Metal-doped hydrogen bond-based organic framework material and preparation method and application thereof

Abstract

The invention belongs to the technical field of electrochemical analysis and detection, and particularly relates to an organic framework material based on metal doped hydrogen bonds and a preparation method and application thereof. The method comprises the following steps: taking melamine, cyanuric acid and 1, 3, 5-benzene tricarboxylic acid as three hydrogen bond monomers, adding a solvent, and synthesizing a hydrogen bond organic framework material through a hydrothermal method; the preparation method comprises the following steps: dispersing a hydrogen bond organic framework material in water, and then adding cobalt salt to obtain HOF (at) Co; and then adding heme to obtain the organic framework material based on the metal doped hydrogen bond. The metal-doped hydrogen bond-based organic framework material provided by the invention can be applied to glucose detection, experimental data shows that the glucose linear detection range is 0.06-6 [mu] M, the detection limit is as low as 53 nM, and the metal-doped hydrogen bond-based organic framework material can be applied to quantitative detection of glucose in a human serum sample.

Description

Technical Field

[0001] This application belongs to the technical field of electrochemical analysis and detection, and specifically relates to a metal-doped hydrogen-bonded organic framework material, its preparation method and application. Background Art

[0002] Glucose is the main energy source of organisms and plays an important role in its physiological and pathological processes. It is reported that abnormal glucose levels in biological fluids are often related to the development of metabolic-related diseases, including hyperglycemia, diabetes and obesity. Therefore, the development of sensitive and selective blood glucose detection methods is of great significance for the diagnosis of related diseases. Currently, a variety of methods have been designed to monitor glucose concentration, including chromatographic detection and spectroscopic analysis, such as colorimetric analysis, chemiluminescence (abbreviated as CL) detection and fluorescence detection. Among the above methods, the CL detection method has attracted much attention due to its rapid reaction, high sensitivity, low background interference and simple instrument operation, and has great potential in bioanalysis applications.

[0003] Enzyme catalysis has the characteristics of high efficiency, specificity, mild reaction conditions, etc., and is widely used in various fields such as chemical engineering, medical and pharmaceutical, food hygiene and agricultural production. However, natural enzymes are difficult to isolate and purify, costly, expensive and have poor stability, which is difficult to meet the needs of glucose detection. Therefore, there is an urgent need for stable and easy-to-prepare mimetic enzymes to meet the requirements of glucose detection. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art, and provide a metal-doped hydrogen-bonded organic framework material, its preparation method and application. The specific technical solutions are as follows: In the first aspect, the present invention provides a preparation method of a metal-doped hydrogen-bonded organic framework material, including the following steps: S1. Using melamine, cyanuric acid, and 1,3,5-benzenetricarboxylic acid as three hydrogen-bond monomers, adding a solvent, and synthesizing a hydrogen-bonded organic framework material by a hydrothermal method; S2. Dispersing the hydrogen-bonded organic framework material in water, then adding a cobalt salt, stirring, washing, and centrifuging to obtain HOF@Co; S3. Dispensing the HOF@Co in N,N-dimethylformamide, then adding hemin, stirring, washing, centrifuging, and drying to obtain the metal-doped hydrogen-bonded organic framework material.

[0005] The present invention uses three hydrogen-bond reaction units, such as melamine (MA), cyanuric acid (CA), and 1,3,5-benzenetricarboxylic acid (H3BTC), to prepare rod-shaped HOF; then, in a mixed solution containing HOF and Co 2+ by ligand H3BTC and Co 2+The direct coordination process between ions was used to prepare HOF@Co. Further, in a mixed solution containing HOF@Co and Heme, the HOF building units and Heme self-organized and assembled through π-π stacking, hydrogen bonding, and electrostatic interactions to form HOF@Co / Heme.

[0006] As a further preferred embodiment, the solvent includes at least one of methanol or N,N-dimethylformamide.

[0007] As a further preferred embodiment, the cobalt salt includes at least one of CoCl2 or Co(NO3)2.

