Low-dimensional Eu-BDC-NO2 / DPA nanomaterials, their synthesis and application in determining alkaline phosphatase activity.

Abstract

The application discloses a kind of low-dimensional Eu-BDC-NO2 / DPA nanomaterial, synthesis and its application in determining alkaline phosphatase activity, belong to metal organic framework material technical field.The technical scheme is as follows: the synthesis method of nanomaterial, specifically as follows:Eu (NO3) 3·6H2O is dissolved in water, 2-nitroterephthalic acid and 2, 6-pyridine dicarboxylic acid are dissolved in ethanol, and triethylamine is added in ethanol, and clear solution is obtained by ultrasonic;Then two kinds of solutions are mixed, heated and reacted;Cool to room temperature, centrifugal collection white solid, wash;Drying, it is Eu-BDC-NO2 / DPA material.The two-dimensional sheet and one-dimensional nanofiber nanomaterial are obtained by changing ligand and solvent respectively, the specific surface area of this low-dimensional MOF is large, more active sites can be provided in practical application, and the method for determining alkaline phosphatase activity has the characteristics of rapid detection, high selectivity and high sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of metal-organic framework materials technology, specifically relating to a low-dimensional Eu-BDC-NO2 / DPA nanomaterial, its synthesis, and its application in determining alkaline phosphatase activity. Background Technology

[0002] Metal-organic frameworks (MOFs) are organic-inorganic hybrid materials with intramolecular pores, formed by the self-assembly of organic ligands and metal ions or clusters through coordination bonds. MOF synthesis methods include solvent evaporation, diffusion (which can be further subdivided into gas-phase diffusion, liquid-phase diffusion, gel diffusion, etc.), hydrothermal or solvothermal methods, and ultrasonic and microwave methods. Compared to three-dimensional MOFs, two-dimensional (2D) MOFs, due to their sheet-like morphology, expose a large number of active sites, which facilitates contact between the substrate molecules and their active sites. This makes them widely used in catalysis, electrochemistry, and optical sensing. Currently, the synthesis of 2D MOFs is divided into bottom-up synthesis and top-down exfoliation methods, both of which have relatively high requirements for reaction conditions and operation. Therefore, developing a simple and time-efficient low-dimensional MOF synthesis method is an urgent problem to be solved.

[0003] Alkaline phosphatase (ALP), an important hydrolase, is widely distributed in the tissues and body fluids of mammals and catalyzes the dephosphorylation of various phosphorylated substrates. In humans, ALP is mainly found in the liver, bones, intestines, and kidneys. The normal range of serum ALP activity in healthy adults is 40-190 U / L. Furthermore, ALP activity is also related to sex, age, and disease type; serum ALP activity in pregnant women and children can reach as high as 500 U / L. Commonly used methods for detecting ALP activity include fluorescence detection, electrochemical detection, electrochemiluminescence, and surface-enhanced Raman spectroscopy. Among these, fluorescence detection has attracted much attention due to its high sensitivity and low detection limit.

[0004] Currently, there are no reports, either domestically or internationally, of synthesizing low-dimensional Eu-MOF nanomaterials using Eu(NO3)3·6H2O, 2-nitroterephthalic acid, and 2,6-pyridinedicarboxylic acid, and of using their ratio fluorescence visualization method to determine alkaline phosphatase activity in human serum. Establishing a low-cost, simple, highly selective, and highly sensitive alkaline phosphatase detection method is of great significance. Summary of the Invention

[0005] This invention provides a low-dimensional Eu-BDC-NO2 / DPA nanomaterial, its synthesis, and its application in the determination of alkaline phosphatase activity. By changing the ligands and solvents, two-dimensional sheet-like and one-dimensional nanofiber-like nanomaterials were obtained. This low-dimensional MOF has a large specific surface area and can provide more active sites in practical applications. The determination of alkaline phosphatase activity has the characteristics of rapid detection, high selectivity, and high sensitivity.

[0006] The technical solution of this invention is as follows:

[0007] Firstly, a method for synthesizing low-dimensional Eu-BDC-NO2 / DPA nanomaterials is disclosed, as follows:

[0008] Eu(NO3)3·6H2O was dissolved in water, 2-nitroterephthalic acid and 2,6-pyridinedicarboxylic acid were dissolved in ethanol, and triethylamine was added to the ethanol. The mixture was sonicated to obtain a clear solution. The two solutions were then mixed and heated to react. The mixture was cooled to room temperature, centrifuged to collect the white solid, washed, and dried to obtain the Eu-BDC-NO2 / DPA material.

