MnO2@UiO-66 composite material, and preparation method and application thereof

Abstract

The application belongs to the field of molecular biology, discloses a kind of MnO2@UiO-66 composite material and its preparation method and application, solve the technical problem that the detection sensitivity and detection range of organic phosphorus compound are relatively narrow.By the ratio fluorescence system of including MnO2@UiO-66 composite material and Ampliflu Red is used for high sensitivity detection of organic phosphorus pesticide and reducing biological molecule.MnO2 Material not only can be used as the fluorescence quenching agent of UiO-66-NH2, can also catalyze Ampliflu Red reagent to generate fluorescent product isophenoxazone, to enhance its fluorescence signal at 590 nm wavelength.When detecting organic phosphorus, the system also includes alkaline phosphatase and L-ascorbic acid-2-sodium phosphate.When the concentration of phoxim is 1×10 ‑6 -9×10 ‑1 mg / mL, fluorescence linear relationship is good, and the detection limit is as low as 0.33×10 ‑6 mg / mL.The system of the application has the characteristics of accuracy, convenience, sensitivity and stability, and can be applied to the detection of organic phosphorus pesticide in complex environment.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and the detection of organophosphorus pesticides, and in particular to a MnO2@UIO-66 composite material and a preparation method and application thereof. Background Art

[0002] Organophosphorus pesticides (OPs) are organic compounds containing phosphorus, primarily used to control plant diseases, insects, and weeds. They are widely used in agriculture, forestry, and animal husbandry worldwide, playing a vital role in increasing agricultural yields. Due to their widespread use, OP residues in the environment and agricultural products pose a threat to human health through exposure to the food chain. The toxic mechanism of OPs is the inhibition of acetylcholinesterase activity in neural tissue and other target organs. This inability to degrade acetylcholine leads to overstimulation of muscarinic and nicotinic receptors, resulting in clinical symptoms such as excessive sweating, salivation, bronchospasm, bronchial edema, bradycardia, hypotension, and decreased consciousness. Even low levels of OPs can pose significant health risks to humans, causing a variety of conditions, including kidney damage, Parkinson's disease, endocrine disruption, memory loss, and proximal muscle weakness. The harmful effects of OPs on human health cannot be ignored. Therefore, the development of a rapid and accurate method for detecting OP residues in the environment is of great importance.

[0003] Biomolecules play a vital role in maintaining normal human life and defending against disease. Reducing small molecules, particularly biothiols and ascorbic acid (AA), play a crucial role in maintaining human health. Glutathione and cysteine, as biothiols, are closely linked to physiological and pathological processes in complex biological environments, and abnormal levels of these compounds are associated with numerous diseases. AA, a crucial nutrient in the human body, possesses excellent reducing properties, effectively reducing oxidative stress and lowering the risk of diseases like cancer. Therefore, highly selective and sensitive detection of small molecules in vivo is crucial for developing treatments for these diseases.

[0004] Currently, the main methods for detecting OPs residues include colorimetry, capillary electrophoresis, thin-layer chromatography, gas-liquid chromatography, high-performance liquid chromatography, electrochemical method, enzyme-linked immunosorbent assay, etc. Although these methods have their own advantages, they rely on expensive instruments used in the detection process, the time-consuming process and the requirement for trained personnel, making these methods still face challenges in practical application. Compared with these methods, fluorescence analysis is favored by more and more researchers due to its high sensitivity, simple operation and low cost. Publication No. CN116660217A discloses a fluorescence detection method for rapid detection of organophosphorus compounds based on UiO-66-NH2. After mixing a water sample containing phosphorus compounds with N-ethylmorpholine, UiO-66-NH2Tris suspension is added, and the fluorescence properties are measured to achieve the detection of organophosphorus. However, in the above technology, the detection limit of paraoxon is 23.8 ng / mL, and the detection range is 0-20 ug / mL. The detection range is relatively narrow and cannot be applied to the detection of mixed solutions with lower organophosphorus content. In addition, the existing technology can only detect a single component of organophosphorus. How to detect reduced biological small molecules sensitively and efficiently is also a problem that needs to be solved urgently. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention proposes a MnO2@UiO-66 composite material and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution of the present invention is implemented as follows:

[0007] A method for preparing a MnO2@UiO-66 composite material, comprising the following steps:

[0008] (1) Mix UiO-66-NH2, ultrapure water and MES buffer to obtain MOF solution;

[0009] (2) KMnO4 solution was added to the MOF solution, stirred at room temperature until the solution turned brown, and incubated to obtain a reaction solution; the reaction solution was desolvated, centrifuged, washed, and dried to obtain the MnO2@UiO-66 composite material.

[0010] In the above step (1), the concentration of UiO-66-NH2 in the MOF solution is 0.1-1 mg / mL; the concentration of the MES buffer is 5 mM, pH = 6; the concentration of the KMnO4 solution in step (2) is 0.1-1 mg / mL; and the volume ratio of the MOF solution to the KMnO4 solution is (1-5):1.

[0011] MnO2@UiO-66 composite material prepared using the above preparation method.

[0012] A ratiometric fluorescence system based on a MnO2@UiO-66 composite material comprises the above-mentioned MnO2@UiO-66 composite material and Ampliflu Red.

[0013] The above ratiometric fluorescence system is used in the highly sensitive detection of organophosphorus or reduced biomolecules. When highly sensitive detection of organophosphorus is performed, the ratiometric fluorescence system further comprises alkaline phosphatase and L-ascorbic acid-2-phosphate sodium solution.

