, it is 15 seconds.

[0027] In step (3) of this invention, the specific method for generating CFME / Au / CB-C≡C / Fc is as follows: a dichloromethane solution of CB-C≡C molecules with a concentration of 2 mM is prepared. After bubbling out the O2 in the solution with N2, the gold-plated carbon fiber electrode prepared is immersed in the above solution under N2 environment to obtain a gold-plated carbon fiber electrode with self-assembled CB-C≡C molecules, named CFME / Au / CB-C≡C; then, by in-situ binding of ferroceneic acid on the electrode surface, a gold-plated carbon fiber electrode with gold-carbene-alkynyl-Fc molecules is generated, named CFME / Au / CB-C≡C / Fc.

[0028] The total concentration of CB-C≡C molecules is 1-20 mM; preferably, it is 2 mM.

[0029] The modification time is 12-48 hours.

[0030] The concentration of the ferrocene carboxylic acid solution is 1-20 mM; the modification time is 12-48 h.

[0031] The present invention also provides a highly stable carbene-alkynyl molecule (CB-C≡C) (also known as a carbene-alkynyl (CB-C≡C) probe molecule) prepared by the above preparation method.

[0032] This invention also provides a highly stable carbene-alkynyl probe molecule self-assembly interface constructed by the above method.

[0033] The present invention also provides the application of the self-assembled interface of the carbene-alkynyl molecule (CB-C≡C) or the carbene-alkynyl probe molecule in the stability study of the interface in different measurement environments containing high concentrations of glutathione (5-10 mM GSH), 25℃-50℃, and pH 2-10.

[0034] The specific steps for studying the interfacial stability of the carbene-alkynyl molecule (CB-C≡C) in different measurement environments are as follows:

[0035] Methods for studying interface stability using differential pulse voltammetry (DPV), XPS, and Raman spectroscopy.

[0036] Taking advantage of the electrochemical activity of the ferrocene groups bonded at the interface, the interface was placed in different experimental environments (high concentration of glutathione (5-10 mM GSH), 25℃-50℃, pH 2-10). The changes in current signal were characterized by differential pulse voltammetry (DPV), the changes in the position and intensity of elemental signals were characterized by XPS, and the changes in Raman signal were characterized by Raman spectroscopy. Thus, the stability of the interface can be obtained based on the changes in the signals.

[0037] The most significant innovations of this invention compared to similar methods are: 1. Designing and synthesizing a highly stable carbene-alkynyl molecule CB-C≡C to construct a gold-carbene-alkynyl tridentate interface; 2. Studying the interface stability under different experimental conditions (high concentration of glutathione (5-10 mM GSH), 25℃-50℃, pH 2-10) using differential pulse voltammetry (DPV), XPS, and Raman spectroscopy; 3. This interface exhibits excellent stability against acids, alkalis, high temperatures, and GSH attack.

[0038] The beneficial effects of this invention are as follows: First, a probe molecule (CB-C≡C) with carbene-alkynyl groups as bonding assembly groups is designed and synthesized. Second, the CB-C≡C molecule is self-assembled onto a gold nanoparticle surface via gold-carbene and gold-alkynyl bonds, forming a gold-carbene-alkynyl tridentate assembly sensing interface. Third, a gold-carbene-alkynyl-Fc molecule self-assembly interface is generated by in-situ binding of ferrocene (Fc) electrochemical responsive groups to the electrode surface. The high stability of the interface is systematically evaluated in different measurement environments containing high concentrations of glutathione (5-10 mM GSH), at temperatures ranging from 25°C to 50°C, and at pH values ​​ranging from 2 to 10. This invention utilizes this novel CB-C≡C self-assembled molecular probe to achieve highly stable measurements for up to 90 days under different measurement environments. Attached Figure Description

[0039] Figure 1 (A) SEM image of the bare carbon fiber electrode CFME of the present invention; (B, C) SEM images of the gold-plated carbon fiber electrode CFME / Au electrode at different magnifications.

