MnO2at-UiO-66 composite material as well as preparation method and application thereof

Abstract

The invention belongs to the field of molecular biology, discloses a MnO2-coated UiO-66 composite material as well as a preparation method and application thereof, and solves the technical problems of relatively narrow detection sensitivity and detection range of organic phosphorus compounds. The ratiometric fluorescence system containing the MnO2 (at) UiO-66 composite material and Ampliflu Red is used for high-sensitivity detection of organophosphorus pesticides and reducing biomolecules. The MnO2 material not only can be used as a fluorescence quenching agent of UiO-66-NH2, but also can be used for catalyzing an Ampliflu Red reagent to generate a fluorescence product isophenoxazolone, so that a fluorescence signal at the wavelength of 590 nm is enhanced. When the organophosphorus is detected, the system further comprises alkaline phosphatase and L-ascorbic acid-2-sodium phosphate. When the concentration of phoxim is 1 * 10 <-6 >-9 * 10 <-1 > mg / mL, the fluorescence linear relation is good, and the detection limit is as low as 0.33 * 10 <-6 > mg / mL. The system has the characteristics of accuracy, convenience, sensitivity, stability and the like, and can be applied to organophosphorus pesticide detection in a complex environment.

Description

Technical Field

[0001] The invention relates to the field of molecular biology and to 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 compound pesticides containing phosphorus. They are mainly used to control plant diseases, insects and weeds. They are widely used in agricultural, forestry and animal husbandry production in countries around the world and play an important role in increasing agricultural production. Due to the widespread use of OPs, OPs residues in the environment and crop products will pose a threat to human health through food chain exposure. The toxic mechanism of OPs is to inhibit the activity of acetylcholinesterase in nerve tissue and other target organs. The inability to decompose acetylcholine leads to excessive stimulation of muscarinic and nicotinic receptors, which in turn leads to clinical symptoms such as excessive sweating, salivation, bronchospasm, bronchial edema, bradycardia, hypotension and decreased consciousness. Even low levels of OPs can cause great harm to human health, leading to kidney damage, Parkinson's disease, endocrine disorders, memory loss and proximal muscle weakness. The harm of OPs to human health cannot be ignored. Therefore, it is of great significance to develop a rapid and accurate method for detecting environmental OPs residues.

[0003] Biomolecules play an important role in maintaining normal life activities of the human body and resisting diseases. In particular, some biomolecules with reducing properties, such as biothiols and ascorbic acid (AA), play an important role in maintaining human health. Glutathione and cysteine, as biothiols, are closely related to physiological and pathological processes in complex biological environments, and their abnormal contents are closely related to many diseases. AA is an important nutrient in the human body with good reducing properties. It can effectively reduce oxidative stress in the body and reduce the incidence of diseases such as cancer. Therefore, highly selective and sensitive detection of small molecule compounds in organisms is very important for the treatment of these diseases.

[0004] At present, the detection methods for OPs residues mainly 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, time-consuming processes and requirements 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 because of 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, by mixing a water sample containing phosphorus compounds with N-ethylmorpholine, adding UiO-66-NH2Tris suspension, and then measuring the fluorescence properties to achieve detection of organophosphorus. However, in the above-mentioned technology, the detection limit of oxonphosphorus is 23.8 ng / mL, the detection range is 0-20 ug / mL, the detection range is narrow, and it 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 organic phosphorus. How to sensitively and efficiently detect reduced biological small molecules 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: A method for preparing a MnO2@UiO-66 composite material, the steps are as follows: (1) Mix UiO-66-NH2, ultrapure water and MES buffer to obtain MOF solution; (2) Add KMnO4 solution to the MOF solution, stir at room temperature until the solution turns brown, and incubate to obtain a reaction solution; the reaction solution is desolventized, centrifuged, washed and dried to obtain a MnO2@UiO-66 composite material.

[0007] 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.

[0008] MnO2@UiO-66 composite material prepared by the above preparation method.

[0009] A ratiometric fluorescence system based on a MnO2@UiO-66 composite material comprises the above-mentioned MnO2@UiO-66 composite material and Ampliflu Red.

[0010] The above ratio fluorescence system is used in the highly sensitive detection of organophosphorus or reduced biological molecules. When the organophosphorus is detected with high sensitivity, the ratio fluorescence system also includes alkaline phosphatase and L-ascorbic acid-2-sodium phosphate solution.

