Ru-n coordinated hydrogen-bonded organic framework nanoscale enzyme and preparation method and application thereof

Abstract

The application discloses a Ru-N coordinated hydrogen-bonded organic framework nanoscale enzyme and a preparation method and application thereof. The nanoscale enzyme is obtained by loading a ruthenium ion on a hydrogen-bonded organic framework material HOFs through a Ru-N coordination bond to obtain a Ru-HOFs nanoscale enzyme. The Ru-HOFs nanoscale enzyme has excellent peroxidase-like activity, good stability and biocompatibility. Based on a cascade reaction colorimetric detection platform constructed by the Ru-HOFs nanoscale enzyme, high-sensitivity and high-selectivity detection of a gastric cancer biomarker D-proline and D-alanine can be realized, and the detection limits are as low as 0.6112 micromoles per liter ‑1 and 2.299 micromoles per liter ‑1 , respectively. In addition, a portable detection method based on a smart phone is also developed, which provides a new technical means for rapid and non-invasive screening of early gastric cancer. The preparation method is simple and the conditions are mild, and the Ru-HOFs nanoscale enzyme has a wide biomedical application prospect.

Description

Technical Field

[0001] This invention relates to nanozymes, specifically Ru-N coordinated hydrogen-bonded organic framework nanozymes (Ru-HOFs), their preparation methods, and applications. Background Technology

[0002] Gastric cancer (GC) is one of the most common malignant tumors, and early diagnosis is crucial for improving patient survival rates.

[0003] Studies have shown that D-amino acids (DAAs) in saliva, especially D-proline (D-Pro) and D-alanine (D-Ala), are associated with early-stage gastric cancer. The DAA levels in the saliva of patients with early-stage gastric cancer are significantly higher than in healthy individuals. The concentration of D-Ala in the saliva of healthy individuals ranges from 5.4 to 7.8 μmol / L. -1 The concentration of D-Pro was 7.3-18.3 μmol / L. -1 The DAA concentration of D-Pro in the saliva of gastric cancer patients ranged from 126.3 to 285.3 μmol / L. -1 The DAA concentration range of D-Ala is 46.1 - 114.5 μmol L. -1 Therefore, accurate D-Pro and D-Ala monitoring can assist in the early diagnosis of gastric GC and can serve as potential biomarkers for the early diagnosis of gastric cancer.

[0004] Currently, methods for detecting these D-amino acids mainly include surface-enhanced Raman scattering (SERS), high-performance liquid chromatography (HPLC), electrochemical methods (ECL), and chemiluminescence. However, these methods usually rely on expensive instruments and equipment, complex sample pretreatment processes, and professional operators, and the detection cycle is long. They are difficult to meet the actual needs of early gastric cancer screening for rapid, convenient, and on-site detection, thus limiting their application in early large-scale screening.

[0005] Colorimetric methods have attracted much attention due to their simplicity, intuitive results, and low cost. In recent years, researchers have developed various nanozymes to construct cascade reaction platforms for the detection of D-amino acids. For example, patent document CN112763438A discloses a carbon dot peroxidase CDs@NC, which has been applied to the detection of D-Ala and D-Pro, but its detection sensitivity still needs improvement. Patent document CN119269482A uses Pt / Ti3C2T... X Nanomaterials are available, but their stability is insufficient and their cost is high.

[0006] Therefore, developing a novel nanozyme with high catalytic activity, good stability, and low cost to construct a sensitive and reliable method for the detection of D-amino acids remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing technologies and provide a novel hydrogen-bonded organic framework nanozyme with high catalytic activity and good stability, as well as its preparation method and application.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A Ru-N coordinated hydrogen-bonded organic framework nanozyme was prepared by anchoring ruthenium ions onto a hydrogen-bonded organic framework material through Ru-N coordination bonds.

[0009] A method for preparing Ru-N coordinated hydrogen-bonded organic framework nanozymes includes the following steps: Step 1: Prepare ruthenium chloride solution (RuCl3); Step 2: Dissolve 1,3,6,8-tetrakis(4-p-aminophenylpyrene) (PyTTA) in methanol (CH3OH) and stir until homogeneous; Step 3: Slowly add ruthenium chloride solution (RuCl3) to the solution while stirring; Step 4: After the reaction is complete, the reaction product is collected using a high-speed centrifuge, washed with deionized water, and then dispersed in purified water for later use to prepare a solution of hydrogen-bonded organic framework nanozymes Ru-HOFs.

