A multi-walled carbon nanotube / covalent organic framework composite material, a preparation method and application thereof

Abstract

The present application relates to the technical fields of preparation and application of functional materials, and provides a multi-walled carbon nanotube / covalent organic framework composite material, a preparation method and application in electrochemical detection of nitric oxide.The multi-walled carbon nanotube / covalent organic framework composite material is prepared by an in-situ growth method, the material contains a large number of M-N4 electrocatalytic active sites arranged in an atomic manner, provides faster nitric oxide transmission / diffusion and a more effective electron transfer path, and solves the problem of random arrangement of active sites.The material is coated on an electrode to form a working electrode, and is used to construct a nitric oxide electrochemical sensor; due to the synergistic effect of the atomic periodic structure characteristics of COF-366-Co and the high conductivity of MWCNTs, the sensor has a wide linear range, high sensitivity, low detection limit and good selectivity.The present application provides a simple and low-cost preparation method of the composite material, and the nitric oxide electrochemical sensor prepared based on the material has stable performance and is convenient for commercial application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of preparation and application of functional materials, in particular to a multi-walled carbon nanotube / covalent organic framework composite material (MWCNTs@COF-366-Co), a preparation method and application thereof in electrochemical detection of nitric oxide. BACKGROUND

[0002] Endogenous nitric oxide (NO) is produced by constitutive and inducible nitric oxide synthase, which can rapidly diffuse into cells or across cell membranes and plays an important role in various physiological and pathological processes. For example, as an endothelium-derived relaxing factor, NO can be involved in vasodilation, immune response regulation, neural communication and blood pressure regulation, etc. Excessive production of NO in cells is associated with a variety of diseases in the body, including cancer, septic shock, transplant rejection and endothelial dysfunction, etc. Therefore, there is a great demand for dynamic quantitative determination of NO released by cells in the field of pathological and medical research. However, due to the low physiological concentration (sub-nanomolar to micromolar) of NO in the organism, the short lifetime (~5 s) and the fast diffusion rate, accurate real-time monitoring of NO is still a challenge. So far, chemiluminescence, spectroscopy, fluorescence and electrochemical techniques have been developed to detect NO. Among them, electrochemical techniques for detecting NO have attracted widespread attention due to their high sensitivity, rapid response and long-term stability, etc., and provide an effective analysis basis for real-time determination of biomarkers secreted by living cells.

[0003] Metalloporphyrins (MPor) with M-N4 structure are similar to natural heme enzymes and exhibit excellent electrocatalytic performance. Based on the large cyclic characteristics and extended π system of MPor, it can realize fast redox process with minimum reorganization energy. In addition, the unique M-N4 molecular structure of MPor can directly expose the metal active site to the analyte molecule, and the short transmission route can realize fast adsorption. However, porphyrin molecules show low affinity to conductive scaffolds, and tend to aggregate with each other, reducing the exposure of active catalytic sites and reducing the charge transfer ability. Fortunately, due to the extended conjugation of COFs in the two-dimensional plane and the periodic cylindrical π array arranged at atomic precision in the vertical direction, it provides a solution to the above problems. In addition, some inherent advantages of COFs, such as high specific surface area, post-modifiable active sites, controllable structure and tight binding ability with analytes, make it have strong adsorption to guest molecules and high electrochemical activity, showing great application potential in the field of electrochemical sensing. However, due to the poor conductivity of COFs, its catalytic activity is low, which limits its application in electrochemical sensing. In order to overcome this challenge while maintaining precise control of active sites, the morphology of COFs is modulated into a thin layer uniformly coated on multi-walled carbon nanotubes (MWCNTs). And because MWCNTs have good conductivity and high chemical stability, they are considered to be an ideal carrier for electrochemical sensors. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art, provide a preparation method of multi-walled carbon nanotubes / covalent organic framework composite material (MWCNTs@COF-366-Co), and on this basis develop a new type of real-time detection of NO electrochemical sensor, which has the advantages of simple operation, high sensitivity and good selectivity.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows.

[0006] The covalent organic framework (COF-366-Co) has a structural formula as shown in formula 1:

[0007] Formula 1.

[0008] Preparation of MWCNTs@COF-366-Co.