[0040] Figure 2 X-ray photoelectron spectroscopy (XPS) of the CFME / Au / CB-C≡C / Fc self-assembled interface, where the high-resolution spectra of gold (A), nitrogen (B), and carbon (C) are shown. a: CFME, b: CFME / Au, c: CFME / Au / CB-C≡C / Fc.

[0041] Figure 3CV curves of the CFME / Au / CB-C≡C / Fc self-assembly interface. a. CV curve of CFME. b. CV curve of CFME / Au. c. CV curve of the CFME / Au / CB-C≡C / Fc self-assembly interface. Scan rate: 0.1V / s -1 .

[0042] Figure 4 The current signal changes of the (AD)CFME / Au / CB-C≡C / Fc self-assembled interface in artificial cerebrospinal fluid (0.1M) with high concentrations of glutathione (5-10mM GSH) (A), 25℃-50℃ (B), pH 2-7 (C), and pH 7-10 (D) for 0, 15, 30, 45, 60, 75, and 90 days. (EH) is a statistical graph of the current signal changes corresponding to (AD).

[0043] Figure 5 Changes in XPS C 1s signal at the (AD) CFME / Au / CB-C≡C / Fc self-assembly interface in artificial cerebrospinal fluid (0.1M) at high concentrations of glutathione (5-10mM GSH) (A), 25℃-50℃ (B), pH 2-7 (C), and pH 7-10 (D) for 0 and 90 days.

[0044] Figure 6 Raman signal changes of the (AD)CFME / Au / CB-C≡C / Fc self-assembled interface in artificial cerebrospinal fluid (0.1M) at high concentrations of glutathione (5-10mM GSH) (A), 25℃-50℃ (B), pH 2-7 (C), and pH 7-10 (D) for 0, 15, 30, 45, 60, 75, and 90 days. (EH) is a statistical graph of Raman signal changes corresponding to (AD). Detailed Implementation

[0045] The invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the invention are all common knowledge and general knowledge in the art, and the invention does not have any particular limitations.

[0046] Example 1: Preparation of carbene-alkynyl molecule CB-C≡C

[0047] Using 3-nitro-4-aminophenol and N-(2-bromoethyl)phthalimide as raw materials, molecule 1-(2-phthalimide)ethoxy-3-nitro-4-aminophenol was synthesized in dichloromethane. The reaction was carried out in tetrahydrofuran solvent under the action of formic acid, iron, and ammonium chloride to generate 5-(2-phthalimide)ethoxybenzimidazole. Then, 3-bromopropyne and cesium carbonate were added to N,N-dimethylformaldehyde. The reaction in an amide solvent yields 1,3-dipropyn-5-(2-phthalimide)ethoxybenzimidazole. Hydrazine hydrate is added, and the reaction proceeds in ethanol to synthesize 1,3-dipropyn-5-(2-phthalimide)ethoxybenzimidazole bromide. Adding a bicarbonate ion exchange resin yields 1,3-dipropyn-5-(2-amino)ethoxybenzimidazole bicarbonate, which is the carbene-alkynyl molecule CB-C≡C.

[0048] Example 2: Construction of a gold-carbene-alkynyl tridentate interface

[0049] Using a three-electrode system containing a reference electrode, a carbon fiber electrode was immersed in a 0.1 M chloroauric acid solution at room temperature. The electrode was connected using an electrochemical workstation, and electrodeposition was performed at a constant potential (-0.1 V) for 15 seconds to obtain a gold-plated carbon fiber electrode. This gold-plated carbon fiber electrode was then immersed in a 2 mM solution of carbene-alkynyl molecule CB-C≡C prepared in Example 1 of this invention. After immersion in an oxygen-free environment at 25°C for 24 hours, followed by air drying for 2 hours, a gold-carbene-alkynyl tridentate interface was obtained. Subsequently, ferrocene-formic acid was in situ bonded to the electrode surface to obtain a CFME / Au / CB-C≡C / Fc self-assembled interface.