[0014] The highly sensitive detection of organophosphorus comprises the following steps:

[0015] (a) Mix different concentrations of phoxim standard solutions and alkaline phosphatase, incubate at 37°C for 1-20 minutes, add L-ascorbic acid-2-phosphate sodium solution, and continue incubation for 1-50 minutes to obtain a mixed solution;

[0016] (b) adding the MnO2@UiO-66 dispersion to the mixed solution obtained in step (a), incubating at 37°C for 1-50 min, then adding ultrapure water and Ampliflu Red solution, and continuing to incubate for 1-30 min to obtain the standard solution I to be tested;

[0017] (c) Perform fluorescence measurement on the standard solution I obtained in step (b). Under excitation wavelengths of 360 nm and 571 nm, record the ratio of the fluorescence intensity at emission wavelengths of 430 nm and 590 nm to establish the ratio fluorescence value F. 430 / F 590 The linear relationship between the concentration of organophosphorus standard solution and the

[0018] The concentration of the phoxim standard solution in step (a) above is 1×10 -6 -9×10 -1 mg / mL. For example, the concentration of phoxim standard solution is 9×10 -1 mg / mL, 1 × 10 -1 mg / mL, 1×10 -2 mg / mL, 1×10 -4 mg / mL, 1 × 10 -5 mg / mL or 1×10 -6 mg / mL.

[0019] The concentration of alkaline phosphatase is 0.1-1 U / mL. For example, the concentration of alkaline phosphatase is 0.1-0.4 U / mL; for example, the concentration of alkaline phosphatase is 0.5-1 U / mL; for example, the concentration of alkaline phosphatase is 0.3-0.8 U / mL; for example, the concentration of alkaline phosphatase is 0.1 U / mL, 0.2 U / mL, 0.3 U / mL, 0.4 U / mL, 0.5 U / mL, 0.6 U / mL, 0.7 U / mL, 0.8 U / mL, 0.9 U / mL, or 1 U / mL.

[0020] The concentration of the aqueous solution of sodium L-ascorbic acid-2-phosphate is 1-4 mM. For example, the concentration of the aqueous solution of sodium L-ascorbic acid-2-phosphate is 1-3 mM; for example, the concentration of the aqueous solution of sodium L-ascorbic acid-2-phosphate is 2-4 mM; for example, the concentration of the aqueous solution of sodium L-ascorbic acid-2-phosphate is 1 mM, 2 mM, 3 mM, or 4 mM.

[0021] The volume ratio of phoxim standard solution, alkaline phosphatase and L-ascorbic acid-2-phosphate sodium solution is (1-9):1:(1-20). For example, the volume ratio of the phoxim standard solution, alkaline phosphatase and L-ascorbic acid-2-sodium phosphate solution is (1-9):1:1; for example, the volume ratio of the phoxim standard solution, alkaline phosphatase and L-ascorbic acid-2-sodium phosphate solution is (1-9):1:20; for example, the volume ratio of the phoxim standard solution, alkaline phosphatase and L-ascorbic acid-2-sodium phosphate solution is 1:1:(1-20); for example, the volume ratio of the phoxim standard solution, alkaline phosphatase and L-ascorbic acid-2-sodium phosphate solution is 9:1:(1-20); for example, the volume ratio of the phoxim standard solution, alkaline phosphatase and L-ascorbic acid-2-sodium phosphate solution is 1:1:1, 1:1:20, 5:1:10, 9:1:1 or 9:1:20.

[0022] Furthermore, in the above step (b), the MnO2@UiO-66 dispersion refers to a MnO2@UiO-66 composite material aqueous solution with a concentration of 0.1-1 mg / mL. For example, the MnO2@UiO-66 dispersion refers to a MnO2@UiO-66 dispersion with a concentration of 0.1-0.6 mg / mL; for example, the MnO2@UiO-66 dispersion refers to a MnO2@UiO-66 dispersion with a concentration of 0.5-1 mg / mL; for example, the MnO2@UiO-66 dispersion refers to a MnO2@UiO-66 dispersion with a concentration of 0.4-0.9 mg / mL; for example, the MnO2@UiO-66 dispersion refers to a MnO2@UiO-66 dispersion with a concentration of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, or 1 mg / mL.

[0023] The Ampliflu Red solution is obtained by dissolving Ampliflu Red in N,N- dimethylformamide, and the concentration of the Ampliflu Red solution is 1-50 µM. For example, the concentration of the Ampliflu Red solution is 1-30 µM; for example, the concentration of the Ampliflu Red solution is 20-50 µM; for example, the concentration of the Ampliflu Red solution is 15-45 µM; for example, the concentration of the Ampliflu Red solution is 1 µM, 10 µM, 20 µM, 25 µM, 30 µM, 40 µM or 50 µM.

[0024] The volume ratio of the mixed solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is (1-30):(1-10):(1-20). For example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is (1-30):1:(1-20); for example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is (1-30):10:(1-20); for example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is 1:1:(1-20); for example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is 30:1:(1-20); for example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is 1:(1-10):1; for example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is 1:(1-10):20; for example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is 1:1:1, 1:10:20, 30:1:20, 30:10:10, 15:1:5 or 3:1:2.

[0025] The above-mentioned step of high-sensitivity detection of a reduced biomolecule is as follows:

[0026] (S1) The reduced biomolecule standard solution and ultrapure water are added to the MnO2@UiO-66 dispersion solution, incubated at 37°C for 1-2 h, then the Ampliflu Red solution is added, and incubated at 37°C in the dark for 1-10 min to obtain a standard solution II to be detected;

[0027] (S2) The standard solution to be detected obtained in step (S1) is subjected to fluorescence determination, and the ratio of the fluorescence intensity at 430 nm and 590 nm is recorded under the excitation wavelength of 360 nm and 571 nm, to establish the ratio fluorescence value F 430 / F 590 linear relationship with the concentration of the reducing biomolecule standard solution.

[0028] In the above step (S1), the reducing biomolecule in the reducing biomolecule standard solution is glutathione, cysteine or ascorbic acid; and the concentration of the reducing biomolecule standard solution is 1-90 µM.