[0008] As a further preferred embodiment, the dosage ratio of HOF@Co, N,N-dimethylformamide, and heme in S3 is 50 mg: 15 mL: 40 mg.

[0009] In a second aspect, the present invention provides a metal-doped hydrogen-bonded organic framework material prepared by the above preparation method.

[0010] In a third aspect, the present invention provides the application of the above metal-doped hydrogen-bonded organic framework material in the quantitative and / or qualitative detection of glucose.

[0011] In a fourth aspect, the present invention provides the application of the above metal-doped hydrogen-bonded organic framework material in the preparation of a product for quantitatively detecting glucose.

[0012] In a fifth aspect, the present invention provides a product for quantitatively detecting glucose, and the product includes the above metal-doped hydrogen-bonded organic framework material.

[0013] In a sixth aspect, the present invention provides a method for quantitatively detecting glucose, including the following steps: Mix the glucose solution to be measured with glucose oxidase, react, then sequentially add a luminol standard solution and the suspension of the above metal-doped hydrogen-bonded organic framework material, and finally detect the CL signal through a chemiluminescence detector (CL detector), and determine the glucose concentration according to the standard curve.

[0014] As a further preferred embodiment, the luminol standard solution is prepared with luminol as the solute and sodium hydroxide solution as the solvent.

[0015] The beneficial effects of the present invention are as follows: (1) In the present invention, heme and Co are loaded in the hydrogen-bonded organic framework 2+HOF@Co / Heme with stable peroxidase-like activity was synthesized for the establishment of a sensitive chemiluminescence (CL) method for glucose detection. Compared with the commonly used horseradish peroxidase (HRP), HOF@Co / Heme has excellent stability and can be stored at room temperature. Radical scavenger experiments and electron paramagnetic resonance (EPR) tests showed that reactive oxygen species (ROS), including 1 O2, OH· and O2· - , were generated during the catalytic decomposition of H2O2 by HOF@Co / Heme, significantly enhancing the light signal of the luminol-H2O2 CL system.

[0016] (2) A CL biosensor based on the tandem catalysis of GOx and HOF@Co / Heme was developed for sensitive glucose detection. GOx can catalyze the oxidation of glucose and provide H2O2 to HOF@Co / Heme to generate ROS and enhance the CL signal. The linear detection range of the GOx-HOF@Co / Heme-based biosensor is 0.06 μM to 6 μM, and the detection limit is as low as 53 nM. In addition, the sensor has high detection stability and can be applied to the quantitative detection of glucose in human serum samples, showing practicality. Description of the Drawings

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 Shown are the schematic diagram for the preparation of metal-doped hydrogen-bonded organic framework materials and the schematic diagram for the detection of glucose by CL method using metal-doped hydrogen-bonded organic framework materials; Figure 2 Shown are the results of radical scavenger experiments and electron paramagnetic resonance (EPR) tests on metal-doped hydrogen-bonded organic framework materials; Figure 3 Shown is the quantitative standard curve for the detection of glucose by CL method using HOF@Co / Heme; Figure 4 Shown are the specificity results for the detection of glucose by CL method using HOF@Co / Heme; Figure 5 Shown are the stability results for the detection of glucose by CL method using HOF@Co / Heme; Figure 6 Shown is the comparison of the enzyme activities of HOF@Co / Heme with HOF@Co and HOF / Heme; Figure 7 Shown is the comparison of the enzyme activity of HOF@Co / Heme with other metal ion-loaded enzymes. Specific implementation manners

[0019] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0020] Example 1 A preparation method of a metal-doped hydrogen-bonded organic framework material (the schematic diagram thereof is as shown in Figure 1 A in the figure) includes the following steps: (1) Synthesis of HOF raw materials: Add 1.2 mM 1,3,5-benzenetricarboxylic acid (H3BTC, 0.2522 g), 1.2 mM melamine (MA, 0.1514 g), and 1.2 mM cyanuric acid (CA, 0.1549 g) to 30 mL of methanol, then ultrasonicate for 2 h, transfer to a high-temperature and high-pressure reaction kettle and heat at 120 °C for 12 h; then, wash three times with methanol and H2O respectively, and collect the product by centrifugation; finally, dry the synthesized HOF in an electrothermal constant-temperature drying oven at 60 °C for 12 h to obtain a white solid powder.