[0050] Figure 1 These are bare carbon fiber electrodes (A) and gold-plated carbon fiber electrodes (B, C). Figure 2 This is the X-ray photoelectron spectroscopy (XPS) of a self-assembled gold-plated carbon fiber electrode of carbene-alkynyl molecule CB-C≡C, where the high-resolution energy dispersive spectra of gold (A), nitrogen (B), and carbon (C) are shown. The signals of nitrogen and carbon demonstrate that the carbene-alkynyl molecule CB-C≡C prepared in Example 1 of this invention has been successfully modified onto the electrode surface.

[0051] Example 3: Electrochemical characterization of the interface

[0052] The CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was used as the working electrode and connected to a three-electrode system. The electrode was inserted into a 10 mL detection cell containing 0.1 M artificial cerebrospinal fluid at pH 7.4. The sensor was characterized using cyclic voltammetry (CV) with a potential window set to -0.1–0.6 V. Representative CV characterization data are shown below. Figure 3The CV plots show that the interfaces all have obvious redox peaks, with peak positions at E... 0’ The voltage was ~0.17 V, and the electrochemical response signal originated from the electroactive ferrocene groups self-assembled on the gold electrode surface. This demonstrates the successful construction of the CFME / Au / CB-C≡C / Fc self-assembled interface, which exhibits a good electrochemical response signal and can be used for subsequent analysis.

[0053] Example 4: Electrochemical Tracking Interface Stability

[0054] Living organisms contain a large number of thiol compounds, and the substitution effect of thiol groups inevitably leads to the instability and unreliability of sensors. Therefore, this invention next evaluated the stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention in a thiol-rich molecular environment. As shown in the figure, after being placed in 5-10 mM glutathione for 90 days, the current value of the CFME / Au / CB-C≡C / Fc self-assembled interface decreased by 7.75 ± 2.95% compared with the initial value, indicating that the CFME / Au / CB-C≡C / Fc self-assembled interface has a strong resistance to thiol interference.

[0055] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was evaluated in an environment of 25-50℃. As shown in the figure, after 90 days in an environment of 25-50℃, the current value of the CFME / Au / CB-C≡C / Fc self-assembled interface decreased by approximately 5.47% compared to the initial value, indicating that the CFME / Au / CB-C≡C / Fc self-assembled interface has strong resistance to high-temperature interference.

[0056] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was then evaluated in an environment of pH 2-7. As shown in the figure, after 90 days in an environment of pH 2-7, the current value of the CFME / Au / CB-C≡C / Fc self-assembled interface decreased by approximately 8.91% compared to the initial value, indicating that the CFME / Au / CB-C≡C / Fc self-assembled interface has strong resistance to acid interference.

[0057] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was then evaluated in an environment with pH 7-10. As shown in the figure, after 90 days in an environment with pH 7-10, the current value of the CFME / Au / CB-C≡C / Fc self-assembled interface decreased by approximately 6.34% compared to the initial value, indicating that the CFME / Au / CB-C≡C / Fc self-assembled interface has strong resistance to alkali interference.

[0058] Example 5: XPS Tracking Interface Stability

[0059] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was then evaluated using XPS in a thiol-rich environment. As shown in the figure, after 90 days of storage in 5-10 mM glutathione, the peak positions and intensities of the CFME / Au / CB-C≡C / Fc self-assembled interface remained essentially unchanged, indicating that the CFME / Au / CB-C≡C / Fc self-assembled interface possesses strong resistance to thiol interference.

[0060] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was evaluated in an environment of 25-50℃. As shown in the figure, after being placed in an environment of 25-50℃ for 90 days, the peak position and intensity of the CFME / Au / CB-C≡C / Fc self-assembled interface remained essentially unchanged, indicating that the CFME / Au / CB-C≡C / Fc self-assembled interface has strong resistance to high-temperature interference.

