Metal-doped hydrogen-bonded organic framework materials and their preparation methods and applications

Abstract

The present application belongs to the field of electrochemical analysis and detection technology, and specifically relates to a metal-doped hydrogen-bonded organic framework material, its preparation method and application. The method uses melamine, cyanuric acid and 1,3,5-benzenetricarboxylic acid as three hydrogen-bonding monomers, adds a solvent, and synthesizes a hydrogen-bonded organic framework material by a hydrothermal method; the hydrogen-bonded organic framework material is dispersed in water, and then a cobalt salt is added to obtain HOF@Co; and heme is added to obtain a metal-doped hydrogen-bonded organic framework material. The present invention provides that the metal-doped hydrogen-bonded organic framework material can be used for glucose detection. The experimental data according to the present invention show that the linear detection range for glucose is 0.06 μM to 6 μM, the detection limit is as low as 53 nM, and it can be applied to the quantitative detection of glucose in human serum samples.

Description

Technical Field

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

[0002] Glucose is the primary energy source for organisms and plays a crucial role in their physiological and pathological processes. Abnormal glucose levels in biological fluids are often associated with the development of metabolic diseases, including hyperglycemia, diabetes, and obesity. Therefore, the development of sensitive and selective glucose detection methods is crucial for the diagnosis of these diseases. Currently, a variety of methods have been developed to monitor glucose concentration, including chromatographic and spectroscopic analysis, such as colorimetric analysis, chemiluminescence (CL), and fluorescence. Among these methods, CL has attracted considerable attention due to its rapid response, high sensitivity, minimal background interference, and simple instrument operation, and holds great potential for bioanalytical applications.

[0003] Enzyme catalysis, characterized by high efficiency, specificity, and mild reaction conditions, is widely used in fields such as chemical engineering, medicine, food hygiene, and agricultural production. However, natural enzymes are difficult to isolate and purify, are costly, expensive, and have poor stability, making them difficult to meet the needs of glucose detection. Therefore, there is an urgent need for stable and easily prepared enzyme mimics to meet the requirements of glucose detection. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide a metal-doped hydrogen-bonded organic framework material and its preparation method and application, specifically adopting the following technical solutions:

[0005] In a first aspect, the present invention provides a method for preparing a metal-doped hydrogen-bonded organic framework material, comprising the following steps:

[0006] S1. Using melamine, cyanuric acid, and 1,3,5-benzenetricarboxylic acid as three hydrogen-bonding monomers and adding a solvent, a hydrogen-bonding organic framework material is synthesized by a hydrothermal method;

[0007] S2, dispersing the hydrogen-bonded organic framework material in water, then adding cobalt salt, stirring, washing, and centrifuging to obtain HOF@Co;

[0008] S3. Dispersing the HOF@Co in N,N-dimethylformamide, then adding hemoglobin, stirring, washing, centrifuging, and drying to obtain the metal-doped hydrogen bond organic framework material.

[0009] The present invention adopts three hydrogen bond reaction units, namely melamine (MA), cyanuric acid (CA) and 1,3,5-benzenetricarboxylic acid (H3BTC), to prepare rod-shaped HOF. Then, in the presence of HOF and Co2+ In the mixed solution, the ligand H3BTC and Co 2+ HOF@Co was prepared through the direct coordination process between ions. Furthermore, in a mixed solution containing HOF@Co and Heme, the HOF constituent units and Heme self-assembled through π-π stacking, hydrogen bonding and electrostatic interactions to form HOF@Co / Heme.

[0010] As a further preferred embodiment, the solvent includes at least one of methanol or N,N-dimethylformamide.

[0011] As a further preferred embodiment, the cobalt salt includes at least one of CoCl2 or Co(NO3)2.

[0012] As a further preferred embodiment, the usage ratio of HOF@Co, N,N-dimethylformamide and hemoglobin in S3 is 50 mg:15 mL:40 mg.

[0013] In a second aspect, the present invention provides a metal-doped hydrogen-bonded organic framework material prepared by the above-mentioned preparation method.

[0014] In a third aspect, the present invention provides the use of the above-mentioned metal-doped hydrogen-bonding organic framework material in the quantitative and / or qualitative detection of glucose.

[0015] In a fourth aspect, the present invention provides the use of the above-mentioned metal-doped hydrogen-bonding organic framework material in the preparation of products for quantitative detection of glucose.

[0016] In a fifth aspect, the present invention provides a product for quantitative detection of glucose, which comprises the above-mentioned metal-doped hydrogen-bonding organic framework material.

[0017] In a sixth aspect, the present invention provides a method for quantitatively detecting glucose, comprising the following steps:

[0018] The glucose test solution is mixed with glucose oxidase to react, and then a luminol standard solution and the above-mentioned suspension of metal-doped hydrogen-bonded organic framework materials are added in sequence. Finally, the CL signal is detected by a chemiluminescence detector (CL detector), and the glucose concentration is determined according to the standard curve.