[0009] Preferably, the molar ratio of Eu(NO3)3·6H2O to the sum of the molar amounts of 2-nitroterephthalic acid and 2,6-pyridinedicarboxylic acid ligands is 1:1.5-2, and the molar ratio of 2-nitroterephthalic acid to 2,6-pyridinedicarboxylic acid is 1:1-3.

[0010] Preferably, the mass-to-volume ratio of Eu(NO3)3·6H2O to water is 20-30 mg:1 mL, the mass-to-volume ratio of the total mass of 2-nitroterephthalic acid and 2,6-pyridinedicarboxylic acid to the total volume of ethanol and triethylamine is 5-6 mg:1 mL, and the volume ratio of ethanol to triethylamine is 100-150 mL:1 mL.

[0011] Preferably, the reaction temperature is 30-50℃, the reaction time is 2-4h, the drying temperature is 40-60℃, and the washing is done with ethanol and water.

[0012] Secondly, the application of the aforementioned low-dimensional Eu-BDC-NO2 / DPA nanomaterials in the determination of alkaline phosphatase activity is disclosed, specifically including:

[0013] 1) Plotting the working curve: Disperse the low-dimensional Eu-BDC-NO2 / DPA nanomaterials in water to obtain an Eu-BDC-NO2 / DPA aqueous solution; transfer the Eu-BDC-NO2 / DPA aqueous solution to a centrifuge tube containing reduced coenzyme I NADH, add ALP solutions with different activities, and finally add HEPES solution to make up the volume. Shake to mix thoroughly and incubate at 30-40℃ for 30-60 min. Then, use a fluorescence spectrometer to test its fluorescence emission spectrum under excitation at 270 nm. Plot the working curve based on the relationship between the ratio of fluorescence intensity at 620 nm and 474 nm and alkaline phosphatase activity.

[0014] 2) Replace the ALP solution with the test sample and add it using the standard addition method. Shake to mix thoroughly and incubate at 30-40℃ for 30-60 min. Then, use a fluorescence spectrometer to test its fluorescence emission spectrum under excitation at 270 nm. Substitute the ratio of fluorescence intensity at 620 nm and 474 nm into the working curve to obtain the alkaline phosphatase content in the test sample.

[0015] Preferably, the Eu-BDC-NO2 / DPA material exhibits a segmented linear range of 0.05-10 U / L and 0.2-0.9 U / mL for the detection of alkaline phosphatase, with a correlation coefficient R0. 2 The values ​​are 0.9981 and 0.9943, respectively.

[0016] Preferably, the detection limits of the Eu-BDC-NO2 / DPA material for detecting alkaline phosphatase in the segmental linear range of 0.05-10 U / L and 0.2-0.9 U / mL are 0.017 U / L and 0.067 U / mL, respectively.

[0017] The principle of alkaline phosphatase detection using the Eu-BDC-NO2 / DPA material described in this invention is as follows: the internal filtration effect of NADH and Eu-BDC-NO2 / DPA leads to a decrease in fluorescence at 620nm under 270nm excitation, while the presence of NADH causes a new peak at 474nm; under alkaline conditions, alkaline phosphatase can react with NADH, NADH is destroyed, the internal filtration effect is relieved, the fluorescence peak at 620nm is enhanced, and the fluorescence peak at 474nm is weakened, forming a ratiometric fluorescence sensing platform.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. The preparation process of the Eu-BDC-NO2 / DPA material of this invention is simple and does not use highly toxic reagents;

[0020] 2. The highly uniform nanoscale cavities and large specific surface area of ​​MOFs play an important role in the specific recognition of targets and also promote the enrichment of targets in the pores. Two-dimensional MOFs have a larger specific surface area and can provide more active sites in reagent reactions. When the synthesized Eu-BDC-NO2 / DPA material is dispersed in an aqueous solution, the highly polar solvent will exfoliate the MOF, and the two-dimensional sheet-like MOF is exfoliated to obtain one-dimensional nanofibers.