[0061] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was then evaluated in an environment with pH 2-7. As shown in the figure, after 90 days of storage in an environment with pH 2-7, the peak positions and intensities of the CFME / Au / CB-C≡C / Fc self-assembled interface remained essentially unchanged, indicating that the CFME / Au / CB-C≡C / Fc self-assembled interface has strong resistance to acid interference.

[0062] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was then evaluated in an environment with pH 7-10. As shown in the figure, after 90 days of storage in an environment with pH 7-10, the peak positions and intensities of the CFME / Au / CB-C≡C / Fc self-assembled interface remained essentially unchanged, indicating that the CFME / Au / CB-C≡C / Fc self-assembled interface has strong resistance to alkali interference.

[0063] Example 6: Raman tracking interface stability

[0064] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was then evaluated in a thiol-rich environment. As shown in the figure, after 90 days in 5-10 mM glutathione, the 2200 cm⁻¹ of the CFME / Au / CB-C≡C / Fc self-assembled interface... -1 The signal peak intensity remained essentially unchanged over time (<8%), indicating that the CFME / Au / CB-C≡C / Fc self-assembly interface has a strong resistance to thiol interference.

[0065] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was evaluated in an environment of 25-50°C. As shown in the figure, after 90 days in an environment of 25-50°C, the stability of the CFME / Au / CB-C≡C / Fc self-assembled interface at 2200 cm⁻¹ was [not specified in the original text]. -1 The signal peak intensity remained essentially unchanged over time (<8%), indicating that the CFME / Au / CB-C≡C / Fc self-assembly interface has a strong resistance to high-temperature interference.

[0066] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was then evaluated in an environment of pH 2-7. As shown in the figure, after 90 days in an environment of pH 2-7, the 2200 cm⁻¹ of the CFME / Au / CB-C≡C / Fc self-assembled interface... -1 The signal peak intensity remained essentially unchanged over time (<8%), indicating that the CFME / Au / CB-C≡C / Fc self-assembly interface has a strong resistance to acid interference.

[0067] The stability of the CFME / Au / CB-C≡C / Fc self-assembled interface prepared in Example 2 of this invention was then evaluated in a pH 8 environment. As shown in the figure, after 90 days of storage in an environment with pH 7-10, the 2200 cm⁻¹ of the CFME / Au / CB-C≡C / Fc self-assembled interface... -1 The signal peak intensity remained essentially unchanged over time (<8%), indicating that the CFME / Au / CB-C≡C / Fc self-assembly interface has a strong resistance to alkali interference.

[0068] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for constructing a high-stability carbene-alkynyl probe molecule self-assembly interface, characterized in that, The specific steps are as follows: Step (1), designing and synthesizing a carbene-alkynyl molecule CB-C≡C; the structure of the carbene-alkynyl molecule CB-C≡C is shown as formula (A): Step (2), electrodepositing gold nanoparticles on a carbon fiber electrode CFME as an electrochemical platform substrate; Step (3), self-assembling the carbene-alkynyl molecule CB-C≡C obtained in step (1) on the gold nanoparticle modified carbon fiber electrode obtained in step (2) through gold-carbene and gold-alkynyl bonds, and then combining a ferrocene Fc electrochemical response group in situ on the electrode surface to generate a CFME / Au / CB-C≡C / Fc self-assembled interface, forming an electrochemical detection platform.