[0029] When the reducing biomolecule is glutathione, the corresponding concentration of the glutathione standard solution is 1 µM, 5 µM, 10 µM, 20 µM, 25 µM, 50 µM, 75 µM and 100 µM, respectively; when the reducing biomolecule is cysteine, the concentration of the cysteine standard solution is 30 µM, 40 µM, 50 µM, 70 µM and 90 µM, respectively; and when the reducing biomolecule is ascorbic acid, the concentration of the ascorbic acid standard solution is 1 µM, 5 µM, 20 µM, 30 µM and 90 µM, respectively.

[0030] In the above step (S1), the volume ratio of the reducing biomolecule standard solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is (0.1-1):1:1. For example, the volume ratio of the reducing biomolecule standard solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is (0.1-0.6):1:1; for example, the volume ratio of the reducing biomolecule standard solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is (0.4-1):1:1; for example, the volume ratio of the reducing biomolecule standard solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is (0.2-0.8):1:1; for example, the volume ratio of the reducing biomolecule standard solution, the MnO2@UiO-66 dispersion solution and the Ampliflu Red solution is 0.1:1:1, 0.4:1:1, 0.7:1:1 or 1:1:1.

[0031] The present application has the following beneficial effects:

[0032] (1) The present invention proposes a method for detecting the concentration of organophosphorus pesticides (OPs) using a ratiometric fluorescence system based on a MnO2@UiO-66 composite material. The system includes a MnO2@UiO-66 composite material, Ampliflu Red (AR), alkaline phosphatase, and L-ascorbic acid-2-phosphate. During the detection process, the MnO2 material in the MnO2@UiO-66 composite material not only acts as a fluorescence quencher for UiO-66-NH2, but also catalyzes the AR reagent to generate a fluorescent product, Resorufin, thereby enhancing its fluorescence signal at a wavelength of 590 nm. When OPs are present, OPs prevent alkaline phosphatase from decomposing L-ascorbic acid-2-phosphate into AA, and the MnO2 coating remains good, so that the fluorescence intensity of UiO-66-NH2 remains stable at a low level. However, AR can be catalyzed by MnO2 to generate Resorufin, resulting in an increase in its fluorescence signal at 590 nm. In the absence of OPs, alkaline phosphatase can decompose sodium L-ascorbic acid-2-phosphate into AA, and MnO2 can be further decomposed into Mn 2+ This results in an increase in the fluorescence intensity of UiO-66-NH2 at 430 nm, while the signal intensity of AR reagent at 590 nm is weakened. This enables highly sensitive detection of OPs. Based on this ratiometric fluorescence detection system, at 9×10 -1 -1×10 -6 The linear relationship is good within the range of mg / mL phoxim concentration, and the logarithm of the concentration is 430 / F 590 The linear relationship between them is: Y=3.257-0.712lgC phoxim (R 2 =0.9935), and the detection limit of phoxim was as low as 0.33×10 -6 The ratiometric fluorescence system designed in the present invention is accurate, convenient, sensitive, and stable, and can be applied to the detection of organophosphorus pesticides in complex environments with high reliability.

[0033] (2) In addition, the present invention also proposes a method for detecting the concentration of reducing biomolecules based on the MnO2@UiO-66 composite material, which includes the MnO2@UiO-66 composite material and Ampliflu Red (AR). When detecting reducing biomolecules, in the presence of glutathione, cysteine, and AA, the MnO2 coating directly decomposes, the fluorescence intensity of UiO-66-NH2 is high, and the fluorescence intensity of AR is low; when the above molecules are not present, the fluorescence intensity of UiO-66-NH2 is low, while the fluorescence intensity of AR is high. Detection of reducing biomolecules is achieved. With the help of PCA, the distinction between GSH, Cys, and AA is achieved. It has the characteristics of accuracy, convenience, sensitivity, and stability, and has been successfully applied to the detection of glutathione in complex environments. It has high reliability and has broad practical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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.

[0035] Figure 1 Characterization of MnO2NS@UiO-66-NH2 and UiO-66-NH2, where (A) is the XRD spectrum; (B) is the full XPS spectrum; (C) is the high-resolution XPS spectrum of Mn 2p; (D) is the FESEM image of UiO-66-NH2; (E) is the FESEM image of MnO2NS@UiO-66-NH2; the insets are photos of the two materials, yellow: UiO-66-NH2; brown: MnO2NS@UiO-66-NH2; (F) is the TEM image of UiO-66-NH2; (G) is the FESEM image of MnO2NS@UiO-66-NH2; (H) is the FIRT spectrum; (I) is the fluorescence lifetime diagram of MnO2NS@UiO-66-NH2 and UiO-66-NH2.

[0036] Figure 2 Schematic diagram of the principle of detecting organophosphorus pesticides and reduced biological molecules using the relative fluorescence system based on MnO2@UiO-66 composite materials. (A) is the preparation process of MnO2@UiO-66 composite materials; (B) is a schematic diagram of the relative fluorescence detection principle; (C) is the PCA analysis of the detection results of GSH, Cys and AA.

[0037] Figure 3 This is the catalytic mechanism of MnO2@UiO-66 composite material.

[0038] Figure 4 Figure 3. Selectivity of the MnO2@UiO-66 composite material-based fluorescence system for detecting organophosphorus pesticides and reduced biomolecules. (A) shows the selectivity for organophosphorus; (B) shows the selectivity for GSH, Cys, and AA. The inset shows the principal component analysis results of GSH, Cys, and AA.

[0039] Figure 5 The results of the detection of phoxim standard solution by MnO2@UiO-66 composite material are shown in Figure 2. (A) is the fluorescence intensity spectrum of UiO-66-NH2 under the action of different concentrations of phoxim; (B) is the fluorescence intensity spectrum of AR under the action of different concentrations of phoxim; (C) is the fluorescence intensity spectrum of F 430 / F 590 Linear plot versus the logarithm of phoxim concentration; all error bars represent the standard deviation of four isolated experiments.