[0011] The above-mentioned highly sensitive detection of organophosphorus comprises the following steps: (a) mixing different concentrations of phoxim standard solutions and alkaline phosphatase, incubating at 37°C for 1-20 min, adding L-ascorbic acid-2-sodium phosphate solution, and continuing to incubate for 1-50 min to obtain a mixed solution; (b) adding MnO2@UiO-66 dispersion to the mixed solution obtained in step (a), incubating at 37°C for 1-50 min, adding ultrapure water and Ampliflu Red solution, and continuing to incubate for 1-30 min to obtain standard solution I to be tested; (c) Performing fluorescence measurement on the standard solution I obtained in step (b), 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 organophosphorus standard solution.

[0012] 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.

[0013] 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.

[0014] The concentration of the sodium L-ascorbic acid-2-phosphate aqueous solution is 1-4 mM. For example, the concentration of the sodium L-ascorbic acid-2-phosphate aqueous solution is 1-3 mM; for example, the concentration of the sodium L-ascorbic acid-2-phosphate aqueous solution is 2-4 mM; for example, the concentration of the sodium L-ascorbic acid-2-phosphate aqueous solution is 1 mM, 2 mM, 3 mM or 4 mM.

[0015] The volume ratio of phoxim standard solution, alkaline phosphatase and L-ascorbic acid-2-sodium phosphate 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.

[0016] Further, in the above step (b), the MnO2@UiO-66 dispersion refers to a MnO2@UiO-66 composite aqueous solution with a concentration of 0.1-1 mg / mL. For example, the MnO2@UiO-66 dispersion refers to a concentration of 0.1-0.6 mg / mL; for example, the MnO2@UiO-66 dispersion refers to a concentration of 0.5-1 mg / mL; for example, the MnO2@UiO-66 dispersion refers to a concentration of 0.4-0.9 mg / mL; for example, the MnO2@UiO-66 dispersion refers to 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.

[0017] The Ampliflu Red solution is prepared 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.

[0018] The volume ratio of the mixed solution, MnO2@UiO-66 dispersion and Ampliflu Red solution is (1-30):(1-10):(1-20). For example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion 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 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 and the Ampliflu Red solution is 1:1:(1-20); for example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion and the Ampliflu Red solution is 30:1:(1-20); for example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion and the Ampliflu Red solution is 1:(1-10):1; for example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion and the Ampliflu Red solution is 1:(1-10): 20; for example, the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion 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.

[0019] The steps for the highly sensitive detection of reduced biomolecules are as follows: (S1) Add the reducing biomolecule standard solution and ultrapure water to the MnO2@UiO-66 dispersion, incubate at 37°C for 1-2 h, add the Ampliflu Red solution, and incubate at 37°C in the dark for 1-10 min 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.

[0020] In the above step (S1), the reducing biomolecule in the reducing biomolecule standard solution is glutathione, cysteine ​​or ascorbic acid; the concentration of the reducing biomolecule standard solution is 1-90 µM.

[0021] When the reducing biological molecule is glutathione, the corresponding glutathione standard solution concentrations are 1 µM, 5 µM, 10 µM, 20 µM, 25 µM, 50 µM, 75 µM, and 100 µM, respectively; when the reducing biological molecule is cysteine, the corresponding cysteine ​​standard solution concentrations are 30 µM, 40 µM, 50 µM, 70 µM, and 90 µM, respectively; when the reducing biological molecule is ascorbic acid, the corresponding ascorbic acid standard solution concentrations are 1 µM, 5 µM, 20 µM, 30 µM, and 90 µM, respectively.

[0022] In the above step (S1), 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. For example, the volume ratio of the reducing biomolecule standard solution, the MnO2@UiO-66 dispersion 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 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 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 and the Ampliflu Red solution is 0.1:1:1, 0.4:1:1, 0.7:1:1 or 1:1:1.

[0023] The beneficial effects produced by the present invention are: (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 can not only serve as a fluorescence quencher for UIO-66-NH2, but also catalyze 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 oxidize 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 In the range of mg / mL phoxim concentration, the linear relationship is good, 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 ratio fluorescence system designed by the present invention has the characteristics of accuracy, convenience, sensitivity, stability, etc., and can be applied to the detection of organophosphorus pesticides in complex environments with high reliability.