[0021] 3. One-dimensional nanofibers generated from the exfoliation of Eu-BDC-NO2 / DPA material are used for the detection of alkaline phosphatase. They exhibit high selectivity, no interference from other ions, and higher accuracy. Within the range of 0.05-10 U / L, they demonstrate good linearity in the detection of alkaline phosphatase, with a linearity result of Y = 0.962X + 12.931 and a correlation coefficient R. 2 =0.9981, with a lower limit of detection of 0.017 U / L; alkaline phosphatase activity was within the range of 0.2-0.9 U / mL, showing good linearity between the detection of alkaline phosphatase activity and the fluorescence intensity ratio. The linearity result was Y = 4.178X - 0.024, with a correlation coefficient R... 2 =0.9943, and the lower limit of detection is 0.067 U / mL. Attached Figure Description

[0022] Figure 1 These are XRD diffraction patterns of Eu-BDC-NO2 / DPA materials with four different ligand ratios in Examples 1-4 of this invention, and the fitted curves of Eu-BDC-NO2 / DPA.

[0023] Figure 2 These are scanning electron microscope images of Eu-BDC-NO2 / DPA materials with four different ligand ratios in Examples 1-4 of this invention, namely, BDC-NO2 / DPA ligand ratios of (A) 1:1 (B) 1:1.5 (C) 1:2 (D) 1:3;

[0024] Figure 3 This is the AFM image of the Eu-BDC-NO2 / DPA material with a BDC-NO2 / DPA ligand ratio of 1:2 in Example 1 of this invention;

[0025] Figure 4 These are TEM images (×15k magnification) of four Eu-BDC-NO2 / DPA materials with different ligand ratios in Examples 1-4 of this invention in methanol, namely, BDC-NO2 / DPA ligand ratios of (A) 1:1 (B) 1:1.5 (C) 1:2 (D) 1:3;

[0026] Figure 5These are TEM images (×15kx) of Eu-BDC-NO2 / DPA materials with four different ligand ratios in Examples 1-4 of this invention in water, which are BDC-NO2 / DPA ligand ratios of (A) 1:1 (B) 1:1.5 (C) 1:2 (D) 1:3;

[0027] Figure 6 These are TEM images (×15k magnification) of Eu-BDC-NO2 / DPA material with a BDC-NO2 / DPA ligand ratio of 1:2 in different solvents, namely (A) methanol, (B) ethylene glycol, (C) dimethyl sulfoxide, and (D) water.

[0028] Figure 7 These are the excitation spectrum and NADH ultraviolet absorption spectrum of the Eu-BDC-NO2 / DPA material in Example 1 of this invention.

[0029] Figure 8 The fluorescence intensity ratio of the Eu-BDC-NO2 / DPA material of the present invention as a function of pH (A), temperature (B), time (C), and Eu-BDC-NO2 / DPA concentration (D);

[0030] Figure 9 The fluorescence spectrum changes of the Eu-BDC-NO2 / DPA and NADH system caused by different concentrations of alkaline phosphatase in Example 5 of the present invention (A) and the linear relationship in the range of 0.05-10 U / L (B);

[0031] Figure 10 The graphs shown in Example 6 of this invention are: fluorescence spectrum changes of Eu-BDC-NO2 / DPA and NADH system caused by different concentrations of alkaline phosphatase (A) and linear relationship (B) in the range of 0.2-0.9 U / ml.

[0032] Figure 11 This is a selective comparison diagram of Embodiment 5 of the present invention. Detailed Implementation

[0033] Example 1: The molar ratio of BDC-NO2 to DPA is 1:2.

[0034] The synthesis of the Eu-BDC-NO2 / DPA material is specifically as follows:

[0035] Eu(NO3)3·6H2O (0.23 g) was dissolved in 10 mL of water, 2-nitroterephthalic acid (0.06 g) and 2,6-pyridinedicarboxylic acid (0.10 g) were dissolved in 30 mL of ethanol, and 278 μl of triethylamine was added to the ethanol. The mixture was sonicated to obtain a clear solution. The two solutions were then mixed and heated in an oil bath at 40 °C for 2 h. After cooling to room temperature, the white solid was collected by centrifugation and washed with ethanol and water. The solid was dried at 40 °C to obtain the Eu-BDC-NO2 / DPA material.

[0036] Example 2: The molar ratio of BDC-NO2 to DPA is 1:1.