[0009] The preparation method of MWCNTs@COF-366-Co, characterized in that it comprises the following steps:

[0010] (1) Cobalt 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (CoTAPP), terephthalaldehyde (TPA) and (MWCNTs) were dispersed in a heat-resistant glass tube containing anhydrous ethanol, 1,2-dichlorobenzene and acetic acid aqueous solution, ultrasonically treated for 2-3 h, then degassed for three cycles, flame-sealed, and reacted at 110-130 °C for 70-74 h; the mass ratio of CoTAPP, TPA and MWCNTs was 1:0.37:0.41, and the total mass was 13.5-40.5 mg; the volume ratio of anhydrous ethanol, 1,2-dichlorobenzene and acetic acid aqueous solution was 1:1:0.2, and the total volume was 0.55-1.65 mL;

[0011] (2) After the reaction is completed, a precipitate is generated at the bottom of the tube. The crude product is obtained by centrifugation. The precipitate is washed with tetrahydrofuran, 1,4-dioxane, and acetone in sequence until it becomes colorless. The precipitate is then vacuum-dried at 50-70 °C for 8-16 h to obtain the product MWCNTs@COF-366-Co.

[0012] The MWCNTs@COF-366-Co prepared above was used to construct an electrochemical sensor.

[0013] The method for constructing the electrochemical sensor is characterized by comprising the following steps:

[0014] (1) 2-6 mg of MWCNTs@COF-366-Co was dispersed in a mixed solution of Nafion (5 wt %), ethanol, and deionized water, and the resulting suspension was ultrasonically dispersed for 2-4 h; the volume ratio of Nafion (5 wt %), ethanol, and deionized water was 1:50:50, and the total volume was 0.5-1.5 mL;

[0015] (2) 3-7 μL of the suspension was added to the pretreated glassy carbon electrode (GCE) and dried at room temperature overnight. After the solvent evaporated, the MWCNTs@COF-366-Co was tightly attached to the GCE surface. An electrochemical sensor was prepared using the MWCNTs@COF-366-Co modified electrode as the working electrode, a platinum sheet as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode.

[0016] The electrochemical sensor constructed above was used to measure NO.

[0017] Compared with the existing technology, the present invention has the following beneficial effects:

[0018] (1) The preparation method of MWCNTs@COF-366-Co provided by the present invention is simple, the reaction conditions are mild, and the post-processing is relatively easy;

[0019] (2) MWCNTs@COF-366-Co contains a large number of M-N4 electrocatalytic active sites arranged atomically, providing faster NO transport / diffusion and more efficient electron transfer pathways, solving the problem of random arrangement of active sites;

[0020] (3) Due to the synergistic effect of the atomic periodic structure characteristics of COF-366-Co and the high conductivity of MWCNTs, the MWCNTs@COF-366-Co-based electrochemical sensor exhibits excellent NO determination performance in a wide linear range of 0.09-400 μM, with a sensitivity of 8.9 μA·μM −1 ·cm −2 , and a detection limit as low as 16 nM;

[0021] (4) The MWCNTs@COF-366-Co-based electrochemical sensor can be widely used for real-time online monitoring of NO molecules released by human umbilical vein endothelial cells (HUVECs), providing broad prospects for continuous dynamic monitoring of active cell activity and its released metabolites. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a synthesis diagram of MWCNTs@COF-366-Co for real-time detection of NO released by living cells;

[0023] Figure 2 is a scanning electron microscope image of MWCNTs@COF-366-Co;

[0024] Figure 3 is a transmission electron microscope image of MWCNTs@COF-366-Co;

[0025] Figure 4 is an X-ray photoelectron spectrogram of MWCNTs@COF-366-Co;

[0026] Figure 5 is a Fourier transform infrared spectrogram of MWCNTs@COF-366-Co;

[0027] Figure 6 is an X-ray diffraction spectrogram of MWCNTs@COF-366-Co;

[0028] Figure 7 is a CV curve of MWCNTs@COF-366-Co (A) for different concentrations of NO and a graph of the relationship between the anodic current peak and the NO concentration (B);

[0029] Figure 8(A) CV curves of MWCNTs@COF-366-Co in the presence of 216 μM NO at different scan rates and the corresponding relationship curves (BC);

[0030] Figure 9 (A) Amperometric response and (B) corresponding linear curve of the MWCNTs@COF-366-Co-based electrochemical sensor when different concentrations of NO were continuously injected.