[0021] (2) Loading of Co on HOF 2+ : First, fully disperse the above solid powder in 30 mL of H2O, then add 1.5 mM CoCl2 (0.3569 g) and stir for 2 h; wash three times with H2O, and collect the prepared HOF@Co by centrifugation. Finally, dry HOF@Co in an electrothermal constant-temperature drying oven at 60 °C for 12 h to obtain a white solid powder; (3) Loading of hemin on HOF@Co: Fully disperse 50 mg of HOF@Co powder in 15 mL of DMF, then add 40 mg of hemin, stir for 3 h, and then wash three times with DMF and H2O respectively, and collect the prepared HOF@Co / Heme by centrifugation. Finally, dry HOF@Co / Heme in an electrothermal constant-temperature drying oven at 60 °C for 12 h to obtain a red solid powder HOF@Co / Heme.

[0022] Example 2 Explore the catalytic decomposition of H2O2 by the metal-doped hydrogen-bonded organic framework material (HOF@Co / Heme) to generate reactive oxygen species. The specific steps are as follows: First, a radical scavenging experiment was carried out. A 100 μL 100 μM H2O2 solution was mixed with 100 μL of 1 mM ascorbic acid, thiourea, tryptophan, and hydroxylamine hydrochloride solutions as the experimental group; a 100 μL 100 μM H2O2 solution was mixed with 100 μL of ultrapure water as the blank group. The above mixtures were transferred to a glass sample cell (φ14 mm × 14 mm). Subsequently, 50 μL of 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately detected using a CL detector. The reactive oxygen free radicals generated were determined based on the signal intensity. Secondly, electron paramagnetic resonance (EPR) analysis was performed. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was used as a scavenger for hydroxyl radicals (OH·) and superoxide anions (O2· - ). 10 μL of 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO), 25 μL of 100 μM H2O2 solution, and 25 μL of 200 μg / mL HOF@Co / Heme suspension were added to the reaction cell, and then the characteristic signal was detected using an electron paramagnetic resonance spectrometer; 2,2,6,6-Tetramethylpiperidine (TEMP) was used as a scavenger for singlet oxygen ( 1 O2). 10 μL of DMPO, 25 μL of 100 μM H2O2 solution, and 25 μL of 200 μg / mL HOF@Co / Heme suspension were added to the reaction cell, and then the characteristic signal was detected using an electron paramagnetic resonance spectrometer.

[0023] The results are as Figure 2 shown. The results of the radical scavenging experiment and EPR analysis show that HOF@Co / Heme can effectively catalyze the decomposition of H2O2 to generate OH·, O2· - and 1 O2, thereby inducing CL and achieving the detection of glucose.

[0024] Example 3 The above-prepared metal-doped hydrogen-bonded organic framework material (HOF@Co / Heme) was used for the detection of glucose by the CL method (the schematic diagram is as shown in Figure 1 B), and the specific steps are as follows: First, 150 μL of glucose solutions with different concentrations (0.06 μM, 0.4 μM, 0.6 μM, 0.9 μM, 2.5 μM, 4 μM, and 6 μM) were mixed with 50 μL of 1 mg / mL GOx solution. After mixing, the mixture was placed in a constant temperature oscillator at 37 °C for 25 min. Then, the above mixture was transferred to a glass sample cell (φ14 mm × 14 mm), and 50 μL of 0.1 mM / L luminol standard solution (prepared with luminol as the solute and 0.1 mM sodium hydroxide solution as the solvent) was added. Subsequently, 50 μL of 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately detected using a CL detector. A standard curve was established based on the relationship between the CL intensity and the glucose concentration as Figure 3 shown, and it can be seen from Figure 3 that there is a linear correlation between the CL intensity and the glucose concentration, ranging from 0.06 μM to 6 μM. Using the regression equation y = 2.96×10 6 C 葡萄糖 + 4.62 × 10 6 (R 2 = 0.9924), the detection limit was 53 nM. The detection limit was calculated using the classical 3σ / k method, where σ is the standard deviation of the blank sample measurement and k is the slope of the calibration curve. Therefore, it can be seen that the CL sensor provided by the present invention shows a good quantitative range, spanning three orders of magnitude.