[0024] (2) In addition, the present invention also proposes a method for detecting the concentration of reduced biomolecules based on the MnO2@UiO-66 composite material, which includes the MnO2@UiO-66 composite material and Ampliflu Red (AR). When detecting reduced biomolecules, in the presence of glutathione, cysteine ​​and AA, the MnO2 coating is directly decomposed, the fluorescence intensity of UiO-66-NH2 is higher, and the fluorescence intensity of AR is lower; when the above molecules are not present, the fluorescence intensity of UiO-66-NH2 is lower, while the fluorescence intensity of AR is higher. The detection of reduced 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, with high reliability and broad practical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0026] 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; where the inset is a photo 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.

[0027] Figure 2 Schematic diagram of the principle of detecting organophosphorus pesticides and reduced biological molecules based on the relative fluorescence system of MnO2@UiO-66 composite materials, where (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.

[0028] Figure 3 This is the catalytic mechanism of MnO2@UiO-66 composite material.

[0029] Figure 4 The selectivity of the fluorescence system based on MnO2@UiO-66 composite material for detecting organophosphorus pesticides and reduced biological molecules, where (A) is the selectivity for organophosphorus; (B) is the selectivity for GSH, Cys and AA, and the inset is the principal component analysis result of GSH, Cys and AA.

[0030] Figure 5 The results of the MnO2@UiO-66 composite material on the standard solution of phoxim, where (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 plots versus the logarithm of phoxim concentration; all error bars represent the standard deviation of four isolated experiments.

[0031] Figure 6 The results of the MnO2@UiO-66 composite material on the standard samples of glutathione, cysteine ​​and ascorbic acid, where (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) is the fluorescence intensity spectrum of UiO-66-NH2 with different concentrations of cysteine; (E) is the 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 F 430 / F 590 Linear relationship with different concentrations of AA, all error bars represent the standard deviation of four independent experiments.

[0032] Figure 7 Figure 2 Performance of the specific fluorescence system based on MnO2@UiO-66 composite materials, where (A) is the recovery rate of thiocyanate in tap water; (B) is the recovery rate of glutathione in 1% human serum; (C) is the stability of GSH detection; (D) is the anti-interference ability of GSH detection. All error bars represent the standard deviation of four isolated experiments. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the 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.

[0034] Example 1 The preparation method of a MnO2@UiO-66 composite material in this embodiment comprises the following steps: (1) Preparation method of UiO-66-NH2, the steps are as follows: 0.81 g 2-aminoterephthalic acid and 10.5 g zirconium tetrachloride are dissolved in 40 mL DMF solvent, and then ultrasonicated until the solute is completely dissolved. The solution is transferred to a hydrothermal reactor, 17 mL glacial acetic acid is added, and then reacted at 120 degrees Celsius for 24 hours. The preparation process is as follows Figure 2 As shown in Figure A.

[0035] (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 after 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.

[0036] 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.

[0037] 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 both MnO2NS@UiO-66-NH2 and UiO-66-NH2, peaks of Zr 3d at 184 eV, C1s at 284 eV, N1s at 400 eV, and O1s at 531 eV can be observed. 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 MnO2 coated UiO-66-NH2, the color of the picture became much darker. Figure 1 The inset 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 dark brown powder. Transmission electron microscopy can also clearly distinguish the morphology of the two materials ( Figure 1 Figure F and Figure 1 (Fig. 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 image, some flakes appear at the edge of the material, indicating that the manganese dioxide nanosheets are successfully covered. FIRT spectrum ( Figure 1 This view is further confirmed by the fact that 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 of UiO-66-NH2 (blue curve) to 0.144 ns of MnO2NS@UiO-66-NH2 (red curve). Figure 1 As shown in Figure I. The above data show that the synthesis of this material is successful.

[0038] Example 2 The preparation method of a MnO2@UiO-66 composite material in this embodiment comprises the following steps: (1) Preparation method of UiO-66-NH2, the steps are as follows: 0.81 g 2-aminoterephthalic acid and 10.5 g zirconium tetrachloride are dissolved in 40 mL DMF solvent, and then ultrasonicated until the solute is completely dissolved. The solution is transferred to a hydrothermal reactor, 17 mL glacial acetic acid is added, and then reacted at 120 degrees Celsius for 24 hours. The preparation process is as follows Figure 2 As shown in Figure A.