2. The construction method of claim 1, wherein, The preparation method of the carbene-alkynyl molecule CB-C≡C comprises the following steps: taking 3-nitro-4-aminophenol and N-(2-bromoethyl)phthalimide as raw materials, synthesizing 1-(2-phthalimido)ethoxy-3-nitro-4-aminophenol in a first organic solvent; then reacting under the action of formic acid, iron and ammonium chloride to generate 5-(2-phthalimido)ethoxybenzoimidazole; then adding 3-bromopropargyl and cesium carbonate to react to obtain 1,3-dipropargyl-5-(2-phthalimido)ethoxybenzoimidazole; continuing to add hydrazine hydrate to react to synthesize 1,3-dipropargyl-5-(2-phthalimido)ethoxybenzoimidazole bromide salt, adding bicarbonate ion exchange resin to obtain 1,3-dipropargyl-5-(2-amino)ethoxybenzoimidazole bicarbonate, which is the carbene-alkynyl molecule CB-C≡C; the reaction process is shown in reaction formula (I):

3. The construction method of claim 2, wherein, The molar ratio of the 3-nitro-4-aminophenol and the N-(2-bromoethyl)phthalimide is (1-2):(1-2); the molar ratio of the 1-(2-phthalimido)ethoxy-3-nitro-4-aminophenol, the formic acid, the iron and the ammonium chloride is (1-2):(1-2):(1-2):(1-2); the molar ratio of the 5-(2-phthalimido)ethoxybenzoimidazole, the 3-bromopropargyl and the cesium carbonate is (1-2):(1-2):(1-2); the molar ratio of the 1,3-dipropargyl-5-(2-phthalimido)ethoxybenzoimidazole and the hydrazine hydrate is (1-2):(1-2); the first organic solvent comprises one or more of dichloromethane, tetrahydrofuran, N,N-dimethylformamide and ethanol; the volume of the first organic solvent is 20-30 mL; in the preparation process of the carbene-alkynyl molecule CB-C≡C, the temperature of all reactions is 25-60 DEG C; in the preparation process of the carbene-alkynyl molecule CB-C≡C, the time of all reactions is 12-24 h.

4. The construction method of claim 1, wherein, In step (2), the preparation method of gold nanoparticles electrodeposited on the carbon fiber electrode is as follows: using a three-electrode system containing a reference electrode, the carbon fiber electrode is immersed in a chloroauric acid solution at room temperature; using an electrochemical workstation to connect the electrode, setting constant potential electrodeposition, obtaining a gold-plated carbon fiber electrode, named CFME / Au.

5. The construction method of claim 4, wherein, The reference electrode in the three-electrode system is an Ag / AgCl electrode, the auxiliary electrode is a platinum wire electrode, and the working electrode is a carbon fiber electrode; the concentration of the chloroauric acid solution is 0.001-0.1M; the potential is set to -0.02 to -0.3V; the deposition time is 10-120 seconds.

6. The construction method of claim 1 wherein, In step (3), the specific method for generating CFME / Au / CB-C≡C / Fc is as follows: prepare a dichloromethane solution of CB-C≡C molecules with a concentration of 2mM, remove O2 in the solution by N2 bubbling, then immerse the gold-plated carbon fiber electrode prepared in step (4) in the above solution in a N2 environment to obtain a gold-plated carbon fiber electrode self-assembled with CB-C≡C molecules, named CFME / Au / CB-C≡C; then combine ferrocene carboxylic acid in situ on the electrode surface to generate a gold-carbene-alkynyl-Fc molecule-plated carbon fiber electrode, named CFME / Au / CB-C≡C / Fc.

7. The construction method of claim 6, wherein, The concentration of the carbene-alkynyl molecule CB-C≡C solution is 1-20mM; the modification time is 12-48h; the concentration of the ferrocene carboxylic acid solution is 1-20mM; the modification time is 12-48h.

8. The carbene-alkynyl molecule CB-C≡C prepared by the method of claim 2 or 3.

9. The carbene-alkynyl probe molecule self-assembled interface prepared by the method of any one of claims 1-7.

10. The use of the carbene-alkynyl molecule CB-C≡C of claim 8 or the carbene-alkynyl probe molecule self-assembled interface of claim 9 in the study of the stability of the interface in different assay environments containing high concentrations of glutathione 5-10mM GSH, 25-50℃, pH 2-10.

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