Preparation method of copper-doped covalent organic framework and application of copper-doped covalent organic framework in epinephrine colorimetric detection

Abstract

The invention belongs to the field of analytical chemistry, relates to material preparation, and particularly discloses a preparation method of a copper-doped covalent organic framework and application of the copper-doped covalent organic framework in adrenaline colorimetric detection. Firstly, 1H-indazole-4, 7-diamine (Iz) and 1, 3, 5-triformyl phloroglucinol (Tp) are used as monomers to synthesize a novel covalent organic framework (Iz-Tp COF) through a Schiff base reaction, copper ions are introduced into the Iz-Tp COF framework to prepare a novel metal covalent organic framework (Iz-Tp (at) Cu), and then according to the phenomenon that the Iz-Tp (at) Cu can catalyze oxidation color development of epinephrine, the metal covalent organic framework (Iz-Tp (at) Cu can catalyze oxidation color development of epinephrine. According to the present invention, the simple, sensitive and efficient adrenaline colorimetric detection method is established, the test conditions are finally optimized, the standard addition method is combined to accurately determine the adrenaline concentration in the urine, and the good practical application value is provided.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry, specifically the field of materials preparation technology, and particularly relates to a method for preparing a novel copper-doped covalent organic framework (Iz-Tp@Cu) and its application in the colorimetric detection of adrenaline. Background Technology

[0002] Pheochromocytoma is a tumor originating in the adrenal medulla, primarily characterized by the excessive release of catecholamine hormones, such as adrenaline, noradrenaline, and dopamine. Sensitive and selective detection of catecholamine hormones (such as adrenaline) in complex body fluids is crucial for the early diagnosis of pheochromocytoma. To date, various methods for detecting adrenaline have been established, including high-performance liquid chromatography (HPLC), capillary electrophoresis, electrochemiluminescence, electrochemical methods, and fluorescence. However, their rapid detection in complex biological systems is limited due to expensive instruments, complex operation, and insufficient sensitivity and selectivity. Now, colorimetric methods offer advantages such as simple operation, low cost, no instrumentation required, rapid reaction, and direct visual observation, making them an effective method to overcome these limitations.

[0003] Laccase, a multi-copper oxidase, has been widely used in environmental pollutant treatment, organic synthesis, and biotechnology processes. However, its inherent drawbacks, such as poor operational stability, high preparation and purification costs, and low recycling and reuse efficiency, severely hinder its practical application. Therefore, biomimetic nanozymes with copper as the core element have attracted widespread attention due to their unique physicochemical properties, high operational stability, efficient recycling, and cost-saving production. For example, Huang et al. used Cu... + / Cu 2+ By coordinating with a cysteine-histidine dipeptide, a novel nanozyme (CH-Cu) with laccase-like activity was constructed. Makam et al. demonstrated that phenylalanine and Cu... 2+ F-Cu nanosheets formed by supramolecular assembly exhibit enhanced laccase-mimicking activity. Although some nanozymes with laccase activity have been designed, designing nanozymes with highly efficient laccase-mimicking activity remains challenging.

[0004] Metal-coated organic frameworks (MOFs) serve as a bridge between metal-organic frameworks (MOFs) and covalent organic frameworks (COFs), and have become an effective strategy to overcome the poor stability of MOFs under harsh conditions and the limitation of COF function due to the lack of metals. However, the development of MCOFs is still in its early stages, and there are few reports on their nanozyme activities. Summary of the Invention

[0005] In view of this, the present invention provides a simple colorimetric detection method for adrenaline to address the shortcomings of existing technologies. First, a metal-covalent organic framework (Iz-Tp@Cu) with a copper ion catalytic active center was prepared. Then, the colorimetric detection of adrenaline was achieved by utilizing the Iz-Tp@Cu-catalyzed adrenaline oxidation colorimetric reaction.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first technical objective of this invention is to provide a method for preparing a copper-doped covalent organic framework (Iz-Tp@Cu). This method involves ultrasonically mixing 20.0 mg of the covalent organic framework Iz-Tp COF with 10.0–30.0 mL of 0.24 mol / L copper acetate, reacting at 120–180 °C for 3–8 hours, washing with deionized water, and finally vacuum drying at 70 °C for 12 hours to obtain the product, which is Iz-Tp@Cu.

[0007] Preferably, the optimal synthesis conditions are: 20 mL of copper acetate, a reaction temperature of 150 °C, and a reaction time of 5 hours.