[0037] The synthesis of the Eu-BDC-NO2 / DPA material is specifically as follows:

[0038] Eu(NO3)3·6H2O (0.23 g) was dissolved in 10 mL of water, 2-nitroterephthalic acid (0.08 g) and 2,6-pyridinedicarboxylic acid (0.10 g) were dissolved in 30 mL of ethanol, and 278 μl of triethylamine was added to the ethanol. The mixture was sonicated to obtain a clear solution. The two solutions were then mixed and heated in an oil bath at 40 °C for 2 h. After cooling to room temperature, the white solid was collected by centrifugation and washed with ethanol and water. The solid was dried at 40 °C to obtain the Eu-BDC-NO2 / DPA material.

[0039] In Example 3, the molar ratio of BDC-NO2 to DPA was 1:1.5.

[0040] The synthesis of the Eu-BDC-NO2 / DPA material is specifically as follows:

[0041] Eu(NO3)3·6H2O (0.23 g) was dissolved in 10 mL of water, 2-nitroterephthalic acid (0.08 g) and 2,6-pyridinedicarboxylic acid (0.10 g) were dissolved in 30 mL of ethanol, and 278 μl of triethylamine was added to the ethanol. The mixture was sonicated to obtain a clear solution. The two solutions were then mixed and heated in an oil bath at 40 °C for 2 h. After cooling to room temperature, the white solid was collected by centrifugation and washed with ethanol and water. The solid was dried at 40 °C to obtain the Eu-BDC-NO2 / DPA material.

[0042] Example 4: The molar ratio of BDC-NO2 to DPA is 1:3.

[0043] The synthesis of the Eu-BDC-NO2 / DPA material is specifically as follows:

[0044] Eu(NO3)3·6H2O (0.23 g) was dissolved in 10 mL of water, 2-nitroterephthalic acid (0.05 g) and 2,6-pyridinedicarboxylic acid (0.12 g) were dissolved in 30 mL of ethanol, and 278 μl of triethylamine was added to the ethanol. The mixture was sonicated to obtain a clear solution. The two solutions were then mixed and heated in an oil bath at 40 °C for 2 h. After cooling to room temperature, the white solid was collected by centrifugation and washed with ethanol and water. The solid was dried at 40 °C to obtain the Eu-BDC-NO2 / DPA material.

[0045] XRD diffraction measurements were performed on the Eu-BDC-NO2 / DPA materials prepared in Examples 1-4, as follows: Figure 1 As shown in the figure, it can be seen that the position of the diffraction peak in the XRD pattern does not change with the change of the ligand ratio, and it is consistent with the position of the diffraction peak in the fitted XRD curve, which proves that we have successfully synthesized the Eu-BDC-NO2 / DPA material.

[0046] The morphology of the Eu-BDC-NO2 / DPA materials prepared in Examples 1-4 was characterized. Specifically, the Eu-BDC-NO2 / DPA materials with different ligand ratios prepared in Examples 1-4 were ground into powder, and their morphology was characterized by scanning electron microscopy. Figure 2 As shown, with the increase of the proportion of DPA in the ligand, the solid morphology changes from granular to plate-like. When the BDC-NO2 / DPA ligand ratio is 1:2, the two-dimensional plate-like morphology is relatively uniform, with an average diameter of about 200 nm.

[0047] The AFM image of the Eu-BDC-NO2 / DPA material with a BDC-NO2 / DPA ligand ratio of 1:2 in Example 1 is shown below. Figure 3 As shown, at this ratio, the thickness of the nanosheet is about 3 nm.

[0048] To investigate the effect of solvent polarity on the morphology of Eu-BDC-NO2 / DPA materials, the solvent was changed. Solvent molecules with high polarity exfoliated the material, thus obtaining one-dimensional nanofibers.

[0049] 1) The four different ligand ratios of Eu-BDC-NO2 / DPA materials from Examples 1-4 were dispersed in methanol, and their TEM images (×15kx) were measured respectively. Figure 4 As shown, with the increase of the proportion of DPA in the ligand, the morphology of the material in methanol changes from granular to flake-like. When methanol, which has lower polarity, is used as the solvent, no exfoliation occurs, and the morphology of Eu-BDC-NO2 / DPA remains unchanged.

[0050] 2) The four different ligand ratios of Eu-BDC-NO2 / DPA materials from Examples 1-4 were dispersed in water, and their TEM images (×15kx) were measured respectively. Figure 5 As shown, when the BDC-NO2 / DPA ligand ratio is 1:1 and 1:1.5, the Eu-BDC-NO2 / DPA material exhibits consistent morphology in both aqueous and methanol solutions, matching the morphology in the SEM images. At these ratios, the highly polar solvent does not exfoliate the material, and the morphology remains unchanged. However, as the proportion of DPA in the ligand increases, when the BDC-NO2 / DPA ligand ratio is 1:2, the highly polar solvent molecules begin to exfoliate the material, changing its morphology from two-dimensional sheets to one-dimensional fibers. Further increases in the proportion of DPA in the ligand result in the same effect from the highly polar solvent molecules.