[0010] The concentration of the ruthenium chloride solution is 1-10 mg / mL; The ratio of 1,3,6,8-tetrakis(4-p-aminophenylpyrene) to methanol is 10-30 mg: 10-20 mL; The amount of ruthenium chloride solution added is 100-1000 μL; The preferred stirring reaction time is 10-24 hours; The centrifuge speed is 8000~12000 rpm / min.

[0011] The above-mentioned Ru-N coordinated hydrogen bond organic framework nanozyme is used in the preparation of reagents for the detection of D-proline and D-alanine.

[0012] A colorimetric method for detecting D-proline and D-alanine includes the following steps: Step A: Contact the sample to be tested with D-amino acid oxidase (DAAO), and the reaction produces hydrogen peroxide; Step B: The reaction product of step A is contacted with the above-mentioned Ru-N coordinated hydrogen-bonded organic framework nanozyme Ru-HOFs and o-phenylenediamine (OPD). The hydrogen-bonded organic framework nanozyme catalyzes the oxidation of o-phenylenediamine by peroxide, resulting in a color change. Step C: Quantitatively or qualitatively detect the content of D-proline and D-alanine in the sample by measuring color changes (e.g., measuring absorbance using an ELISA reader, or capturing and analyzing the RGB values ​​of an image using a smartphone).

[0013] Preferably, the colorimetric detection range for D-proline is 2-150 μmol / L. -1 The detection limit is 0.6112 μmol L / L. -1 The colorimetric detection range for D-alanine is 5-100 μmol / L. -1 The detection limit is 2.299 μmol L / L. -1 .

[0014] This invention discloses a Ru-N coordination interface-regulated hydrogen-bonded organic framework for inducing peroxidase activity in the detection of gastric cancer biomarkers. The nanozyme is prepared using 1,3,6,8-tetrakis(4-p-aminophenylpyrene) (PyTTA) as a monomer, synthesizing an innovative Ru-HOF nanozyme through Ru-N coordination. The synthesized Ru-HOF nanozyme exhibits superior peroxidase activity, and the HOFs can convert Ru... 3+ The confinement of the reaction area stabilizes the catalytic activity to a certain extent. This method has advantages such as simple operation, low material synthesis difficulty, and mild reaction conditions, and exhibits excellent selectivity and a low detection limit. Notably, this invention successfully developed a novel sensor integrating multiple functions such as loading, catalysis, and sensing. This invention demonstrates great application potential in the field of biomedical point-of-care diagnostics. Attached Figure Description

[0015] Figure 1 This is a flowchart illustrating the preparation process of Ru-HOFs nanozymes in the examples.

[0016] Figure 2 The image shows a characterization diagram of the Ru-HOFs nanozyme in the examples. The white scale bar in the image represents 1 μm. Where A is the TEM image, B is the HAADF-STEM image, and CE is the EDX-mapping elemental analysis image.

[0017] Figure 3 The XPS spectra of HOFs and Ru-HOFs nanozymes in the examples are shown.

[0018] Figure 4This is a schematic diagram illustrating the principle of the Ru-HOFs nanozyme-based cascade reaction for detecting D-Pro and D-Ala according to the present invention.

[0019] Figure 5 This is a schematic diagram illustrating the use of D-Pro and D-Ala colorimetric detection in conjunction with a smartphone as an example.

[0020] Figure 6 The UV-Vis absorption spectra are for the peroxidase activity of (A) Ru-HOFs nanozymes and the feasibility verification of (B) D-Pro and (C) D-Ala cascade reactions in the examples.

[0021] Figure 7 The following is a diagram showing the results of detecting D-Pro and D-Ala using the Ru-HOFs nanozyme colorimetric method in an example. Where A and C are the UV-Vis absorption spectra of D-Pro and D-Ala at different concentrations, and B and D are the corresponding response curves and linear fitting plots.