[0008] Preferably, the covalent organic framework (Iz-Tp COF) is prepared using the following process: 1H-indazole-4,7-diamine (Iz) and 1,3,5-tricarboxymethylphloroglucinol (Tp) were added to a solvent and a catalyst. The mixed solution was subjected to three freeze-pump thaw cycles. After being flame-sealed under vacuum, the mixture was reacted at 120 °C for 3 days. The resulting solid was washed three times each with N,N-dimethylacetamide, mesitylene, and ethanol, and then vacuum-dried at 70 °C for 12 hours. The product obtained was Iz-Tp COF.

[0009] Preferably, the molar ratio of 1H-indazole-4,7-diamine (Iz) and 1,3,5-tricarboxymethylphloroglucinol (Tp) is 1:1 to 2:1, and the masses of the two used are 8.93–17.86 mg and 12.6 mg, respectively.

[0010] Preferably, the reaction solvent is N,N-dimethylacetamide and mesitylene in a volume ratio of 1:1 to 4:1, with a total volume of 2 mL; the catalyst is 0.1–0.5 mL of acetic acid at a concentration of 6 mmol / L.

[0011] The second technical objective of this invention is to provide an application of the copper-doped covalent organic framework (Iz-Tp@Cu) as described above in the colorimetric detection of adrenaline.

[0012] Furthermore, the copper-doped covalent organic framework (Iz-Tp@Cu) was used in the colorimetric detection of adrenaline using the following process: In a Tris-HAc buffer solution at pH 6.0–9.0, a certain amount of adrenaline was added to 40 μL of Iz-Tp@Cu (2.0 mg / mL), mixed well, and reacted at 25–50 °C for 30–60 minutes; subsequently, the absorption spectrum at 350–700 nm was measured using a UV-Vis absorption spectrometer. The concentration of adrenaline is calculated using formula (1): A 485 =0.0014 c + 0.0059 (1) in, A 485 - The absorption peak intensity at 485 nm after the addition of adrenaline; c – Adrenaline concentration; linear correlation coefficient R 2 = 0.9972.

[0013] Preferably, the optimal reaction pH is 7.0, and the concentration range of adrenaline is 5.0 – 500.0 μM.

[0014] The beneficial effects of the above-mentioned preferred technical solution are: within this concentration range, the intensity of the characteristic absorption peak at 485 nm shows a good linear relationship with the adrenaline concentration, which enables quantitative analysis of adrenaline and ensures the accuracy of the detection results.

[0015] Preferably, adding one or more of bovine serum albumin, valine, threonine, leucine, starch, cysteine, ascorbic acid, sodium chloride, potassium chloride, calcium chloride, zinc nitrate, and magnesium nitrate to Tris-HAc buffer solution and Iz-Tp@Cu mixed solution can verify the effect of common interfering substances on adrenaline detection.

[0016] Preferably, adrenaline can also be detected semi-quantitatively by observing color with the naked eye, or quantitatively by using a smartphone.

[0017] The advantages of the above-mentioned preferred technical solution are: visual observation does not rely on instruments and equipment at all, and smartphones make the colorimetric detection of xanthine unnecessary to be carried out in the laboratory, making it suitable for on-site analysis.

[0018] Preferably, in the above applications, the concentration of adrenaline in urine is determined by spiking.

[0019] The beneficial effect of the above-mentioned preferred technical solution is that when the urine concentration does not reach the test concentration, the spiked method is used for testing, making the method adaptable to urine detection.

[0020] By means of the above solution, compared with the prior art, the present invention has the following beneficial effects: 1) This invention provides a method for preparing a copper-doped covalent organic framework (Iz-Tp@Cu) and its application in the colorimetric detection of adrenaline. The Iz-Tp@Cu obtained by introducing copper ions into the covalent organic framework (Iz-Tp COF) skeleton with 1H-indazole-4,7-diamine (Iz) and 1,3,5-tricarboxymethylphloroglucinol (Tp) as monomers has a copper ion catalytic center similar to that of natural laccase, which can efficiently catalyze the oxidation of adrenaline to generate brown products, thus realizing a simple, sensitive and efficient colorimetric detection of adrenaline.

[0021] 2) The spiked method is used to further apply it to make it suitable for colorimetric detection of trace amounts of adrenaline in urine, and to ensure detection accuracy, thus having high application value. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely embodiments of the present invention, and those skilled in the art can obtain other drawings based on the provided drawings without any creative effort.

[0023] Figure 1 Fourier-infrared spectra of Iz-Tp COF, Iz-Tp@Cu, and monomers.