[0025] Example 4 Detection of Serum Samples for Glucose by CL Method Based on Metal-Doped Hydrogen Bonded Organic Framework Material (HOF@Co / Heme) To confirm the effectiveness of HOF@Co / Heme for the CL method in determining the glucose concentration in human serum samples, in this example, each serum sample was diluted 10,000 times, and then 1.0 μM and 4.0 μM glucose standard solutions were added. Subsequently, 50 μL of 1 mg / mL GOx solution was mixed with 150 μL of the serum sample added with the glucose standard solution, and after mixing, it was placed in a constant temperature oscillator at 37 °C for reaction for 25 min; then the above mixed solution was transferred to a glass sample cell (φ14 mm × 14 mm), and then 50 μL of 0.1 mM / L luminol standard solution (prepared with luminol as the solute and 0.1 mM sodium hydroxide solution as the solvent) was added; subsequently, 50 μL of 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately detected with a CL detector. The glucose content was determined according to the standard curve. The results are shown in Table 1, with a recovery rate of 95.7% to 99.7% and a relative standard deviation (RSD) of 3.7% to 6.6%, indicating the practical feasibility of the CL sensor based on HOF@Co / Heme.

[0026] Table 1 Analysis of Glucose in Diluted Human Serum Samples by Chemiluminescence Method Example 5 Detection of Urine Samples for Glucose by CL Method Based on Metal-Doped Hydrogen Bonded Organic Framework Material (HOF@Co / Heme) To confirm the effectiveness of HOF@Co / Heme for the CL method in determining the glucose concentration in human urine samples, in this example, each urine sample was diluted 100 times, and then 1.0 μM and 4.0 μM glucose standard solutions were added. Subsequently, 50 μL of 1 μg / mL GOx solution was mixed with 150 μL of the urine sample added with the glucose standard solution, and after mixing, it was placed in a constant temperature oscillator at 37 °C for reaction for 25 min; then the above mixed solution was transferred to a glass sample cell (φ14 mm × 14 mm), and then 50 μL of 0.1 mM / L luminol standard solution (prepared with luminol as the solute and 0.1 mM sodium hydroxide solution as the solvent) was added; subsequently, 50 μL of 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately detected with a CL detector. The glucose content was determined according to the standard curve. The results are shown in Table 2, with a recovery rate of 94% to 109% and a relative standard deviation (RSD) of 2.7% to 5.4%, indicating the practical feasibility of the CL sensor based on HOF@Co / Heme.

[0027] Table 2 Analysis of Glucose in Diluted Human Urine Samples by Chemiluminescence Method Example 6 Selectivity and Stability Analysis of Metal-Doped Hydrogen Bonded Organic Framework Material (HOF@Co / Heme) for Glucose Detection by CL Method (1) To test the specific selectivity of the experimental method, 150 μL of 4 μM glucose solution and various interfering substances in serum (water, leucine, threonine, aspartic acid, lysine, glycine, sodium ion, calcium ion, potassium ion, and glutathione) were mixed with 50 μL of 1 mg / mL GOx solution, and the mixture was placed in a constant temperature oscillator at 37 °C for 25 min; then the above mixture was transferred to a glass sample cell (φ14 mm × 14 mm), and 50 μL of 0.1 mM / L luminol standard solution (prepared with luminol as the solute and 0.1 mM sodium hydroxide solution as the solvent) was added; subsequently, 50 μL of 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately detected with a CL detector to explore the influence of different substances on the chemiluminescence signal intensity.