[0051] 3) The Eu-BDC-NO2 / DPA material with a ligand ratio of 1:2 prepared in Example 1 was dispersed in solvents of (A) methanol, (B) ethylene glycol, (C) dimethyl sulfoxide, and (D) water, and TEM images (×15kx) were measured. Figure 6 As shown, the polarity of the four solvents, from smallest to largest, is methanol, ethylene glycol, dimethyl sulfoxide, and water. As the solvent polarity increases, the degree of exfoliation of the material by the solvent molecules gradually increases, and the material gradually peels away from the sheet-like structure to obtain one-dimensional nanofibers.

[0052] To investigate the changes in fluorescence intensity of the Eu-BDC-NO2 / DPA material with pH, ​​time, temperature, and Eu-BDC-NO2 / DPA concentration, experiments were conducted to study the changes in fluorescence intensity with pH, ​​time, temperature, and Eu-BDC-NO2 / DPA concentration, as detailed below:

[0053] Fluorescence intensity changes with pH: 1) The Eu-BDC-NO2 / DPA material prepared in Example 1 was dispersed in an aqueous solution to prepare an aqueous solution of Eu-BDC-NO2 / DPA material with a concentration of 0.8 mg / mL.

[0054] 2) Transfer 50 μL of Eu-BDC-NO2 / DPA aqueous solution to a centrifuge tube containing 100 μL of 0.05 mM NADH, and add 100 μL of 1 U / L ALP solution. Finally, add HEPES solutions with pH values ​​of 7, 7.5, 8, 8.5, and 9 respectively until the total volume of the mixed solution is 1 mL. Shake to mix thoroughly. Incubate at 37°C for 30 min, and then use a fluorescence spectrometer to measure its fluorescence emission spectrum under excitation at 270 nm. Plot the spectrum with the buffer pH value on the x-axis and the fluorescence intensity ratio on the y-axis. Figure 8Figure A shows the fluorescence intensity of the Eu-BDC-NO2 / DPA material corresponding to the addition of alkaline phosphatase as a function of temperature. The figure shows that the fluorescence intensity ratio at 620 nm and 474 nm gradually changes with increasing pH, and the ratio gradually increases when the pH is between 7.0 and 8.5. Furthermore, the fluorescence intensity ratio gradually stabilizes between 8.5 and 9.0, with the highest response value at 8.5. Therefore, we selected 8.5 as the optimal pH for detecting alkaline phosphatase.

[0055] Fluorescence intensity changes with temperature: 1) The Eu-BDC-NO2 / DPA material prepared in Example 1 was dispersed in an aqueous solution to prepare an aqueous solution of Eu-BDC-NO2 / DPA material with a concentration of 0.8 mg / mL.

[0056] 2) Transfer 50 μL of Eu-BDC-NO2 / DPA aqueous solution to a centrifuge tube containing 100 μL of 0.05 mM NADH, add 100 μL of 1 U / L ALP solution, and finally add HEPES solution (pH=8) to a final volume of 1 mL. Shake to mix thoroughly. Incubate at 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃ for 30 min each. Measure the fluorescence emission spectrum using a fluorescence spectrometer at 270 nm excitation. Plot the spectrum with temperature on the x-axis and fluorescence intensity ratio on the y-axis. Figure 8 Figure B shows the fluorescence intensity of the Eu-BDC-NO2 / DPA material corresponding to the addition of alkaline phosphatase as a function of temperature. As the reaction temperature increases, the ratio of fluorescence intensity at 620 nm to 474 nm gradually increases, reaching its optimal value at approximately 40 °C. Considering the physiological environment, a reaction temperature of 37 °C was chosen as the optimal condition for detecting alkaline phosphatase.

[0057] Changes in fluorescence intensity over time: 1) The Eu-BDC-NO2 / DPA material prepared in Example 1 was dispersed in an aqueous solution to prepare an aqueous solution of Eu-BDC-NO2 / DPA material with a concentration of 0.8 mg / mL.