[0024] Figure 2 PXRD patterns of Iz-Tp COF and Iz-Tp@Cu.

[0025] Figure 3 SEM and EDS images of Iz-Tp@Cu.

[0026] Figure 4 This is the absorption spectrum of Iz-Tp@Cu catalyzed adrenaline oxidation.

[0027] Figure 5 The effect of pH on adrenaline detection.

[0028] Figure 6 This is a linear curve for adrenaline detection.

[0029] Figure 7 The effects of common interfering substances on adrenaline detection. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1 Preparation and characterization of Iz-Tp COF: In a 10 mL glass tube, 13.4 mg of 1H-indazole-4,7-diamine, 12.6 mg of 1,3,5-tricarboxymethylresorcinol, 1.5 mL of N,N-dimethylacetamide, 0.5 mL of mesitylene, and 0.1 mL of acetic acid (6 mmol / L) were added sequentially. The mixture was sonicated for 10-15 minutes to obtain a homogeneous solution. This solution was subjected to three freeze-pump thaw cycles and flame-sealed under vacuum. After heating at 120 °C for 3 days, the mixture was cooled to room temperature. The resulting precipitate was washed 3-5 times each with N,N-dimethylacetamide, mesitylene, and ethanol, and then dried under vacuum at 70 °C for 12 hours to obtain Iz-Tp COF.

[0032] Example 2 Preparation and characterization of Iz-Tp COF: In a 10 mL glass tube, 8.93 mg of 1H-indazole-4,7-diamine, 12.6 mg of 1,3,5-tricarboxymethyl phloroglucinol, 1.0 mL of N,N-dimethylacetamide, 1.0 mL of mesitylene, and 0.1 mL of acetic acid (6 mmol / L) were added sequentially. The mixture was sonicated for 10-15 minutes to obtain a homogeneous solution. This solution was subjected to three freeze-pump thaw cycles and flame-sealed under vacuum. After heating at 100 °C for 3 days, the mixture was cooled to room temperature. The resulting precipitate was washed 3-5 times each with N,N-dimethylacetamide, mesitylene, and ethanol, and then dried under vacuum at 70 °C for 12 hours to obtain Iz-Tp COF.

[0033] Example 3 Preparation and characterization of Iz-Tp COF: In a 10 mL glass tube, 13.4 mg of 1H-indazole-4,7-diamine, 12.6 mg of 1,3,5-tricarboxymethylresorcinol, 1.0 mL of N,N-dimethylacetamide, 1.0 mL of mesitylene, and 0.2 mL of acetic acid (6 mmol / L) were added sequentially. The mixture was sonicated for 10-15 minutes to obtain a homogeneous solution. This solution was subjected to three freeze-pump thaw cycles and flame-sealed under vacuum. After heating at 120 °C for 3 days, the mixture was cooled to room temperature. The resulting precipitate was washed 3-5 times each with N,N-dimethylacetamide, mesitylene, and ethanol, and then dried under vacuum at 70 °C for 12 hours to obtain Iz-Tp COF.

[0034] The prepared Iz-Tp COF was analyzed, and the results are shown in the attached figure. Figure 1 As shown, with the formation of Iz-Tp COF, at 1597 cm⁻¹ - A new C=N vibrational peak appeared at ¹, while Iz was at 3316 cm⁻¹. - ¹The NH vibration peak and Tp are at 1652 cm⁻¹ - The disappearance of the C=O vibration peak in ¹ indicates that Iz-Tp COF was successfully synthesized via the Schiff base reaction. The peak of Iz-Tp COF at 4.64° corresponds to the diffraction peak of the (100) crystal plane (see attached diagram). Figure 2 This indicates that Iz-Tp COF has good crystallinity.

[0035] Example 4 Preparation and characterization of Iz-Tp@Cu: After ultrasonically mixing 20.0 mg Iz-Tp COF and 15 mL copper acetate (0.24 mol / L), the mixture was reacted at 120 °C for 5 hours, washed three times with deionized water, and finally dried under vacuum at 70 °C to obtain the product, which is Iz-Tp@Cu.

[0036] Example 5 Preparation and characterization of Iz-Tp@Cu: After ultrasonically mixing 20.0 mg Iz-Tp COF and 20 mL copper acetate (0.24 mol / L), the mixture was reacted at 150 °C for 3 hours, washed three times with deionized water, and finally dried under vacuum at 70 °C to obtain the product, which is Iz-Tp@Cu.