[0022] Figure 8 The following is a diagram showing the results of detecting D-Pro and D-Ala using Ru-HOF nanozymes combined with a smartphone colorimetric method, as an example. Where A and C are the RGB value response curves of D-Pro and D-Ala at different concentrations, and B and D are the corresponding linear fitting graphs and color charts. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments and accompanying drawings, but this is not intended to limit the scope of the invention.

[0024] Example 1: Preparation of Ru-HOF nanozymes, referring to... Figure 1 It includes the following steps: Step 1: Prepare a 5 mg / mL ruthenium chloride solution (RuCl3); Step 2: Weigh 20 mg of 1,3,6,8-tetra(4-p-aminophenylpyrene) (PyTTA), dissolve it in 16 mL of methanol (CH3OH) and stir well; Step 3: Slowly add 800 μL of RuCl3 solution to the well-stirred solution and continue stirring for 12 hours. Step 4: After the reaction is complete, the reaction product is collected using a centrifuge at 10,000 rpm / min, washed twice with deionized water, and then dispersed in 12 mL of purified water for later use to prepare a hydrogen-bonded organic framework nanozyme Ru-HOFs solution.

[0025] The structure of PyTTA in step 2 is: .

[0026] The structural formula of Ru-HOFs in step 4 is: .

[0027] The Ru-HOFs nanozyme prepared by the method of this invention exhibits peroxidase-like activity and can maintain stable catalytic activity, catalyzing the oxidation of OPD by H2O2 and inducing a color change.

[0028] Example 2: Characterization of Ru-HOF nanozymes

[0029] Reference Figure 2 The prepared Ru-HOFs nanozymes were characterized. 2(A) is a TEM image of Ru-HOFs, showing that the nanozymes are rod-shaped.

[0030] 2(B) is a HAADF-STEM image of Ru-HOFs, with a bar-shaped pattern.

[0031] 2 (CE) shows the elemental analysis of Ru-HOFs using EDX-mapping, where C, N, and Ru are uniformly distributed.

[0032] Figure 3 XPS characterization of Ru-HOFs verifies that the material is an innovative nanozyme formed through Ru-N coordination bonds. The XPS full spectrum in image 3(A) shows the presence of Ru compared to HOFs, preliminarily proving the successful synthesis of Ru-HOFs.

[0033] 3(B) The high-resolution spectrum of Ru 3p shows that Ru 3p appears at 463.59 eV, 467.18 eV and 485.78 eV, 489.55 eV, respectively. 3 / 2 and Ru 3p 1 / 2 Characteristic peaks.

[0034] The N 1s high-resolution spectra of 3(C)HOFs and Ru-HOFs show that 399.78 eV is the -NH2 in HOFs; while in the N 1s high-resolution spectrum of Ru-HOFs, a characteristic peak of Ru-N is observed at a binding energy of 400.53 eV. The presence of Ru-N bonds indicates that Ru atoms are firmly anchored by N functional groups, which can stabilize metal sites and regulate electronic properties.

[0035] The C 1s high-resolution spectrum of 3(D)HOFs shows characteristic peaks at CC / C=C and CN.

[0036] In the high-resolution spectra of 3(E)C 1s and Ru 3d, distinct peaks are observed at binding energies of 282.34 eV and 286.57 eV, respectively, which can be attributed to Ru 3d.5 / 2 and Ru 3d 3 / 2 It partially overlaps with the C1s peak.

[0037] Example 3: Verification of the peroxidase activity and cascade reaction feasibility of Ru-HOF nanozymes

[0038] Based on the Ru-HOFs cascade reaction detection platform, colorimetric analysis of D-Pro and D-Ala provides a detection platform for the early diagnosis of gastric cancer, such as... Figure 4 The detection principle is as follows: DAAO can catalyze the hydrolysis of D-AA to produce H2O2. Since Ru-HOFs have excellent peroxidase activity, they can catalyze the colorless OPD to turn into yellow DAP. Based on the color change of OPD, D-Pro and D-Ala can be detected colorimetrically.

[0039] Figure 5 To detect the analysis process of D-Pro and D-Ala, the content of D-Pro and D-Ala was determined linearly by extracting RGB data from a smartphone.

[0040] Figure 6 Feasibility analysis of UV-Vis absorption spectra for verifying the peroxidase activity of Ru-HOFs; among them, 6(A) verifies the peroxidase activity of Ru-HOFs. It can be seen from the figure that when Ru-HOFs, OPD and H2O2 coexist, the absorbance increases significantly, indicating that Ru-HOFs have excellent peroxidase activity.