[0040] Figure 6 The results of the MnO2@UiO-66 composite material test on glutathione, cysteine ​​and ascorbic acid standard samples are shown in Figure 2. (A) is the fluorescence intensity spectrum of UiO-66-NH2 under the action of different concentrations of glutathione; (B) is the fluorescence intensity spectrum of AR under the action of different concentrations of glutathione; (C) is the fluorescence intensity spectrum of F 430 / F 590 Linear relationship with different concentrations of glutathione; (D) Fluorescence intensity spectrum of UiO-66-NH2 with different concentrations of cysteine; (E) Fluorescence intensity spectrum of AR under the action of different concentrations of cysteine; (F) F 430 / F 590 Linear relationship with different concentrations of cysteine; (G) is the fluorescence intensity spectrum of UiO-66-NH2 with different concentrations of ascorbic acid; (H) is the fluorescence intensity spectrum of AR under the action of different concentrations of AA; (I) is the fluorescence intensity spectrum of F 430 / F 590 The linear relationship with different concentrations of AA was observed, and all error bars represent the standard deviation of four independent experiments.

[0041] Figure 7 Figure 2 Performance of the specific fluorescence system based on the MnO2@UiO-66 composite material, where (A) is the recovery rate of thioctic phosphate in tap water; (B) is the recovery rate of glutathione in 1% human serum; (C) is the stability of GSH detection; and (D) is the anti-interference ability of GSH detection. All error bars represent the standard deviation of four isolated experiments. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] The preparation method of a MnO2@UiO-66 composite material of this embodiment comprises the following steps:

[0045] (1) Preparation of UiO-66-NH2: 0.81 g of 2-aminoterephthalic acid and 10.5 g of zirconium tetrachloride were dissolved in 40 mL of DMF solvent, and then sonicated until the solute was completely dissolved. The solution was transferred to a hydrothermal reactor, 17 mL of glacial acetic acid was added, and then the reaction was carried out at 120 °C for 24 hours. Figure 2 As shown in Figure A.

[0046] (2) 10 mg of UiO-66-NH2, 8.75 mL of ultrapure water and 1.25 mL of MES buffer (5 Mm, pH = 6) were mixed in a reaction tube to obtain a MOF solution; 2 mL of 1 mg / mL KMnO4 solution was slowly added to the stirring MOF solution and stirred at room temperature for a period of time; the solution was incubated for a period of time until the color turned brown; the solvent in the solution was removed and the powder was collected by centrifugation at 1000 rpm; the powder was washed three times with deionized water and dried at 60 °C under vacuum overnight to obtain a MnO2@UiO-66 composite material, named MnO2NS@UiO-66-NH2.

[0047] The crystal structure, element existence state, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and functional group analysis of the MnO2@UiO-66 composite material (MnO2NS@UiO-66-NH2) prepared in Example 1 and the raw material UiO-66-NH2 were performed. The results are as follows: Figure 1 shown.

[0048] In order to prove that the material was successfully synthesized, a series of characterization experiments were carried out, and the results are as follows Figure 1 shown. Figure 1Figure A is the XRD spectrum. In the UiO-66-NH2 spectrum (blue curve), the peaks are located at 7.36, 8.48, 12.04, 14.15, 17.08, 22.25, 25.68 and 33.12, corresponding to the crystal planes of (111), (002), (022), (113), (004), (115), (224) and (137), respectively, indicating the successful synthesis of UIO-66-NH2. After covering with MnO2, even if the intensity is reduced, the crystal structure does not change significantly (red curve). In XPS analysis ( Figure 1 In Figure B), the peaks of Zr 3d at 184 eV, C1s at 284 eV, N1s at 400 eV, and O1s at 531 eV can be observed in both MnO2NS@UiO-66-NH2 and UiO-66-NH2. Figure 3 Figure C) shows that Mn 3+ and Mn 4+ Also present in the MnO2 coating. Figure 1 Figures D and E in the middle are FESEM images of UiO-66-NH2 and MnO2NS@UiO-66-NH2, respectively. It can be seen from the figure that after MO2 is coated on UiO-66-NH2, the color of the picture becomes much darker. Figure 1 The illustration between Figure D and Figure E is a photo of the two materials. It can be seen that after MnO2 coating, the light yellow powder of UiO-66-NH2 turns into a dark brown powder. Transmission electron microscopy can also clearly distinguish the morphology of the two materials ( Figure 1 Figure F and Figure 1 Figure G). Figure 1 As shown in Figure F, UiO-66-NH2 is a typical octahedral morphology with smooth edges, but Figure 1 In the middle G figure, some flakes appeared at the edge of the material, indicating that the manganese dioxide nanosheets were successfully covered. FIRT spectrum ( Figure 1 Figure H in the middle further confirms this view. Compared with UiO-66-NH2 (blue curve), the −1 The absorption peak of Mn-O appears at (red curve). The fluorescence lifetime is shortened from 0.247 ns (blue curve) of UiO-66-NH2 to 0.144 ns (red curve) of MnO2NS@UiO-66-NH2. Figure 1 As shown in Figure I. The above data show that the synthesis of the material is successful.

[0049] Example 2

[0050] The preparation method of a MnO2@UiO-66 composite material of this embodiment comprises the following steps:

[0051] (1) Preparation of UiO-66-NH2: 0.81 g of 2-aminoterephthalic acid and 10.5 g of zirconium tetrachloride were dissolved in 40 mL of DMF solvent, and then sonicated until the solute was completely dissolved. The solution was transferred to a hydrothermal reactor, 17 mL of glacial acetic acid was added, and then the reaction was carried out at 120 °C for 24 hours. Figure 2 As shown in Figure A.