[0019] As a further preferred embodiment, the luminol standard solution is prepared with luminol as the solute and sodium hydroxide solution as the solvent.

[0020] The beneficial effects of the present invention are:

[0021] (1) The present invention loads heme and Co in a hydrogen bond organic framework 2+HOF@Co / Heme with stable peroxidase-like activity was synthesized and used to establish a sensitive glucose chemiluminescence detection method. Compared with the commonly used horseradish peroxidase (HRP), HOF@Co / Heme has excellent stability and can be stored at room temperature. Free radical scavenger experiments and electron paramagnetic resonance (EPR) tests showed that during the catalytic decomposition of H2O2 by HOF@Co / Heme, reactive oxygen species (ROS) were generated, including 1 O2, OH· and O2· - , significantly enhancing the optical signal of the luminol-H2O2CL system.

[0022] (2) A CL biosensor based on GOx and HOF@Co / Heme tandem catalysis was developed for sensitive detection of glucose. GOx catalyzed the oxidation of glucose and provided H2O2 to HOF@Co / Heme to generate ROS and enhance the CL signal. The GOx-HOF@Co / Heme-based biosensor had a linear detection range of 0.06 μM to 6 μM, with a detection limit as low as 53 nM. In addition, the sensor had high detection stability and could be applied to the quantitative detection of glucose in human serum samples, showing practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Shown are schematic diagrams of the preparation of metal-doped hydrogen-bonding organic framework materials and the use of metal-doped hydrogen-bonding organic framework materials for the detection of glucose by CL method;

[0025] Figure 2 Shown are the results of free radical scavenger experiments and electron paramagnetic resonance (EPR) tests on metal-doped hydrogen-bonded organic framework materials;

[0026] Figure 3 Shown is the quantitative standard curve of HOF@Co / Heme used in the CL method for glucose detection;

[0027] Figure 4 Shown are the specific results of HOF@Co / Heme for glucose detection by CL method;

[0028] Figure 5 Shown are the stability results of HOF@Co / Heme for glucose detection using the CL method;

[0029] Figure 6 Shown is the comparison of enzyme activities between HOF@Co / Heme, HOF@Co, and HOF / Heme;

[0030] Figure 7 Shown is the comparison of enzyme activity of HOF@Co / Heme with other metal ion loadings. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0032] Example 1

[0033] A preparation method based on metal-doped hydrogen-bonded organic framework materials (schematic diagram as shown in FIG Figure 1 As shown in A in FIG), comprising the following steps:

[0034] (1) Synthesis of HOF raw materials: 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) were added to 30 mL of methanol, then ultrasonicated for 2 h, moved to a high-temperature and high-pressure reactor and heated at 120 °C for 12 h; then, washed with methanol and H2O three times, respectively, and the product was collected by centrifugation; finally, the synthesized HOF was dried in an electric constant temperature drying oven at 60 °C for 12 h to obtain a white solid powder.

[0035] (2) HOF loaded with Co 2+ First, the solid powder was fully dispersed in 30 mL of H2O. Subsequently, 1.5 mM CoCl2 (0.3569 g) was added and stirred for 2 h. The mixture was then washed three times with H2O and the prepared HOF@Co was collected by centrifugation. Finally, the HOF@Co was dried in an electric constant temperature drying oven at 60 °C for 12 h to obtain a white solid powder.

[0036] (3) HOF@Co loaded with heme: 50 mg of HOF@Co powder was fully dispersed in 15 mL of DMF, and then 40 mg of heme was added. After stirring for 3 h, the mixture was washed with DMF and H2O three times, respectively, and the prepared HOF@Co / Heme was collected by centrifugation. Finally, HOF@Co / Heme was dried in an electric constant temperature drying oven at 60 °C for 12 h to obtain red solid powder HOF@Co / Heme.

[0037] Example 2

[0038] The metal-doped hydrogen-bonded organic framework material (HOF@Co / Heme) was used to catalyze the decomposition of H2O2 to produce reactive oxygen radicals. The specific steps are as follows:

[0039] First, a free radical scavenging experiment was conducted. 100 μL of a 100 μM H2O2 solution was mixed with 100 μL of a 1 mM ascorbic acid, thiourea, tryptophan, and hydroxylamine hydrochloride solution as the experimental group. A blank control was prepared by mixing 100 μL of a 100 μM H2O2 solution with 100 μL of ultrapure water. The mixture was transferred to a glass sample cell (φ14 mm × 14 mm). Subsequently, 50 μL of a 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately detected using a CL detector. The generated reactive oxygen species were determined based on the signal intensity. Electron spin resonance (EPR) analysis was then performed, using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the scavenger for hydroxyl radicals (OH·) and superoxide anions (O2·). - ) capture agent, 10 μL 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 25 μL 100 μM H2O2 solution and 25 μL 200 μg / mL HOF@Co / Heme suspension were added to the reaction pool, and then added to the electron spin resonance instrument to detect the characteristic signal; 2,2,6,6-tetramethylpiperidine (TEMP) was used as the singlet oxygen ( 1 O2) capture agent, 10 μL DMPO, 25 μL 100 μM H2O2 solution and 25 μL 200 μg / mL HOF@Co / Heme suspension were added to the reaction cell, and then added to the electron spin resonance instrument to detect the characteristic signal.

[0040] The results are as follows Figure 2 As shown in the figure, the free radical scavenging experiment and EPR analysis results show that HOF@Co / Heme can effectively catalyze the decomposition of H2O2 to generate OH·, O2· - and 1 O2, thereby inducing CL and realizing the detection of glucose.

[0041] Example 3

[0042] The metal-doped hydrogen-bonded organic framework material (HOF@Co / Heme) prepared above was used for the CL method to detect glucose (the schematic diagram is shown in Figure 1 The specific steps are as follows:

[0043] First, 150 μL of glucose solutions of 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 and placed in a constant temperature oscillator at 37 °C for 25 min. The mixture was then 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 solute and 0.1 mM sodium hydroxide solution as 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 CL intensity and glucose concentration. Figure 3 shown by Figure 3 It can be seen that there is a linear correlation between CL intensity and 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), with a detection limit of 53 nM. The detection limit was calculated using the classic 3σ / k method, where σ is the standard deviation of the blank sample measurement and k is the slope of the calibration plot. Therefore, the CL sensor provided by the present invention exhibits a good quantitative range, spanning three orders of magnitude.

[0044] Example 4

[0045] Detection of glucose in serum samples based on metal-doped hydrogen-bonded organic framework (HOF@Co / Heme) using CL method

[0046] To confirm the effectiveness of HOF@Co / Heme for the CL method for determining glucose concentration in human serum samples, each serum sample was diluted 10,000-fold and then spiked with 1.0 μM and 4.0 μM glucose standards. Subsequently, 150 μL of the serum sample with the glucose standard solution was mixed with 50 μL of a 1 mg / mL GOx solution. The mixture was then incubated in a 37°C constant-temperature shaker for 25 minutes. The mixture was then transferred to a glass sample cell (φ14 mm × 14 mm), and 50 μL of a 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 a 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately measured using a CL detector. The glucose content was determined based on the standard curve. The results are shown in Table 1 , with recoveries ranging from 95.7% to 99.7% and relative standard deviations (RSDs) ranging from 3.7% to 6.6%, indicating the practical feasibility of the HOF@Co / Heme-based CL sensor.

[0047] Table 1 Analysis of glucose in diluted human serum samples using chemiluminescence

[0048]

[0049] Example 5

[0050] Detection of glucose in urine samples using CL method based on metal-doped hydrogen-bonded organic framework (HOF@Co / Heme)

[0051] To confirm the effectiveness of HOF@Co / Heme for the CL method for determining glucose concentration in human urine samples, each urine sample was diluted 100-fold and spiked with 1.0 μM and 4.0 μM glucose standards. Subsequently, 150 μL of the urine sample spiked with the glucose standards was mixed with 50 μL of a 1 μg / mL GOx solution. The mixture was then incubated in a 37°C constant-temperature oscillator for 25 minutes. The mixture was then transferred to a glass sample cell (φ14 mm × 14 mm) and 50 μL of a 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 a 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately measured using a CL detector. The glucose content was determined based on the standard curve. The results are shown in Table 2 , with recoveries ranging from 94% to 109% and relative standard deviations (RSDs) ranging from 2.7% to 5.4%, indicating the practical feasibility of the HOF@Co / Heme-based CL sensor.

[0052] Table 2 Analysis of glucose in diluted human urine samples using chemiluminescence

[0053]

[0054] Example 6

[0055] Selective and stable analysis of glucose detection by CL method based on metal-doped hydrogen-bonded organic framework (HOF@Co / Heme)

[0056] (1) In order 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 ions, calcium ions, potassium ions and glutathione) were mixed with 50 μL of 1 mg / mL GOx solution and 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 solute and 0.1 mM sodium hydroxide solution as solvent) was added; then, 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 effect of different substances on the chemiluminescence signal intensity.

[0057] The results are as follows Figure 4 As shown in Figure 2, the results demonstrate a clear distinction between the signal intensities of the target glucose and the interfering substances; the high signal value for glucose indicates the high specificity of the present invention. These findings not only confirm the selectivity of the present invention but also highlight its potential for practical applications in measuring glucose levels in biological samples.