[0058] 2) Transfer 50 μL of Eu-BDC-NO2 / DPA aqueous solution to a centrifuge tube containing 100 μL of 0.05 mM NADH, add 100 μL of 1 U / L ALP solution, and finally add HEPES solution (pH=8) to a final volume of 1 mL. Shake to mix thoroughly. Incubate at 37°C for 0 min, 10 min, 20 min, 30 min, 40 min, 60 min, and 70 min, respectively. Measure the fluorescence emission spectrum using a fluorescence spectrometer at 270 nm excitation. Plot a graph with time on the x-axis and fluorescence intensity on the y-axis. Figure 8 Figure C shows the fluorescence intensity of the Eu-BDC-NO2 / DPA material corresponding to the addition of alkaline phosphatase over time. The fluorescence intensity gradually increases with increasing reaction time, and then stabilizes after 30 minutes. Therefore, a reaction temperature of 30 minutes was selected as the optimal condition for detecting alkaline phosphatase.

[0059] Fluorescence intensity variation with Eu-BDC-NO2 / DPA concentration: 1) The Eu-BDC-NO2 / DPA material prepared in Example 1 was dispersed in an aqueous solution to prepare aqueous solutions of Eu-BDC-NO2 / DPA material with concentrations 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, 0.9 mg / mL, and 1.0 mg / mL.

[0060] 2) Transfer 50 μL 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, 0.9 mg / mL, and 1.0 mg / mL Eu-BDC-NO2 / DPA aqueous solutions to centrifuge tubes containing 100 μL of 0.05 mM NADH, add 100 μL of 1 U / L ALP solution, and finally add HEPES solution at pH=8 to a final volume of 1 mL. Shake to mix thoroughly. Incubate at 37°C for 30 min, and then measure the fluorescence emission spectra using a fluorescence spectrometer at 270 nm excitation. Plot the Eu-BDC-NO2 / DPA concentration on the x-axis and the fluorescence intensity ratio on the y-axis. Figure 8Figure D shows the fluorescence intensity of the Eu-BDC-NO2 / DPA material corresponding to the addition of alkaline phosphatase as a function of Eu-BDC-NO2 / DPA concentration. When the Eu-BDC-NO2 / DPA concentration is between 0 and 0.8 mg / mL, the fluorescence intensity ratio at 620 nm and 474 nm gradually increases. Furthermore, the fluorescence ratio gradually stabilizes between 0.8 and 1.0 mg / mL, with the highest response value observed at 0.8 mg / mL. Therefore, a Eu-BDC-NO2 / DPA concentration of 0.8 mg / mL is selected as the optimal concentration for detecting alkaline phosphatase.

[0061] Example 5

[0062] The Eu-BDC-NO2 / DPA material prepared in Example 1 was applied to the sensitive detection of alkaline phosphatase, and a standard curve was plotted. The specific steps are as follows:

[0063] 1) Disperse the Eu-BDC-NO2 / DPA material prepared in Example 1 into an aqueous solution to prepare an aqueous solution of Eu-BDC-NO2 / DPA material with a concentration of 0.8 mg / mL.

[0064] 2) Transfer 50 μL of Eu-BDC-NO2 / DPA aqueous solution to a centrifuge tube containing 100 μL of 0.05 mM NADH, and add 100 μL of ALP solutions at concentrations of 0.1 U / L, 0.5 U / L, 1 U / L, 5 U / L, 10 U / L, 30 U / L, 50 U / L, 70 U / L, and 100 U / L, respectively. Finally, add HEPES solution at pH 8.5 to each tube until the total volume of the mixed solution is 1 mL. Shake to mix thoroughly. Incubate at 37°C for 30 min, and then measure the fluorescence emission spectrum using a fluorescence spectrometer at 270 nm excitation. Figure 9 As shown in Figure A, a curve of ALP concentration versus fluorescence intensity was plotted based on the changes in its spectrum, as detailed below. Figure 9 As shown in B.

[0065] Figure 7 The excitation spectrum of Eu-BDC-NO2 / DPA and the ultraviolet absorption spectrum of NADH are shown in Example 1 of this invention. When 270 nm is selected as the optimal excitation wavelength for the experiment, NADH also has strong ultraviolet absorption at 270 nm. The overlap between the excitation spectrum of Eu-BDC-NO2 / DPA and the ultraviolet absorption spectrum of NADH leads to the generation of a fluorescence internal filtering effect.