[0037] From the appendix Figure 2 As can be seen, after the introduction of copper ions, Iz-Tp@Cu still exhibits a distinct diffraction peak at 4.64°, indicating that the introduction of copper ions did not alter the crystal structure of Iz-Tp COF. Meanwhile, from the attached... Figure 3The scanning electron microscope images show that Iz-Tp@Cu has a spherical structure with a diameter of about 200 nm, and N, S and Cu elements are uniformly distributed on the surface of Iz-Tp@Cu.

[0038] Example 6 Iz-Tp@Cu-based colorimetric detection of adrenaline: In a Tris-HAc buffer solution at pH 6.0–9.0, 200 µM epinephrine was added to 40 μL of Iz-Tp@Cu (2.0 mg / mL), mixed well, and reacted at 30 °C for 30 minutes. Finally, the absorption spectrum from 300–700 nm was measured using a UV-Vis absorption spectrometer. (From the attached...) Figure 4 As can be seen, after Iz-Tp@Cu reacted with adrenaline for 30 minutes, a characteristic absorption peak of the adrenaline oxidation product appeared at 485 nm, indicating that Iz-Tp@Cu successfully catalyzed the oxidation reaction of adrenaline.

[0039] Example 7 Determining the optimal pH: This embodiment investigated the effect of pH on the colorimetric detection of adrenaline, as shown in the attached figure. Figure 5 As shown, the ability of Iz-Tp@Cu to catalyze the oxidation of adrenaline gradually increases within the pH range of 3.0–7.0, and the optimal catalytic oxidation ability is obtained at pH 7.0. Subsequently, the catalytic oxidation ability of Iz-Tp@Cu weakens with further increase in pH. Therefore, pH 7.0 was selected as the optimal pH.

[0040] Example 8 Plotting the standard curve of adrenaline: A series of concentrations were designed and detected between 0 and 500 µM. The detection results are as follows: Figure 6 As shown, the intensity of the characteristic absorption peak at 485 nm gradually increases with increasing adrenaline concentration. Within the adrenaline concentration range of 5.0–500.0 µM, the absorption intensity at 485 nm exhibits a good linear relationship with the adrenaline concentration, with a linear correlation coefficient of [value missing]. R 2 = 0.9972, the linear equation is A 485 =0.0014 c + 0.0059 (wherein, A 485 The absorption peak intensity is at 485 nm. c (This refers to the concentration of adrenaline). In addition to ultraviolet-visible absorption spectrometry, quantitative detection of adrenaline can also be achieved via smartphones.

[0041] Example 9 The influence of coexisting substances To investigate whether Iz-Tp@Cu exhibits good selectivity for the detection of adrenaline, common coexisting substances in biological samples were examined in the experiment. For example... Figure 7 As shown, apart from adrenaline, other coexisting substances, such as bovine serum albumin, valine, threonine, leucine, starch, cysteine, ascorbic acid, sodium chloride, potassium chloride, calcium chloride, zinc nitrate, and magnesium nitrate, do not cause any color change in the solution, even at concentrations 10 times that of adrenaline, and the absorption at 485 nm also shows no significant change. This indicates that Iz-Tp@Cu has good selectivity for adrenaline.

[0042] Example 10 Detection of adrenaline levels in urine samples: Urine sample processing: Urine from healthy individuals is first centrifuged at 10,000 rpm for 45 minutes to obtain the supernatant, which is the urine sample. The specific procedure for determining the adrenaline working curve in urine samples is as follows: In a 1.5 mL centrifuge tube, add 50 μL Tris-HAc buffer, 40 μL Iz-Tp@Cu (2.0 mg / mL), 20 μL urine sample, and a certain amount of adrenaline (10, 25, 50, 75, 100, 150, or 200 μmol / L), bring the volume to 1 mL, mix well, and react at 30 °C for 45 minutes. Finally, measure the absorption spectrum from 350 to 700 nm using a UV-Vis absorption spectrometer. Based on the absorption intensity at 485 nm and the adrenaline concentration, the working curve of adrenaline can be obtained as follows: A 485 =0.0009 c + 0.0138 ( R 2 = 0.9928).

[0043] The specific procedure for detecting adrenaline in urine samples is as follows: (1) In three centrifuge tubes, add 50 μL Tris-HAc buffer solution, 40 μL Iz-Tp@Cu (2.0 mg / mL), 20 μL urine sample, and epinephrine of unknown concentration. Make up to 1 mL, mix well, and react at 30 °C for 45 minutes. Finally, measure the absorption spectrum of the solution using a UV-Vis absorption spectrometer and read the absorbance at 485 nm. A 1.