[0028] The results are as Figure 4 shown. The results confirm that there are obvious differences in the signal intensities of the target glucose and interfering substances; the high signal value of glucose indicates that the present invention has high specificity. These research results not only confirm the selectivity of the present invention but also highlight its potential in measuring glucose levels in biological samples in practical applications.

[0029] (2) To test the stability of the experimental method, 150 μL of 4 μM glucose solution was mixed with 50 μL of 1 mg / mL GOx solution, and the mixture was placed in a constant temperature oscillator at 37 °C for 25 min; then the above mixture was transferred to a glass sample cell (φ14 mm × 14 mm), and 50 μL of 0.1 mM / L luminol standard solution (prepared with luminol as the solute and 0.1 mM sodium hydroxide solution as the solvent) was added; subsequently, 50 μL of 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately detected with a CL detector, and the detector was used once every 5 days within 30 days.

[0030] The results are as Figure 5 shown. During the one-month research period, the CL intensity remained almost the same with very little change. The results show that the HOF@Co / Heme material has excellent stability and catalytic performance, and the CL sensor provided by the present invention has good reproducibility in glucose detection.

[0031] Example 7 Determination of Glucose Content in Unknown Samples by CL Method Based on Metal-Doped Hydrogen Bonded Organic Framework Material (HOF@Co / Heme) To determine the practicality of this method for detecting unknown samples, the unknown serum was diluted 1000 times. 150 μL of the serum sample was mixed with 50 μL of 1 μg / mL GOx solution, and the mixture was placed in a constant temperature oscillator at 37 °C for 25 min. Then, the above mixture was transferred to a glass sample cell (φ14 mm × 14 mm), and 50 μL of 0.1 mM / L luminol standard solution (prepared with luminol as the solute and 0.1 mM sodium hydroxide solution as the solvent) was added. Subsequently, 50 μL of 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately detected with a CL detector. The glucose content was determined according to the standard curve. The detection result showed that the glucose content of the unknown sample was 5.53 mM, and the standard deviation from the hospital detection result was 6.2%.

[0032] Comparative Example 1 Comparison of Enzyme Activities between Metal-Doped Hydrogen Bonded Organic Framework Material (HOF@Co / Heme) and Those Only Loaded with Co 2+ and Those Only Loaded with Heme (HOF / Heme) To determine the beneficial effect of loading Co 2+ and heme on peroxidase activity, 150 μL of 4 μM glucose solution was mixed with 50 μL of 1 mg / mL GOx solution, and the mixture was placed in a constant temperature oscillator at 37 °C for 25 min. Then, the above mixture was transferred to a glass sample cell (φ14 mm × 14 mm), and 50 μL of 0.1 mM / L luminol standard solution (prepared with luminol as the solute and 0.1 mM sodium hydroxide solution as the solvent) was added. Subsequently, 50 μL of 200 μg / mL HOF@Co / Heme, HOF, HOF@Co, and HOF / Heme suspensions were quickly added, and the CL signal was immediately detected with a CL detector. The strength of the enzyme activity was determined by the signal intensity. The results are as Figure 6 shown. The peroxidase activity is the strongest after loading Co 2+ and heme, indicating that loading Co 2+ and heme is beneficial to peroxidase activity.