[0052] (2) 1 mg of UiO-66-NH2, 8.75 mL of ultrapure water and 1.25 mL of MES buffer (5 Mm, pH = 6) were mixed in a reaction tube to obtain a MOF solution; 10 mL of 0.1 mg / mL KMnO4 solution was slowly added to the stirring MOF solution and stirred at room temperature for a period of time; the solution was incubated for a period of time until the color turned brown; the solvent in the solution was removed and the powder was collected by centrifugation at 1000 rpm; the powder was washed three times with deionized water and dried at 60 °C under vacuum overnight to obtain a MnO2@UiO-66 composite material, named MnO2NS@UiO-66-NH2.

[0053] Example 3

[0054] The preparation method of a MnO2@UiO-66 composite material of this embodiment comprises the following steps:

[0055] (1) Preparation of UiO-66-NH2: 0.81 g of 2-aminoterephthalic acid and 10.5 g of zirconium tetrachloride were dissolved in 40 mL of DMF solvent, and then sonicated until the solute was completely dissolved. The solution was transferred to a hydrothermal reactor, 17 mL of glacial acetic acid was added, and then the reaction was carried out at 120 °C for 24 hours. Figure 2 As shown in Figure A.

[0056] (2) 5 mg of UiO-66-NH2, 8.75 mL of ultrapure water and 1.25 mL of MES buffer (5 Mm, pH = 6) were mixed in a reaction tube to obtain a MOF solution; 5 mL of 5 mg / mL KMnO4 solution was slowly added to the stirring MOF solution and stirred at room temperature for a period of time; the solution was incubated for a period of time until the color turned brown; the solvent in the solution was removed and the powder was collected by centrifugation at 1000 rpm; the powder was washed three times with deionized water and dried at 60 °C under vacuum overnight to obtain a MnO2@UiO-66 composite material, named MnO2NS@UiO-66-NH2.

[0057] Implementation effect examples

[0058] The MnO2@UiO-66 composite material can be applied to the detection of organic phosphorus and reducing biomolecules, and the detection principle is as shown in Figure 2 . Specifically, the technical principle of the detection method is as shown in Figure 2 B of the drawings, specifically: the MnO2 material can not only be used as a fluorescence quencher of UiO-66-NH2, but also can catalyze the Ampliflu Red (AR) reagent to generate a fluorescent product, Resorufin, so as to enhance the fluorescence signal thereof at a wavelength of 590 nm. When the organic phosphorus (OPs) exists, the OPs prevent the alkaline phosphatase from decomposing the L-ascorbic acid-2-phosphate sodium into ascorbic acid (AA), and the MnO2 coating still remains good, so that the fluorescence intensity of the UiO-66-NH2 is kept at a lower level. However, the AR can be catalytically oxidized by the MnO2 to generate the Resorufin, which causes the fluorescence signal thereof at 590 nm to increase. When the OPs does not exist, the alkaline phosphatase can decompose the L-ascorbic acid-2-phosphate sodium into the AA, and the MnO2 is further decomposed into Mn 2+ , which causes the fluorescence intensity of the UiO-66-NH2 at a wavelength of 430 nm to increase, and the signal intensity of the AR reagent at a wavelength of 590 nm to decrease, so as to realize the high-sensitivity detection of the OPs. For the detection of reducing biomolecules, when the glutathione (GSH), cysteine (Cys) and ascorbic acid (AA) exist, the MnO2 coating is directly decomposed, the fluorescence intensity of the UiO-66-NH2 is high, and the fluorescence intensity of the AR is low. When the above molecules do not exist, the fluorescence intensity of the UiO-66-NH2 is low, and the fluorescence intensity of the AR is high, so as to realize the detection of the reducing biomolecules. Figure 2 C of the drawings is a PCA analysis of the detection results of the GSH, Cys and AA.

[0059] In addition, the catalytic mechanism of the MnO2@UiO-66 composite material is that the AR is catalyzed by the MnO2@UiO-66 to generate two free radicals. Then the free radical anion is oxidized to form a resorcinol. At the same time, the free radical cation is deacetylated, and then exchanges with an alcohol ketone to generate the Resorufin (as shown in Figure 3 ).

[0060] Selective detection: different kinds of organic phosphorus (OPs) have different hindering effects on the alkaline phosphatase activity, and the reducing properties of the GSH, Cys and AA are different, which causes the fluorescence intensities at 430 nm, 590 nm and F 430 / F 590 to be different, and the target species can be distinguished. Therefore, the principal component analysis is selected to analyze the selectivity. The specific test process and results are as shown in Figure 4 .

[0061] The above-mentioned organophosphorus-specific detection includes the following steps:

[0062] (a) 9 μL of a 1×10 -1 Mix 1 μg / mL of different organophosphorus standard solutions (phoxim, chlorpyrifos, parathion-methyl, and dimethoate) with 1 μL of 1 U / mL alkaline phosphatase, incubate at 37°C for 20 min, then add 20 μL of 4 mM sodium L-ascorbic acid-2-phosphate solution and continue incubation for 50 min to obtain a mixed solution.

[0063] (b) Add 10 μL of 1 mg / mL MnO2@UiO-66 dispersion to the mixed solution obtained in step (a). Incubate at 37°C for 50 min. Then, add 140 μL of ultrapure water and 20 μL of 50 μM Ampliflu Red solution (solvent: N,N-dimethylformamide, solute: Ampliflu Red). Continue incubation for 30 min to obtain the standard solution I to be tested.