[0031] Figure 10 It is the selectivity of MWCNTs@COF-366-Co based electrochemical sensor towards NO and interfering substances;

[0032] Figure 11 is the repeatability of MWCNTs@COF-366-Co based electrochemical sensor, inset: sensor stability;

[0033] Figure 12 Detection of NO released from cells by the MWCNTs@COF-366-Co-based electrochemical sensor under (A) different conditions and (B) different doses of L-Arg stimulation. Inset: corresponding amperometric current. Specific implementation methods

[0034] The following will be combined with the accompanying drawings in the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. However, the content of the present invention is not limited to the following embodiments. The details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0035] It should be understood that the terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention. The methods in the following examples where no specific conditions are specified are generally performed under conventional conditions or the conditions recommended by the manufacturers.

[0036] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention are consistent with the prior art knowledge and description of the present invention by those skilled in the art. The present invention can also be implemented using any methods, equipment and materials in the prior art that are similar or equivalent to the methods, equipment and materials described in the embodiments of the present invention.

[0037] Example 1 Synthesis and Characterization of MWCNTs@COF-366-Co

[0038] 1.1 Synthesis of MWCNTs@COF-366-Co

[0039] (1) Figure 1 is a synthesis scheme of MWCNTs@COF-366-Co for real-time detection of NO released by living cells; 15.2 mg of CoTAPP, 5.6 mg of TPA and 6.0 mg of MWCNTs were first dispersed in a heat-resistant glass tube containing 0.5 mL of anhydrous ethanol, 0.5 mL of 1,2-dichlorobenzene and 0.1 mL of an aqueous acetic acid solution, ultrasonic treatment for 3 h, then three cycles of degassing treatment, flame sealing, reaction at 120 °C for 72 h;

[0040] (2) After the reaction was completed, a precipitate was generated at the bottom of the tube, the product was obtained by centrifugation, and the precipitate was washed with tetrahydrofuran, 1,4-dioxane and acetone in turn until it was colorless, and then dried at 60 °C under vacuum for 12 h to obtain the product MWCNTs@COF-366-Co;

[0041] 1.2 Characterization of MWCNTs@COF-366-Co

[0042] (1) Figure 2 and Figure 3 are scanning electron microscope and transmission electron microscope images of MWCNTs@COF-366-Co, respectively; it can be seen from the images that MWCNTs@COF-366-Co exhibits an interwoven and rough network morphology, and the average diameter is 39 nm, which ensures the electronic conduction capacity;

[0043] (3) Figure 4 is an X-ray photoelectron spectroscopy image of MWCNTs@COF-366-Co; it can be seen from Figure 4 that MWCNTs@COF-366-Co corresponds to the binding energies of C 1s, N 1s, O 1s and Co 2p at 283.9, 398.1, 532.1 and 781.2 eV, respectively;

[0044] (2) Figure 5 and Figure 6 are Fourier transform infrared spectroscopy and X-ray diffraction spectroscopy images of MWCNTs@COF-366-Co, respectively; the synthesis of MWCNTs@COF-366-Co is confirmed by infrared and X-ray diffraction.

[0045] Example 2 Construction of an electrochemical sensor

[0046] (1) 4 mg of MWCNTs@COF-366-Co was dispersed in a mixed solution containing 10 μL of Nafion (5wt %), 500 μL of ethanol and 500 μL of deionized water, and then the obtained suspension was ultrasonic dispersed for 3 h;

[0047] (2) 5 μL of the suspension was dropped onto the pretreated GCE and dried at room temperature overnight. After the solvent was evaporated, the MWCNTs@COF-366-Co was tightly attached to the surface of the GCE. The MWCNTs@COF-366-Co modified electrode was used as the working electrode, a platinum sheet as the counter electrode, and a saturated SCE as the reference electrode to prepare an electrochemical sensor.