[0041] 6(B) Feasibility analysis of D-Pro cascading: When Ru-HOFs, DAAO and D-Pro coexist, the OD value increases significantly, verifying the feasibility of cascading detection of D-Pro.

[0042] 6(C) Feasibility analysis of D-Ala cascade: When Ru-HOFs, DAAO and D-Ala coexist, the OD value increases significantly, verifying the feasibility of cascade detection of D-Ala.

[0043] Example 4: Colorimetric determination of D-Pro and D-Ala based on Ru-HOF nanozymes

[0044] D-Pro and D-Ala were detected using the Ru-HOF nanozyme colorimetric method. Different concentrations of D-Pro and D-Ala solutions were prepared first. Specifically, the D-Pro concentrations were: 0, 2, 10, 20, 40, 50, 100, 150, 200, 300, 400, 600, and 700 μmol / L. -1 ; The specific D-Ala concentrations were: 0, 5, 10, 30, 50, 75, 100, 150, 200, 300, 400, 500, 700, and 800 μmol / L. -1 .

[0045] Prepare 7.5 mmol L -1 For the OPD solution, weigh 0.00162 g of OPD, add 2 mL of water to dissolve it, and then ultrasonically disperse it evenly.

[0046] The detection method includes the following steps: Step A: In a 0.5 mL centrifuge tube, add 10 µL DAAO, 30 µL of D-Pro or D-Ala at different concentrations, and 60 µL Tris-HCl buffer (200 mmol / L pH=8.3), and incubate at 42℃ for 60 min before removing the tube. Step B: Add 100 µL NaAc-HAc buffer (200 mmol / L pH=4), 25 µL Ru-HOFs, and 25 µL LOPD, mix well, and incubate at 42℃ for 60 min; Step C: Measure the change in optical density (OD value) using a microplate reader, such as... Figure 7 As shown, at D-Pro concentrations of 2-700 μmol / L... -1 Within the range, absorbance increases with increasing D-Pro concentration, as shown in 7A (the inset shows the corresponding colorimetric solution).

[0047] 7(B) Response curves of ∆OD at 450 nm as a function of different concentrations of D-Pro (inset shows linear fitting plots of different concentrations of D-Pro); 7(C) indicates that the D-Ala concentration is 5-800 μmol / L. -1 Within the range, absorbance increases with increasing D-Ala concentration (the inset shows the corresponding colorimetric solution).

[0048] 7(D) Response curves of ∆OD at 450 nm as a function of different concentrations of D-Ala (inset shows linear fitting plots of different concentrations of D-Ala).

[0049] Example 5: Portable detection of D-Pro and D-Ala based on Ru-HOF nanozymes and smartphones

[0050] This embodiment utilizes a portable sensing platform based on a smartphone, comprising Ru-HOF nanozymes prepared using the method of this invention, as well as DAAO, D-Pro and D-Ala, Tris-HCl buffer, OPD, H2O2, and NaAc-HAc solutions. Specific steps: The reaction is carried out according to the method in Example 4. Then, the color of the reaction solution is photographed using a smartphone under the same lighting conditions. RGB values ​​are extracted using color analysis software, and the relationship between Δ(G+B) / R value and D-amino acid concentration is established. The linear range and detection limit are then calculated.

[0051] Figure 8 This example illustrates the results of Ru-HOFs nanozymes combined with smartphone colorimetric methods for detecting D-Pro and D-Ala. The RGB values ​​of the images were extracted using color analysis software, and the data was analyzed to draw conclusions.

[0052] The examples evaluated enzyme activity by monitoring changes in Δ(G+B) / R, with 8(A) at D-Pro concentrations ranging from 0.5 to 800 μmol / L. -1 Within this range, the value of Δ(G+B) / R increases with increasing D-Pro concentration, and gradually reaches a plateau. The illustration in 8A is a colorimetric chart extracted from the RGB image of a smartphone.

[0053] Analysis of 8(B) showed that the D-Pro concentration was between 2-50 μmol / L. -1 Within the range, Δ(G+B) / R and C D-Pro It exhibits good linearity, with the linear equation being y = 0.00882x + 0.00217 (R²). 2 =0.9945, n=3), LOD is 0.9399 μmol L -1 (3σ / K, n=9).