[0066] Figure 9A is a graph showing the fluorescence intensity changes of the Eu-BDC-NO2 / DPA system under optimal reaction conditions in Example 5 of this invention. From bottom to top, the graphs show the fluorescence curves corresponding to alkaline phosphatase activities ranging from 0 to 100 U / L. As the alkaline phosphatase activity increases, the fluorescence intensity of the Eu-BDC-NO2 / DPA system at 620 nm also increases.

[0067] Figure 9 B represents alkaline phosphatase activity in the range of 0.05-10 U / L. Example 5 demonstrates that the detection of alkaline phosphatase exhibits good linearity. The linearity result is Y = 0.962X + 12.931, with a correlation coefficient R. 2 =0.9981, with a minimum detection limit of 0.017 U / L.

[0068] Example 6

[0069] Using the Eu-BDC-NO2 / DPA material prepared in Example 1, and by changing the concentration of added NADH, sensitive detection of alkaline phosphatase was achieved, as detailed below:

[0070] 1) Disperse Eu-BDC-NO2 / DPA material in water to obtain a 0.8 mg / mL Eu-BDC-NO2 / DPA aqueous solution; transfer 50 μL of the Eu-BDC-NO2 / DPA aqueous solution to a centrifuge tube containing 100 μL of 2 mM NADH, and add 100 μL of 0.01 U / mL, 0.05 U / mL, 0.1 U / mL, 0.2 U / mL, 0.4 U / mL, 0.6 U / mL, 0.8 U / mL, 1 U / mL, and 5 U / mL solutions respectively. Finally, add HEPES solution at pH 8.5 to a total volume of 1 mL, shake to mix thoroughly, and incubate at 37°C for 30 min. Then, measure the fluorescence emission spectrum using a fluorescence spectrometer under excitation at 270 nm. Figure 10 As shown in Figure A, a curve of ALP concentration versus fluorescence intensity was plotted based on the changes in its spectrum, as detailed below. Figure 10 As shown in B.

[0071] Figure 10 A is a graph showing the fluorescence intensity changes of the Eu-BDC-NO2 / DPA system under optimal reaction conditions in Example 6 of this invention. From bottom to top, the graphs represent the fluorescence curves corresponding to 0 to 5 U / mL alkaline phosphatase. As the alkaline phosphatase activity increases, the ratio of the fluorescence intensity of the Eu-BDC-NO2 / DPA system at 620 nm and 474 nm increases accordingly.

[0072] Figure 10B represents alkaline phosphatase activity in the range of 0.2-0.9 U / mL. Example 6 of this invention demonstrates a good linear range between the detection of alkaline phosphatase activity and the fluorescence intensity ratio. The linearity result is Y = 4.178X - 0.024, and the correlation coefficient R0 is [missing value]. 2 =0.9943, and the lower limit of detection is 0.067 U / mL.

[0073] To demonstrate the selectivity of the Eu-BDC-NO2 / DPA material of this invention for alkaline phosphatase, a control was set up. The fluorescence intensity change of the material was measured in the presence of interfering substances. The specific procedure was as follows:

[0074] Eu-BDC-NO2 / DPA material was dispersed in water to obtain a 0.8 mg / mL Eu-BDC-NO2 / DPA aqueous solution. Fourteen 50 μL portions of the Eu-BDC-NO2 / DPA aqueous solution were transferred to centrifuge tubes containing 100 μL of 2 mM NADH. One of these tubes contained 100 μL of 1 U / mL ALP solution. Six other tubes contained 100 μL of 10 mM NaCl, KCl, CaCl2, MgCl2, FeCl3, and FeCl2 solutions, respectively. Finally, 100 μL of each of the remaining six tubes was added to the remaining six tubes. A solution of 0.25 mg / mL glutamic acid, glycine, arginine, glutathione, bovine serum albumin, and glucose oxidase was prepared, with the last sample serving as a blank control. HEPES solution (pH 8.5) was added to each sample until the total volume of the mixture was 1 mL. The mixture was shaken to ensure thorough mixing and incubated at 37°C for 30 min. The fluorescence emission spectra were then measured using a fluorescence spectrometer at 270 nm excitation. The ratio of fluorescence intensity was plotted on the x-axis as the concentration of different interfering substances. 620 / I 474 Plot a graph on the ordinate, such as Figure 11 As shown, the results indicate that the fluorescence intensity of the material only changes in the presence of the analyte, and other interfering substances have little effect on the change in fluorescence intensity.