[0064] (c) Perform fluorescence measurement on the standard solution I obtained in step (b). Under excitation wavelengths of 360 nm and 571 nm, record the ratio of the fluorescence intensity at emission wavelengths of 430 nm and 590 nm. Record F 430 / F 590 Ratio fluorescence value. Figure 4 As can be seen in Figure A, at the same concentration, the detection system has a good response to different organophosphorus (phoxim, chlorpyrifos, methyl parathion and dimethoate) (F 430 / F 590 ), indicating that the system is responsive to a wide range of OPs.

[0065] In addition, reducing substrates (GSH, Cys, and AA) and oxidative amino acids (such as arginine (Arg), tryptophan (Try), and L-glutamate (Gly)) were selected to test the selectivity of the detection system based on the MnO2@UiO-66 composite material.

[0066] The above-mentioned specific detection of reduced biological small molecules includes the following steps:

[0067] (S1) 20 μL of 50 μM standard solutions of different reducing biomolecules (GSH, Cys, and AA) and 145 μL of ultrapure water were added to 20 μL of 1 mg / mL MnO2@UiO-66 dispersion. After incubation at 37°C for 2 h, 20 μL of Ampliflu Red solution (50 μM) was added and incubated at 37°C in the dark for 10 min to obtain standard solution II.

[0068] (S2) 20 μL of 100 μM standard solutions of different types of oxidative biomolecules (arginine, glutamate, and tryptophan) and 145 μL of ultrapure water were added to 20 μL of 1 mg / mL MnO2@UiO-66 dispersion. After incubation at 37°C for 2 h, 20 μL of Ampliflu Red solution (50 μM) was added and incubated at 37°C in the dark for 10 min to obtain the standard solution III to be tested.

[0069] (S3) Performing fluorescence measurement on the different standard solutions II to be tested obtained in step (S1) and the different standard solutions III to be tested obtained in step (S2), recording the ratio of the fluorescence intensity at the emission wavelengths of 430 nm and 590 nm under the excitation wavelengths of 360 nm and 571 nm, and recording F 430 / F 590 Ratiometric fluorescence values.

[0070] The results are as follows Figure 4 As shown in Figure B, it shows that the reducing substrate is 430 / F 590 The detection system showed good response when , while Arg, Try and Gly had a good response when F 430 / F 590 The detection results of the sites showed no significant changes compared to the blank control. The inset shows the principal component analysis (PCA) results of GSH, Cys, and AA at the same concentration, showing very clear differences, which means that this detection system can be used to distinguish different reducing substrates.

[0071] Application Examples

[0072] 1. Organophosphorus Pesticide (OPs) Detection

[0073] (1) 9 μL of phoxim standard solution with different concentrations (concentrations were 9×10 -1 mg / mL, 1 × 10 -1 mg / mL, 1×10 -2 mg / mL, 1×10 -4 mg / mL, 1 × 10 -5 mg / mL and 1×10 -6mg / mL) was mixed with 1 µL alkaline phosphatase (ALP, 1 U / mL) and allowed to react at 37°C for 20 min. Then, 20 µL sodium L-ascorbic acid-2-phosphate (AAP, 4 mM) was added and allowed to react at 37°C for 50 min.

[0074] (2) Add 10 µL of MnO2@UiO-66 dispersion (1 mg / mL) to the solution in step (1) and react at 37°C for 50 min. Finally, add 140 µL of ultrapure water and 20 µL of Ampliflu Red solution (AR, 50 µM) and react at 37°C for 30 min to obtain the test solution. Here, MnO2@UiO-66 dispersion refers to the aqueous dispersion of MnO2@UiO-66 composite material. Ampliflu Red solution refers to the solution obtained by dissolving Ampliflu Red in N,N-dimethylformamide.

[0075] (3) The test solution after the reaction in step (2) is subjected to fluorescence measurement of the fluorescence intensity at 430 nm and 590 nm at excitation wavelengths of 360 nm and 571 nm.

[0076] Figure 5 This is the test result of MnO2@UiO-66 composite material on phoxim standard solution. Figure 5 In Figure A, the blue fluorescence intensity at 430 nm increases with the decrease of phoxim concentration, while the red fluorescence intensity at 590 nm decreases with the decrease of phoxim concentration ( Figure 5 (Figure B). Figure 5 Figure C in the middle shows that based on 3σ / S, the concentration of phoxim increased from 1×10 -6 mg / mL to 9×10 -1 mg / mL, the logarithm of its concentration is related to F 430 / F 590 In the case of a good linear relationship, Y=3.257-0.712 lgC(R 2 =0.9935), and the detection limit of phoxim was 0.33×10 -6 mg / mL.

[0077] 2. Detection of reduced biomolecules (GSH, Cys, AA)

[0078] (1) 20 μL of different concentrations of glutathione (1 μM, 5 μM, 10 μM, 20 μM, 25 μM, 50 μM, 75 μM, 100 μM), cysteine ​​(30 μM, 40 μM, 50 μM, 70 μM, 90 μM), and ascorbic acid standard solution (1 μM, 5 μM, 20 μM, 30 μM, 50 μM) were added to 145 μL of ultrapure water and 20 μL of 1 mg / mL MnO2@UiO-66 dispersion and incubated at 37°C for 2 h.

[0079] (2) Add 20 μL of 50 μM Ampliflu Red solution (AR) in step (1) and incubate at 37°C in the dark for 10 min to obtain the test solution. Subsequently, the test solution is subjected to fluorescence measurement at 430 nm and 590 nm under excitation wavelengths of 360 nm and 571 nm.

[0080] Figure 6 These are the test results of MnO2@UiO-66 composite materials on standard samples of glutathione, cysteine ​​and ascorbic acid.