[0039] (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; when the solution color turned brown, it was incubated for a period of time; 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.

[0040] Example 3 The preparation method of a MnO2@UiO-66 composite material in this embodiment comprises the following steps: (1) Preparation method of UiO-66-NH2, the steps are as follows: 0.81 g 2-aminoterephthalic acid and 10.5 g zirconium tetrachloride are dissolved in 40 mL DMF solvent, and then ultrasonicated until the solute is completely dissolved. The solution is transferred to a hydrothermal reactor, 17 mL glacial acetic acid is added, and then reacted at 120 degrees Celsius for 24 hours. The preparation process is as follows Figure 2 As shown in Figure A.

[0041] (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; when the solution color turned brown, it was incubated for a period of time; 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.

[0042] Implementation effect example MnO2@UiO-66 composite materials can be used for the detection of organophosphorus and reduced biomolecules. The detection principle is as follows: Figure 2Specifically, the technical principle of the detection method is as follows Figure 2 As shown in Figure B, specifically: MnO2 material can not only serve as a fluorescence quencher for UiO-66-NH2, but also catalyze the Ampliflu Red (AR) reagent to generate the fluorescent product isophene oxazolone (Resorufin), thereby enhancing its fluorescence signal at a wavelength of 590 nm. When organophosphorus (OPs) are present, OPs prevent alkaline phosphatase from decomposing L-ascorbic acid-2-phosphate into ascorbic acid (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 and oxidized by MnO2 to generate Resorufin, resulting in an increase in its fluorescence signal at 590 nm. When OPs are not present, alkaline phosphatase can decompose L-ascorbic acid-2-phosphate into AA, and MnO2 is further decomposed into Mn 2+ This results in an increase in the fluorescence intensity of UiO-66-NH2 at a wavelength of 430 nm, while the signal intensity of the AR reagent at a wavelength of 590 nm is weakened, thereby achieving highly sensitive detection of OPs. For the detection of reduced biomolecules, when glutathione (GSH), cysteine ​​(Cys) and ascorbic acid (AA) are present, the MnO2 coating is directly decomposed, and the fluorescence intensity of UiO-66-NH2 is higher and the fluorescence intensity of AR is lower. When the above molecules are not present, the fluorescence intensity of UiO-66-NH2 is lower, while the fluorescence intensity of AR is higher, thereby achieving the detection of reduced biomolecules. Figure 2 Figure C in the middle is the PCA analysis of the detection results of GSH, Cys and AA.

[0043] In addition, the catalytic mechanism of the MnO2@UiO-66 composite material is as follows: MnO2@UiO-66 catalyzes AR to generate two free radicals. The free radical anion is then oxidized to form resorufin. At the same time, the free radical cation is deacetylated and then exchanged with alcohol ketone to generate Resorufin (such as Figure 3 ).

[0044] Selective detection: Different types of organophosphates (OPs) have different inhibitory effects on alkaline phosphatase activity, and the reducibility of GSH, Cys and AA is also different, resulting in different detection at 430 nm, 590 nm and F 430 / F 590 The fluorescence intensity at different locations is different, which can be used to distinguish target species. Therefore, principal component analysis is used to analyze its selectivity. The specific test process and results are as follows Figure 4 shown.

[0045] The above-mentioned organophosphorus specific detection comprises the following steps: (a) 9 µL of a 1×10 -1Mix 1 μL of 1U / mL alkaline phosphatase with different organophosphorus standard solutions (phoxim, chlorpyrifos, parathion-methyl and dimethoate) and incubate at 37°C for 20 min, then add 20 μL of 4 mM sodium L-ascorbic acid-2-phosphate solution and continue incubating for 50 min to obtain a mixed solution. (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 is N,N-dimethylformamide, solute is Ampliflu Red), and continue incubation for 30 min to obtain the standard solution I to be tested; (c) Perform fluorescence measurement on the standard solution I obtained in step (b), and 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 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.

[0046] 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 examine the selectivity of the detection system based on the MnO2@UiO-66 composite material.