[0048] Example 3 Performance test of the electrochemical sensor on NO

[0049] (1) In order to verify the sensitivity of MWCNTs@COF-366-Co to NO oxidation, the CV curves of different concentrations (0-432 μΜ) of NO were recorded; Figure 7 is the CV curves of MWCNTs@COF-366-Co (A) for different concentrations of NO and (B) the relationship diagram of the anodic current peak and the NO concentration. As can be seen from the figure, with the gradual addition of NO, the anodic oxidation current also gradually increases, and shows a good linear relationship with the change of NO concentration, which indicates that MWCNTs@COF-366-Co is very sensitive to the change of NO concentration;

[0050] (2) The electrocatalytic behavior of MWCNTs@COF-366-Co on NO was studied by changing the scan rate of CV; Figure 8 is the CV curves of MWCNTs@COF-366-Co at different scan rates (A) containing 216 μM NO and (B-C) the corresponding relationship curves; from Figure 8 A can be seen that the peak potential changes with the increase of the scan rate, indicating that the kinetics of MWCNTs@COF-366-Co electrocatalytic oxidation of NO is irreversible, from Figure 8 B can be seen that the peak current and the square root of the scan rate are linearly related, indicating that MWCNTs@COF-366-Co has a typical diffusion-controlled process and fast electron transfer kinetics, from Figure 8 C can be seen that the peak potential E p changes with the change of the scan rate l g , which further illustrates the irreversibility of the electrocatalytic process;

[0051] (3) The concentration range, sensitivity and detection limit of the MWCNTs@COF-366-Co-based electrochemical sensor for quantitative detection of NO were discussed; Figure 9 is the amperometric response (A) and the corresponding linear curve (B) of the MWCNTs@COF-366-Co electrochemical sensor when different concentrations of NO were continuously injected; from Figure 9As can be seen, the current on the MWCNTs@COF-366-Co stably increases with the injection of NO and reaches a steady state within 2.5 s, which indicates that the sensor has a fast electrochemical response, from Figure 9 As can be seen, the amperometric response shows a good linear relationship in the range of 0.09-400 μM, and the linear regression equation is J (μA / cm 2 ) = 45.9 + 8.9 c (μM), the correlation linear coefficient R 2 = 0.998, the sensitivity is known from the slope 8.9 μA·μM −1 ·cm −2 , and the detection limit of the sensor for NO is low, being 16 nM;

[0052] (4) In order to test the selectivity of the MWCNTs@COF-366-Co-based electrochemical sensor, the response curves of the sensor to 36 μM of NO and potential interfering substances were analyzed; Figure 10 is the selectivity of the MWCNTs@COF-366-Co electrochemical sensor to NO and interfering substances; as can be seen from the figure, the MWCNTs@COF-366-Co-based sensor has an obvious current response to NO, while the current change of the interfering substances is not obvious, indicating that the MWCNTs@COF-366-Co-based electrochemical sensor has high selectivity to NO;

[0053] (5) Finally, the repeatability and stability of the MWCNTs@COF-366-Co-based electrochemical sensor to 36 μM of NO were explored; Figure 11 is the repeatability of the MWCNTs@COF-366-Co electrochemical sensor, and the inset is the stability of the sensor; as can be seen from Figure 11 , the repeatability measurement of the same MWCNTs@COF-366-Co-based sensor to NO has a relative standard deviation (RSD) of only 6.39%, and as can be seen from Figure 11 the inset, the response current of the MWCNTs@COF-366-Co-based electrochemical sensor to NO is still 98% of the initial current after the sensor is stored at room temperature for 20 days, and the above results show that the MWCNTs@COF-366-Co-based electrochemical sensor has good repeatability and stability.

[0054] Example 4 Real-time detection of NO released by living cells

[0055] (1) Using HUVECs as a model cell line to investigate whether the MWCNTs@COF-366-Co-based sensor can detect NO in a cell environment, L-Arg is selected as a common stimulant to stimulate HUVECs to produce NO, because it can be enzymatically oxidized by nitric oxide synthase to generate NO; Figure 12 is the detection of NO released by cells under different conditions (A) and the detection of NO released by cells under different doses of L-Arg stimulation (B) by the MWCNTs@COF-366-Co electrochemical sensor, and the inset is the corresponding amperometric current;

[0056] (2) As shown in Figure 12 A, when 0.25 mM of L-Arg is added to the solution without HUVECs, no obvious current response is observed, on the contrary, when 0.25 mM of L-Arg is injected into the HUVECs suspension, the current response is observed to gradually rise, in order to prove that the above-mentioned current signal response is caused by NO release, L-Arg and L-NAME (a specific NOS inhibitor) are added at the same time, and no obvious current signal is generated, as shown in Figure 12 B, further study the amperometric response of the prepared sensor to simulated HUVECs with different concentrations of L-Arg, apparently, when the concentration of L-Arg is low, the level of NO secretion by HUVECs is linearly related to the concentration of the drug, however, the current peak is not completely linearly related to the high concentration of L-Arg, which may be related to the complex mechanism of NO biosynthesis in HUVECs;

[0057] (3) Therefore, as a proof of concept, the MWCNTs@COF-366-Co-based electrochemical sensor can be used to detect the NO molecules released by HUVECs in real time.