[0044] (2) Obtained from the working curve of adrenaline in urine samples AThe adrenaline concentration corresponding to 1 is used to calculate the adrenaline content in urine.

[0045] The absorbance intensities of the two urine samples measured using this method were 0.0574 and 0.1035, respectively. According to the linear equation... A 485 = 0.0009 c + 0.0138 ( R 2 = 0.9928) The adrenaline concentrations in the urine samples were calculated to be 48.4 μM and 99.7 μM, respectively. The spiked recovery rates were calculated by comparing with blank urine samples, and the results are shown in the table below: Table 1. Results of adrenaline content determination in urine samples

[0046] As shown above, because the concentration of adrenaline in the urine sample measured in this example was lower than the detection limit of the method provided by this invention, it could not be directly measured. Therefore, a spiked method was used to verify the applicability of this method in urine samples. Table 1 shows that the method of this invention can detect the adrenaline content in urine samples, with spiked recoveries of 96.8% and 99.7%, and relative standard deviations of 4.21 and 3.89, respectively. This indicates that the detection method of this invention has good accuracy and can be used for the colorimetric detection of adrenaline.

[0047] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a copper-doped covalent organic framework (Iz-Tp@Cu), characterized in that, 20.0 mg of covalent organic framework Iz-Tp COF and 10.0–30.0 mL of 0.24 mol / L copper acetate were ultrasonically mixed and reacted at 120–180 °C for 3–8 hours. The mixture was then washed with deionized water and finally dried under vacuum at 70 °C for 12 hours to obtain the product, which is Iz-Tp@Cu.

2. The method for preparing the copper-doped covalent organic framework (Iz-Tp@Cu) according to claim 1, characterized in that, The covalent organic framework Iz-Tp COF was prepared using the following process: Using 1H-indazole-4,7-diamine (Iz) and 1,3,5-tricarboxymethyl phloroglucinol (Tp) as monomers in a molar ratio of 1:1 to 2:1, the mixture obtained by the Schiff base reaction is cooled to room temperature, washed 3-5 times each with N,N-dimethylacetamide (DMAC), mesitylene, and ethanol, and then vacuum dried at 70°C for 12 hours to obtain the product Iz-Tp COF.

3. The method for preparing the copper-doped covalent organic framework (Iz-Tp@Cu) according to claim 2, characterized in that, The Schiff base reaction conditions are: Using N,N-dimethylacetamide (DMAC) and mesitylene as solvents in a volume ratio of 1:1 to 4:1, and 0.1 to 0.5 mL of acetic acid at a concentration of 6 mmol / L as catalyst, the monomer, solvent, and catalyst were mixed thoroughly and subjected to three freeze-pump thaw cycles. The mixture was then flame-sealed under vacuum and heated at 100 to 150 °C for 1 to 5 days.

4. The application of the copper-doped covalent organic framework (Iz-Tp@Cu) prepared according to any one of claims 1-3 in the colorimetric detection of adrenaline, characterized in that, Iz-Tp@Cu can catalyze the oxidation of adrenaline to produce a brown product. Based on the color changes caused by different concentrations of adrenaline, a colorimetric detection method for adrenaline can be established.

5. The application according to claim 4, characterized in that, The following process was used for the colorimetric detection of adrenaline using a copper-doped covalent organic framework (Iz-Tp@Cu): In a Tris-HAc buffer solution at pH 6.0–9.0, a certain amount of adrenaline was added to 40 μL of Iz-Tp@Cu at a concentration of 2.0 mg / mL. After mixing, the mixture was reacted at 25–50 °C for 30–60 minutes. Subsequently, the absorption spectrum of 300-700 nm was measured using a UV-Vis absorption spectrometer; The concentration of adrenaline is calculated using formula (1): A 485 =0.0014 c + 0.0059（1） in, A 485 - The absorption peak intensity at 485 nm after the addition of adrenaline; c – Adrenaline concentration; linear correlation coefficient R 2 = 0.9972.

6. The application according to claim 5, characterized in that, The concentration range of the adrenaline is 5.0 – 500.0 μM.

7. The application according to claim 5, characterized in that, One or more of the following ingredients, a mixture of bovine serum albumin, valine, threonine, leucine, starch, cysteine, ascorbic acid, sodium chloride, potassium chloride, calcium chloride, zinc nitrate, and magnesium nitrate, were added to Tris-HAc buffer and Iz-Tp@Cu mixed solution to verify the effect of common interfering substances on adrenaline detection.

8. The application according to claim 5, characterized in that, The concentration of adrenaline in urine was determined using a spiking method.

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