[0047] The above-mentioned specific detection of reduced biological small molecules includes the following steps: (S1) 20 μL of 50 μM standard solutions of different types of 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, 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 II to be tested; (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, 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.

[0048] (S3) Performing fluorescence measurement on the different standard solutions II to be detected obtained in step (S1) and the different standard solutions III to be detected 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.

[0049] 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 Arg, Try and Gly were 430 / F 590 The detection results of the sites showed no significant changes compared with the blank control. The inset shows the principal component analysis (PCA) results of GSH, Cys and AA at the same concentration, and very obvious differences can be seen, which means that the detection system can be used to distinguish different reducing substrates.

[0050] Application Examples 1. Organophosphorus Pesticides (OPs) Detection (1) 9 μL of phoxim standard solutions 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 -6 mg / mL) was mixed with 1 µL alkaline phosphatase (ALP, 1 U / mL) and reacted at 37°C for 20 min. Then 20 µL L-ascorbic acid-2-phosphate (AAP, 4 mM) was added and reacted at 37°C for 50 min.

[0051] (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, 50µM) 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. (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.

[0052] 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 as the concentration of phoxim decreases, while the red fluorescence intensity at 590 nm decreases as the concentration of phoxim decreases ( Figure 5 (Fig. B). Figure 5 Figure C 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.

[0053] 2. Detection of reduced biomolecules (GSH, Cys, AA) (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. (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 fluorescence intensity at 430 nm and 590 nm of the test solution was measured at excitation wavelengths of 360 nm and 571 nm.

[0054] Figure 6 These are the test results of MnO2@UiO-66 composite materials against standard samples of glutathione, cysteine ​​and ascorbic acid.

[0055] Figure 6 In Figure A, the blue fluorescence intensity at 430 nm increases with the increase of glutathione concentration, while Figure 6 In Figure B, the red fluorescence intensity at 590 nm decreased with the increase of glutathione concentration. Figure 6 Figure C shows that based on 3σ / S, the concentration of glutathione ranged from 1 to 100 µM and its relationship with F 430 / F 590 The linear relationship between them is: Y=0.2477C GSH +0.5181(R 2 =0.9938), the detection limit of the sensor is 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 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.

[0056] 3. Detection of organophosphorus in real samples and glutathione in 1% human serum In order to verify the practicality and reliability of the ratio fluorescence system based on MnO2@UiO-66 composite materials, the recovery effect of the ratio fluorescence system on the spiked organic phosphorus in real samples and glutathione in 1% human serum was tested. The specific detection process and results are as follows: The steps for testing the above-mentioned actual sample of phoxim are as follows: (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 mg / mL). Take 9 µL of the above solution and mix with 1 µL of 1 U / mL alkaline phosphatase, incubate at 37°C for 0-20 min, add 20 µL of 4 mM sodium L-ascorbic acid-2-phosphate solution, and continue incubating for 0-50 min to obtain a mixed solution; (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 incubating for 30 min to obtain the standard solution I to be tested; (c) Performing fluorescence measurement on the standard solution I obtained in step (b), 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 ratio fluorescence value was 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%.

[0057] The steps for the actual sample detection of the above-mentioned reducing molecules are as follows: (S1) 20 µL of glutathione standard solutions of different concentrations (30 µM, 50 µM, 75 µM, 100 µM) and 1% human serum solution (145 µL) were 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 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 F 430 / F 590 The ratio fluorescence value was used to calculate the recovery rate. Figure 7As shown in Figure B, the recovery of GSH in 1% human serum ranged from 91.18% to 104.2%, with an RSD within 4.54% ( Figure 6 B). The above results indicate that this test system has great potential in the detection of organophosphates (OP) and GSH in real samples.

[0058] The stability of the ratiometric fluorescence system was also evaluated using GSH ( Figure 7 MnO2 / UIO-66-NH2 was dispersed in ultrapure water to 1 mg / mL and stored in the dark at 4°C. There was no significant decrease in the recovery from day 1 to day 12, and a recovery of 107.9% and an RSD of 6.34% were maintained on day 12, which means that the system was stable for at least 12 days.