[0054] 8(C) at D-Ala concentrations of 5-700 μmol / L -1 Within this range, the value of Δ(G+B) / R increases with increasing D-Ala concentration, and gradually reaches a plateau. The illustration in 8(C) is a colorimetric chart extracted from RGB data by a smartphone.

[0055] Analysis of 8(D) showed that the D-Ala concentration was between 5-50 μmol / L. -1 Within the range, Δ(G+B) / R and C D-Ala It exhibits good linearity, with the linear equation being y = 0.00429x + 0.05109 (R²). 2 =0.9862, n=3), LOD is 2.694 μmol L -1 (3σ / K, n=9).

[0056] The results showed that Ru-HOF nanozymes are suitable for portable detection of D-Pro and D-Ala, with the detection range for D-Pro being 2-50 μmol / L. -1 The detection limit is 0.9399 μmol L / L. -1 The detection range of D-Ala is 5-50 μmol / L. -1 The detection limit is 2.694 μmol L / L. -1 .

[0057] This invention successfully prepared a novel Ru-HOF nanozyme, which stably anchors ruthenium ions to the HOF backbone via Ru-N coordination bonds, exhibiting excellent peroxidase-like activity and stability. Based on this, a colorimetric detection platform was constructed that can sensitively and specifically detect the gastric cancer-related biomarkers D-Pro and D-Ala, and a portable detection mode based on a smartphone was successfully developed. The materials, methods, and applications provided by this invention have enormous application potential in the fields of biosensing and early disease diagnosis.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Ru-N coordinated hydrogen-bonded organic framework nanozyme, characterized in that, The nanozyme stabilizes ruthenium ions on a hydrogen-bonded organic framework via Ru-N coordination bonds.

2. A method for preparing the Ru-N coordinated hydrogen-bonded organic framework nanozyme of claim 1, characterized in that, Ruthenium ions are loaded onto hydrogen-bonded organic framework materials (HOFs) via Ru-N coordination bonds to obtain Ru-HOF nanozymes.

3. The preparation method according to claim 2, characterized in that, The Ru-HOFs nanozyme was prepared by dissolving 1,3,6,8-tetrakis(4-p-aminophenylpyrene) in methanol, stirring and mixing it evenly, adding ruthenium chloride solution, continuing to stir, and centrifuging and washing after the reaction was completed to obtain the Ru-HOFs nanozyme.

4. The preparation method according to claim 3, characterized in that, The concentration of the ruthenium chloride solution is 1-10 mg / mL; The ratio of 1,3,6,8-tetrakis(4-p-aminophenylpyrene) to methanol is 10-30 mg: 10-20 mL; The amount of ruthenium chloride solution added is 100-1000 μL; The stirring reaction time is 10-24 hours; The centrifuge operates at a speed of 8000-12000 rpm / min.

5. The Ru-N coordinated hydrogen-bonded organic framework nanozyme of claim 1 or 2, in the preparation of reagents for the detection of D-proline and D-alanine.

6. A colorimetric method for detecting D-proline and D-alanine, characterized in that, Includes the following steps: Step A: Contact the sample to be tested with D-amino acid oxidase, and the reaction produces hydrogen peroxide; Step B: The reaction product of step A is contacted with the Ru-N coordinated hydrogen-bonded organic framework nanozyme and o-phenylenediamine as described in claim 1 or 2. The Ru-N coordinated hydrogen-bonded organic framework nanozyme catalyzes the oxidation of o-phenylenediamine by peroxide, resulting in a color change. Step C: Quantitatively or qualitatively detect the content of D-proline and D-alanine in the sample by measuring color changes.

7. The colorimetric method according to claim 6, characterized in that, The method for measuring color change in step C is to use an ELISA reader to measure absorbance and to use a smartphone to capture and analyze the RGB values ​​of the image.

8. The colorimetric method according to claim 6 or 7, characterized in that, The colorimetric detection range for D-proline is 2-150 μmol / L. -1 The detection limit is 0.6112 μmol L / L. -1 The colorimetric detection range for D-alanine is 5-100 μmol / L. -1 The detection limit is 2.299 μmol L / L. -1 .

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