[0081] Figure 6 In Figure A, the blue fluorescence intensity at 430 nm increased with the increase of glutathione concentration, while Figure 6 In panel B, the red fluorescence intensity at 590 nm decreased with increasing glutathione concentration. Figure 6 Figure C shows that based on 3σ / S, the concentration of glutathione ranged from 1 to 100µM and its concentration was related to F 430 / F 590 The linear relationship between them is: Y=0.2477C GSH +0.5181(R 2 =0.9938), and the detection limit of the sensor was 0.33 μM. Figure 6 Figures D to F show the relationship between cysteine ​​concentrations from 30 µM to 90 µM and F. 430 / F 590 The linear relationship between them. Based on 3σ / S, the linear equation is Y=0.08337C Cys -1.628 (R 2 =0.9922) with a detection limit of 10 μM. Figure 6 Figures G to I in the middle are the concentrations of AA from 1 µM to 50 µM and F 430 / F 590 The linear relationship between them is based on 3σ / S, and the linear equation is Y=0.05633C AA +0.5022 (R 2=0.9983), with a detection limit of 0.33 μM.

[0082] 3. Detection of organophosphorus in real samples and glutathione in 1% human serum

[0083] To verify the practicality and reliability of the ratiometric fluorescence system based on the MnO2@UiO-66 composite material, the recovery of spiked organophosphorus in real samples and glutathione in 1% human serum was tested. The specific detection process and results are as follows:

[0084] The steps for testing the above-mentioned actual sample of phoxim are as follows:

[0085] (a) Dilute the 1 mg / mL phoxim standard solution with tap water to different concentrations (1 × 10 -2 mg / mL, 1×10 - 4 mg / mL, 1 × 10 -5 mg / mL, 1×10 -6 9 µL of the above solution was mixed with 1 µL of 1 U / mL alkaline phosphatase, incubated at 37°C for 0-20 min, and then 20 µL of 4 mM sodium L-ascorbic acid-2-phosphate solution was added. The mixture was incubated for 0-50 min to obtain a mixed solution.

[0086] (b) Add 10 μL of 1 mg / mL MnO2@UiO-66 dispersion to the mixed solution obtained in step (a), incubate at 37°C for 50 min, then add 140 μL of ultrapure water and 20 μL of 50 μM Ampliflu Red solution, and continue incubation for 30 min to obtain the test standard solution I.

[0087] (c) Perform fluorescence measurement on the standard solution I obtained in step (b), record the ratio of the fluorescence intensity at the emission wavelengths of 430 nm and 590 nm under the excitation wavelengths of 360 nm and 571 nm, and establish the F 430 / F 590 The ratio fluorescence value is used to calculate the recovery rate. Figure 7 As shown in Figure A, the detection recoveries of phoxim in tap water ranged from 98.60% to 110.7%, and the relative standard deviation (RSD) was within 9.20%.

[0088] The steps for detecting the actual sample of the above-mentioned reducing molecules are as follows:

[0089] (S1) 20 μΐ, different concentrations of glutathione standard solution (30 μΜ, 50 μΜ, 75 μΜ, 100 μΜ), 1% human serum solution (145 μΐ,) were added to 20 μΐ, 1 mg / mL MnO2@UiO-66 dispersion, incubated at 37°C for 2 h, then Ampliflu Red solution (20 μΐ, 50 μΜ) was added, and incubated at 37°C for 10 min in the dark, to obtain the standard solution II to be detected;

[0090] (S2) The fluorescence of the standard solution II to be detected obtained in step (S1) was determined, and the ratio of the fluorescence intensity at 430 nm and 590 nm was recorded at an excitation wavelength of 360 nm and 571 nm, to establish the F 430 / F 590 ratio fluorescence value, and the recovery rate was calculated. The results are shown in FIG. B of Figure 7 , which shows that the detection recovery rate of GSH in 1% human serum is between 91.18% and 104.2%, and the RSD is within 4.54% (n = 3) Figure 6 B). The above results show that this test system has great potential in the detection of organophosphorus (OP) and GSH in real samples.

[0091] The stability of the ratio fluorescence system was also evaluated by GSH (FIG. C of Figure 7 ). MnO2 / UIO-66-NH2 was dispersed in ultrapure water to 1 mg / mL and stored in the dark at 4°C. From the 1st day to the 12th day, the recovery rate did not decrease significantly, and on the 12th day it maintained a recovery rate of 107.9% and an RSD of 6.34%, which means that this system is stable for at least 12 days.

[0092] In addition, the anti-interference ability of GSH detection was tested with 50 μΜ and 75 μΜ GSH respectively, and the specific test process was as follows:

[0093] (S1) 20 μΐ, different concentrations of glutathione standard solution (50 μΜ, 75 μΜ) were added to 20 μΐ, 1 mg / mL MnO2@UiO-66 dispersion containing K + , Mg 2+ , Ca 2+ ultrapure water solution (145 μΐ,) respectively, incubated at 37°C for 2 h, then Ampliflu Red solution (20 μΐ, 50 μΜ) was added, and incubated at 37°C for 10 min in the dark, to obtain the standard solution II to be detected; wherein the concentrations of K + , Mg 2+ , Ca 2+ in the ultrapure water solution were 10 μΜ respectively;

[0094] (S2) Performing fluorescence measurement on the standard solution II obtained in step (S1), recording the ratio of the fluorescence intensity at the emission wavelengths of 430 nm and 590 nm under the excitation wavelengths of 360 nm and 571 nm, and establishing the F 430 / F 590 The recovery rate was calculated based on the ratio fluorescence value.

[0095] (S3) 20 μL of glutathione standard solution (50 μM, 75 μM) with different concentrations of K + Mg 2+ , Ca 2+ Ultrapure water solution (145 μL) was added to 20 μL of 1 mg / mL MnO2@UiO-66 dispersion, incubated at 37°C for 2 h, and then Ampliflu Red solution (20 μL, 50 μM) was added and incubated at 37°C in the dark for 10 min to obtain the standard solution III to be tested; K + Mg 2+ , Ca 2+ The concentration in ultrapure aqueous solution was 30 μM;

[0096] (S4) Performing fluorescence measurement on the standard solution III obtained in step (S3), recording the ratio of the fluorescence intensity at the emission wavelengths of 430 nm and 590 nm under the excitation wavelengths of 360 nm and 571 nm, and establishing the F 430 / F 590 The recovery rate was calculated based on the ratio fluorescence value.