[0059] In addition, the anti-interference ability of GSH detection was tested using 50 µM and 75 µM GSH, respectively. The specific test process is as follows: (S1) 20 µL of glutathione standard solutions (50 µM, 75 µM) with K + Mg 2+ , Ca 2+ Ultrapure aqueous solution (145 µL) was added to 20 µL, 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 II to be tested; K + Mg 2+ , Ca 2+ The concentrations in ultrapure aqueous solution were 10 µM; (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.

[0060] (S3) 20 µL of glutathione standard solution (50 µM, 75 µM) with different concentrations of K + Mg 2+ , Ca 2+ Ultrapure aqueous solution (145 µL) was added to 20 µL, 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; among them, K+ Mg 2+ , Ca 2+ The concentration in ultrapure aqueous solution was 30 µM; (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.

[0061] like Figure 7 As shown in Figure D, when only one ion is present, the recovery of 50 µM GSH is between 87.09% and 92.01% with an RSD of 1.68% (blue), and the recovery of 75 µM GSH is between 91.14% and 96.28% with an RSD of 4.18% (red). When more than two ions are present, the recovery of 50 µM GSH is 89.52% with an RSD of 0.46%, and the recovery of 75 µM GSH is 97.48% with an RSD of 4.42%. This result shows that the proposed ratiometric fluorescence system has good anti-interference ability.

[0062] In summary, the present invention designs a ratiometric fluorescence system based on MnO2@UiO-66 composite materials, and it has been successfully applied to the determination of OPs and reduced biomolecules. With the help of PCA, the distinction between GSH, Cys and AA was achieved. The system is accurate, convenient, sensitive and stable, and has been successfully applied to the detection of phoxim and glutathione in complex environments. It has high reliability and broad practical application prospects.

[0063] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a MnO2@UiO-66 composite material, characterized in that: Here are the steps: (1) Mix UiO-66-NH2, ultrapure water and MES buffer to obtain MOF solution; (2) Add KMnO4 solution to the MOF solution, stir at room temperature until the solution turns brown, and incubate to obtain a reaction solution; the reaction solution is desolventized, centrifuged, washed and dried to obtain a MnO2@UiO-66 composite material.

2. The preparation method according to claim 1, characterized in that: 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, and the pH is 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. A MnO2@UiO-66 composite material prepared by the preparation method described in claim 1 or 2.

4. A ratiometric fluorescence system based on MnO2@UiO-66 composite material, characterized in that: The ratio fluorescence system comprises the MnO2@UiO-66 composite material described in claim 3 and Ampliflu Red.

5. Use of the ratio fluorescence system according to claim 4 in highly sensitive detection of organophosphorus or reduced biomolecules, characterized in that: When highly sensitive detection of organophosphates is desired, the ratiometric fluorescence system further comprises alkaline phosphatase and sodium L-ascorbic acid-2-phosphate solution.

6. The use according to claim 5, characterized in that: The highly sensitive detection of organophosphorus comprises the following steps: (a) mixing different concentrations of phoxim standard solutions and alkaline phosphatase, incubating at 37°C, adding L-ascorbic acid-2-sodium phosphate solution, and continuing to incubate to obtain a mixed solution; (b) adding 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 standard solution I to be tested; (c) Performing fluorescence measurement on the standard solution I obtained in step (b), 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 organophosphorus standard solution.

7. The use according to claim 6, 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-sodium phosphate aqueous solution is 1-4 mM; the volume ratio of phoxim standard solution, alkaline phosphatase and L-ascorbic acid-2-sodium phosphate solution is (1-9):1:(1-20).

8. The use according to claim 7, characterized in that: The MnO2@UiO-66 dispersion in step (b) refers to a MnO2@UiO-66 composite material aqueous solution with a concentration of 0.1-1 mg / mL; the Ampliflu Red solution is obtained by dissolving AmplifluRed in N,N-dimethylformamide, and the concentration of Ampliflu Red is 1-50 µM; the volume ratio of the mixed solution, the MnO2@UiO-66 dispersion and the Ampliflu Red solution is (1-30):(1-10):(1-20).

9. The use according to claim 5, characterized in that: The steps of highly sensitive detection of reduced biomolecules are as follows: (S1) Add the reducing biomolecule standard solution and ultrapure water to the MnO2@UiO-66 dispersion, incubate at 37°C, add the Ampliflu Red solution, and incubate 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.

10. The use according to claim 9, characterized in that In the 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.

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