[0058] In summary, the NO electrochemical sensor based on MWCNTs@COF-366-Co has low detection limit, wide linear range, high sensitivity, and good selectivity to common coexisting interfering substances, and can be flexibly applied to real-time determination of NO released by biological systems.

[0059] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. A highly sensitive nitric oxide electrochemical sensor, characterized in that: The following steps are involved: (1) 2-6 mg of multi-walled carbon nanotube / covalent organic framework composite MWCNTs@COF-366-Co was dispersed in a mixed solution of Nafion, ethanol, and deionized water, and the resulting suspension was ultrasonically dispersed for 2-4 h; wherein the volume ratio of Nafion, ethanol, and deionized water was 1:50:50, and the total volume was 0.5-1.5 mL; (2) 3-7 μL of the suspension was added dropwise to the pretreated glassy carbon electrode (GCE) and dried at room temperature overnight. After the solvent evaporated, a uniform and dense composite film layer was formed to obtain a MWCNTs@COF-366-Co modified electrode. (3) A three-electrode nitric oxide electrochemical sensor was constructed using the MWCNTs@COF-366-Co modified electrode as the working electrode, a platinum sheet as the counter electrode, and a saturated calomel electrode as the reference electrode. The preparation method of MWCNTs@COF-366-Co comprises the following steps: (1) 5,10,15,20-tetrakis(4-aminophenyl)porphyrin cobalt CoTAPP, terephthalaldehyde TPA and high-purity unfunctionalized multi-walled carbon nanotubes MWCNTs were dispersed in a heat-resistant glass tube containing anhydrous ethanol, 1,2-dichlorobenzene and acetic acid aqueous solution, ultrasonically treated for 2-3 hours, and then degassed for three cycles, flame-sealed, and reacted at 110-130°C for 70-74 hours; wherein the mass ratio of CoTAPP, TPA and MWCNTs was 1:0.37:0.41, and the total mass was 13.5-40.5 mg; the volume ratio of anhydrous ethanol, 1,2-dichlorobenzene and acetic acid aqueous solution was 1:1:0.2, and the total volume was 0.55-1.65 mL; (2) After the reaction is completed, the precipitate is collected and centrifuged to obtain a crude product, which is washed with tetrahydrofuran, 1,4-dioxane, and acetone in sequence until colorless, and then dried in a vacuum at 50-70°C for 8-16 h to obtain a MWCNTs@COF-366-Co composite material; (3) The MWCNTs@COF-366-Co is a non-covalent composite structure. The COF-366-Co skeleton is in situ generated on the surface of MWCNTs and is tightly combined with MWCNTs through π–π stacking to form a three-dimensional network composite material. The material presents an interwoven and rough network morphology with an average diameter of 35–45 nm, ensuring axial electron conduction ability and stability.

2. The highly sensitive nitric oxide electrochemical sensor according to claim 1, wherein: The constructed sensor combines the ordered structure of COF-366-Co with the electrocatalytic activity of the Co center and the efficient electron transport network constructed by MWCNTs. The relationship between nitric oxide concentration and current response is linear in the range of 0-432 μM, reaching a steady state within 2.5 s. The linear range is 0.09-400 μM and the sensitivity is 8.9 μA·μM. -1 cm -2 , with a detection limit of 16nM, and good selectivity, repeatability and stability.

3. The highly sensitive nitric oxide electrochemical sensor according to claim 1, wherein: It is suitable for detecting nitric oxide molecules released by human umbilical vein endothelial cells (HUVECs) under physiological or stimulation conditions. It has good biocompatibility and can achieve real-time quantitative analysis of nitric oxide at the cellular level.

Images