[0097] like Figure 7 As shown in Figure D, when only one ion is present, the recovery of 50 µM GSH ranges from 87.09% to 92.01% with an RSD of 1.68% (blue), while the recovery of 75 µM GSH ranges from 91.14% to 96.28% with an RSD of 4.18% (red). When two or more ions are present, the recovery of 50 µM GSH is 89.52% with an RSD of 0.46%, while the recovery of 75 µM GSH is 97.48% with an RSD of 4.42%. These results demonstrate the excellent interference resistance of the proposed ratiometric fluorescence system.

[0098] In summary, this paper presents a ratiometric fluorescence system based on the MnO2@UiO-66 composite material, which has been successfully applied to the determination of OPs and reduced biomolecules. With the help of PCA, GSH, Cys, and AA were distinguished. This system is accurate, convenient, sensitive, and stable, and has been successfully applied to the detection of phoxim and glutathione in complex environments. It demonstrates high reliability and has broad practical application prospects.

[0099] The above description is only 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 in the scope of protection of the present invention.

Claims

1. A ratiometric fluorescence system based on MnO2@UiO-66 composite material, characterized in that: The ratiometric fluorescence system includes a MnO2@UiO-66 composite material and Ampliflu Red. The preparation method of the MnO2@UiO-66 composite material comprises the following steps: (1) Mix UiO-66-NH2, ultrapure water and MES buffer to obtain MOF solution; (2) KMnO4 solution was added to the MOF solution, stirred at room temperature until the solution turned brown, and incubated to obtain a reaction solution; the reaction solution was desolvated, centrifuged, washed, and dried to obtain the MnO2@UiO-66 composite material.

2. The ratio fluorescence system according to claim 1, wherein: The concentration of UiO-66-NH2 in the MOF solution in step (1) is 0.1-1 mg / mL; the concentration of the MES buffer is 5 mM, pH = 6; the concentration of the KMnO4 solution in step (2) is 0.1-1 mg / mL; and the volume ratio of the MOF solution to the KMnO4 solution is (1-5):

1.

3. Use of the ratiometric fluorescence system according to claim 1 or 2 in highly sensitive detection of organophosphorus, characterized in that: When highly sensitive detection of organophosphorus is performed, the ratiometric fluorescence system further comprises alkaline phosphatase and L-ascorbic acid-2-sodium phosphate solution; the highly sensitive detection of organophosphorus comprises the following steps: (a) Mixing organophosphorus standard solutions of varying concentrations with alkaline phosphatase, incubating at 37°C, adding sodium L-ascorbic acid-2-phosphate solution, and continuing the incubation to obtain a mixed solution; (b) adding the MnO2@UiO-66 dispersion to the mixed solution obtained in step (a), incubating at 37°C, adding ultrapure water and Ampliflu Red solution, and continuing to incubate to obtain the standard solution I to be tested; (c) Perform fluorescence measurement on the standard solution I obtained in step (b). Under excitation wavelengths of 360 nm and 571 nm, record the ratio of the fluorescence intensity at emission wavelengths of 430 nm and 590 nm to establish the ratio fluorescence value F. 430 / F 590 The linear relationship between the concentration of organophosphorus standard solution and the 4. The use according to claim 3, characterized in that The concentration of the phoxim standard solution in step (a) is 1×10 -6 -9×10 -1 mg / mL, the concentration of alkaline phosphatase is 0.1-1 U / mL; the concentration of L-ascorbic acid-2-phosphate sodium aqueous solution is 1-4 mM; the volume ratio of phoxim standard solution, alkaline phosphatase and L-ascorbic acid-2-phosphate sodium solution is (1-9):1:(1-20).

5. The use according to claim 3, characterized in that In step (b), the MnO2@UiO-66 dispersion refers to an aqueous solution of MnO2@UiO-66 composite material with a concentration of 0.1-1 mg / mL; the Ampliflu Red solution is obtained by dissolving Ampliflu Red in N,N-dimethylformamide, and the concentration of Ampliflu Red is 1-50 µM; the volume ratio of the mixed solution, MnO2@UiO-66 dispersion, and Ampliflu Red solution is (1-30):(1-10):(1-20).

6. The use according to claim 3, characterized in that The organophosphorus is selected from phoxim, chlorpyrifos, methyl parathion or dimethoate.

7. Use of the ratiometric fluorescence system according to claim 1 in highly sensitive detection of reduced biomolecules, characterized in that: The steps of highly sensitive detection of reduced biomolecules are as follows: (S1) Adding a reducing biomolecule standard solution and ultrapure water to the MnO2@UiO-66 dispersion, incubating at 37°C, adding Ampliflu Red solution, and incubating at 37°C in the dark to obtain the standard solution II to be tested; (S2) Performing fluorescence measurement on the standard solution II obtained in step (S1), recording the ratio of the fluorescence intensity at the emission wavelengths of 430 nm and 590 nm under the excitation wavelengths of 360 nm and 571 nm, and establishing the ratio fluorescence value F 430 / F 590 Linear relationship with the concentration of reduced biomolecule standard solution.

8. The use according to claim 7, characterized in that In step (S1), the concentration of the reducing biomolecule standard solution is 1-90 μM; the volume ratio of the reducing biomolecule standard solution, the MnO2@UiO-66 dispersion and the Ampliflu Red solution is (0.1-1):1:

1.

9. The use according to claim 7, characterized in that The reducing biomolecule is selected from glutathione, cysteine ​​or ascorbic acid.

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