[0033] Comparative Example 2 Comparison of Enzyme Activities between Metal-Doped Hydrogen Bonded Organic Framework Material (HOF@Co / Heme) and Those Loaded with Other Metal Ions To determine that the peroxidase activity of loading Co 2+ is better, the peroxidase activities of loading Co 2+ and other metals (Cu2+ , Zn 2+ , Cr 3+ , Hg 2+ ), the enzyme activity was determined by mixing 150 μL of 4 μM glucose solution with 50 μL of 1 mg / mL GOx solution, and then reacting the mixture in a constant temperature oscillator at 37 °C for 25 min; then the above mixture was transferred to a glass sample cell (φ14 mm × 14 mm), and 50 μL of 0.1 mM / L luminol standard solution (prepared with luminol as the solute and 0.1 mM sodium hydroxide solution as the solvent) was added; subsequently, 50 μL of 200 μg / mL HOF@Co / Heme, HOF@Cu / Heme, HOF@Zn / Heme, HOF@Cr / Heme, HOF@Hg / Heme suspension was quickly added, and the CL signal was immediately detected with a CL detector, and the enzyme activity was determined by the signal intensity. The results are as Figure 7 shown. The peroxidase activity was the strongest after loading with Co 2+ and heme, indicating that the loading of Co 2+ had the best effect.

[0034] Comparative Example 3 Comparison of Metal-Doped Hydrogen Bonded Organic Framework Material (HOF@Co / Heme) with the Prior Art To determine the application value of HOF@Co / Heme for detecting glucose, a comparison was made with the prior art. GOx / MOF-919@ZIF-8 was used as a probe for detecting glucose. The detection process was the same as that in Example 2. Its linear range was 0.05 - 1 mM, and the detection limit was 0.027 mM. The present invention had a lower detection limit (53 nM), indicating better performance of the present invention.

[0035] The embodiments of the present application have been described above with reference to the accompanying drawings. Specific examples have been used herein to elaborate on the principle and implementation manner of the present application. The description of the above embodiments is only used to help understand the core idea of the present application. However, the present application is not limited to the above specific implementation manners. The above specific implementation manners are only illustrative and not restrictive. Those of ordinary skill in the art, under the inspiration of the present application, without departing from the purpose of the present application and the scope protected by the claims, can also make many forms, all of which belong to the protection scope of the present application.

Claims

1. A method for preparing a metal-doped hydrogen-bonded organic framework material, characterized in that: The following steps are involved: S1. Using melamine, cyanuric acid and 1,3,5-benzenetricarboxylic acid as three hydrogen-bonding monomers, adding solvent, and synthesizing hydrogen-bonding organic framework materials by hydrothermal method; S2, dispersing the hydrogen-bonded organic framework material in water, then adding cobalt salt, stirring, washing, and centrifuging to obtain HOF@Co; S3. Dispersing the HOF@Co in N,N-dimethylformamide, then adding heme, stirring, washing, centrifuging, and drying to obtain the metal-doped hydrogen bond organic framework material.

2. The preparation method according to claim 1, characterized in that: The solvent includes at least one of methanol or N,N-dimethylformamide.

3. The preparation method according to claim 1, characterized in that: The cobalt salt includes at least one of CoCl2 or Co(NO3)2.

4. The preparation method according to claim 1, characterized in that: The usage ratio of HOF@Co and N,N-dimethylformamide to neutralize heme described in S3 is 50 mg:15 mL:40 mg.

5. A metal-doped hydrogen-bonded organic framework material, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the metal-doped hydrogen-bonded organic framework material according to claim 5 in quantitative and / or qualitative detection of glucose.

7. Use of the metal-doped hydrogen-bonded organic framework material according to claim 5 in the preparation of products for quantitative detection of glucose.

8. A product for quantitative detection of glucose, characterized in that: The product includes the metal-doped hydrogen-bonding organic framework material according to claim 5.

9. A method for quantitatively detecting glucose, characterized in that: The following steps are involved: The glucose test solution is mixed with glucose oxidase to react, and then a luminol standard solution and a suspension of the metal-doped hydrogen-bonded organic framework material according to claim 5 are added in sequence. Finally, the CL signal is detected by a CL detector, and the glucose concentration is determined according to the standard curve.

10. The method according to claim 9, characterized in that The luminol standard solution is prepared by taking luminol as solute and sodium hydroxide solution as solvent.

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