[0058] (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. After mixing, the mixture was placed in a constant temperature oscillator at 37 °C for 25 min. The mixture was then 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 solute and 0.1 mM sodium hydroxide solution as 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, and the detector was detected once every 5 days within 30 days.

[0059] The results are as follows Figure 5As shown in the figure, the CL intensity remained almost consistent with minimal changes during the one-month study period. 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.

[0060] Example 7

[0061] Detection of glucose content in unknown samples using CL method based on metal-doped hydrogen-bonded organic framework (HOF@Co / Heme)

[0062] To determine the practicality of this method for testing unknown samples, an unknown serum sample was diluted 1000-fold. 150 μL of the serum sample was mixed with 50 μL of a 1 μg / mL GOx solution and incubated at 37°C in a thermostatted oscillator for 25 minutes. The mixture was then transferred to a glass sample cell (φ14 mm × 14 mm), and 50 μL of a 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 a 200 μg / mL HOF@Co / Heme suspension was quickly added, and the CL signal was immediately measured using a CL detector. The glucose content of the unknown sample was determined using the standard curve. The glucose content was 5.53 mM, with a standard deviation of 6.2% compared to the hospital test result.

[0063] Comparative Example 1

[0064] Metal-doped hydrogen-bonded organic framework materials (HOF@Co / Heme) and Co-only loaded 2+ Comparison of enzyme activity between HOF@Co and heme-only loaded HOF / Heme

[0065] In order to determine the load Co 2+ Heme is beneficial to the activity of peroxidase. 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. The mixture was then 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 solute and 0.1 mM sodium hydroxide solution as 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 to determine the enzyme activity by the signal intensity. The results are shown in Figure 2. Figure 6 As shown, the load Co 2+The peroxidase activity after loading with hemoglobin was the strongest, indicating that 2+ and heme are beneficial to peroxidase activity.

[0066] Comparative Example 2

[0067] Comparison of enzyme activity between metal-doped hydrogen-bonded organic frameworks (HOF@Co / Heme) and those loaded with other metal ions

[0068] In order to determine the load Co 2+ The peroxidase activity of the loaded Co 2+ and other metals (Cu 2+ 、Zn 2+ Cr 3+ 、Hg 2+ ) enzyme activity, 150 μL 4 μM glucose solution was mixed with 50 μL 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 0.1 mM / L luminol standard solution (prepared with luminol as solute and 0.1 mM sodium hydroxide solution as solvent) was added; subsequently, 50 μL 200 μg / mL HOF@Co / Heme, HOF@Cu / Heme, HOF@Zn / Heme, HOF@Cr / Heme, and HOF@Hg / Heme suspensions were quickly added, and the CL signal was immediately detected with a CL detector to determine the enzyme activity by the signal intensity. The results are shown in Figure 2. Figure 7 As shown, the load Co 2+ The peroxidase activity after loading with hemoglobin was the strongest, indicating that 2+ The effect is best.

[0069] Comparative Example 3

[0070] Comparison between metal-doped hydrogen-bonded organic framework materials (HOF@Co / Heme) and existing technologies

[0071] In order to determine the application value of HOF@Co / Heme in detecting glucose, GOx / MOF-919@ZIF-8 was used as a probe to detect glucose, and the detection process was the same as that in Example 2. The 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 that the present invention has better performance.

[0072] The embodiments of the present application are described above in conjunction with the accompanying drawings. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the core idea of the present application, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present application and the claims, all of which are within the protection of the present application.

Claims

1. An application of a metal-doped hydrogen-bonded organic framework material in the quantitative and / or qualitative detection of glucose; characterized in that: The quantitative and / or qualitative detection method is the CL method; The preparation method of the metal-doped hydrogen-bonded organic framework material comprises the following steps: S1. Using melamine, cyanuric acid, and 1,3,5-benzenetricarboxylic acid as three hydrogen-bonding monomers and adding a solvent, a hydrogen-bonding organic framework material is synthesized by a 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 hemoglobin, stirring, washing, centrifuging, and drying to obtain the metal-doped hydrogen bond organic framework material.

2. The use according to claim 1, characterized in that The solvent includes at least one of methanol and N,N-dimethylformamide.

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

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

5. Use of the metal-doped hydrogen-bonding organic framework material according to claim 1 in the preparation of a product for quantitative detection of glucose.

6. A product for quantitative detection of glucose, characterized in that: The product includes the metal-doped hydrogen bond organic framework material according to claim 1.

7. 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 1 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.

8. The method according to claim 7, characterized in that The luminol standard solution is prepared by taking luminol as solute and sodium hydroxide solution as solvent.

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