[0075] Example 7

[0076] The activity curve of alkaline phosphatase in the range of 0.2-0.9 U / mL was used to apply the standard addition method for the detection of alkaline phosphatase in human serum, as detailed below:

[0077] Three human serum samples were taken, and alkaline phosphatase with a concentration of 0 was added to each sample for recovery experiments.

[0078] Specifically as follows:

[0079] The Eu-BDC-NO2 / DPA material prepared in Example 1 was applied to the sensitive detection of alkaline phosphatase. The specific steps are as follows:

[0080] 1) Disperse the Eu-BDC-NO2 / DPA material prepared in Example 1 into an aqueous solution to prepare an aqueous solution of Eu-BDC-NO2 / DPA material with a concentration of 0.8 mg / mL.

[0081] 2) Transfer 50 μL of Eu-BDC-NO2 / DPA aqueous solution to a centrifuge tube containing 100 μL of 2 mM NADH and 100 μL of human serum, add 100 μL of ALP solution with a concentration of 0 U / mL, and finally add HEPES solution with pH=8.5 to a total volume of 1 mL. Shake to mix thoroughly and incubate at 37 °C for 30 min. Then, use a fluorescence spectrometer to test the fluorescence emission spectrum at 270 nm excitation. Substitute the ratio of fluorescence intensity at 620 nm and 474 nm into the working curve to obtain the alkaline phosphatase content in the sample and calculate the recovery rate, as shown in Table 1.

[0082] Example 8

[0083] Unlike Example 6, in Example 7, the concentration of alkaline phosphatase added to human serum was 0.2 U / mL.

[0084] Example 9

[0085] Unlike Example 6, in Example 8, the concentration of alkaline phosphatase added to human serum was 0.3 U / mL.

[0086] Example 10

[0087] Unlike Example 6, in Example 9, the concentration of alkaline phosphatase added to human serum was 0.4 U / mL.

[0088] Table 1

[0089]

[0090]

[0091] As can be seen from Table 1, the recovery rates in Examples 7-10 were in the range of 97.3%-103%, and the relative standard deviation (n=3) was less than 5%, indicating that the Eu-BDC-NO2 / DPA material has high accuracy and precision in detecting alkaline phosphatase in human serum samples.

[0092] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of low-dimensional Eu-BDC-NO2 / DPA nanomaterials in the determination of alkaline phosphatase activity, characterized in that, Specifically, it includes: 1) Plotting the working curve: Disperse the low-dimensional Eu-BDC-NO2 / DPA nanomaterials in water to obtain an Eu-BDC-NO2 / DPA aqueous solution; transfer the Eu-BDC-NO2 / DPA aqueous solution to a centrifuge tube containing reduced coenzyme I NADH, add ALP solutions with different activities, and finally add HEPES solution to make up the volume. Shake to mix thoroughly and incubate at 30-40℃ for 30-60 min. Then, use a fluorescence spectrometer to test its fluorescence emission spectrum under excitation at 270 nm. Plot the working curve based on the relationship between the ratio of fluorescence intensity at 620 nm and 474 nm and alkaline phosphatase activity. 2) Replace the ALP solution with the test sample and add it using the standard addition method. Shake to mix thoroughly and incubate at 30-40℃ for 30-60 min. Then, use a fluorescence spectrometer to test its fluorescence emission spectrum under excitation at 270 nm. Substitute the ratio of fluorescence intensity at 620 nm and 474 nm into the working curve to obtain the alkaline phosphatase content in the test sample. The synthesis method of low-dimensional Eu-BDC-NO2 / DPA nanomaterials is as follows: Eu(NO3)3·6H2O was dissolved in water, 2-nitroterephthalic acid and 2,6-pyridinedicarboxylic acid were dissolved in ethanol, and triethylamine was added to the ethanol. The mixture was sonicated to obtain a clear solution. The two solutions were then mixed and heated to react. The mixture was cooled to room temperature, centrifuged to collect the white solid, washed, and dried to obtain the Eu-BDC-NO2 / DPA material. The ratio of the amount of Eu(NO3)3·6H2O to the sum of the amounts of 2-nitroterephthalic acid and 2,6-pyridinedicarboxylic acid ligands is 1:1.5-2,2-nitroterephthalic acid and 2,6-pyridinedicarboxylic acid are 1:1-3. The detection limits of the Eu-BDC-NO2 / DPA material for detecting alkaline phosphatase in the segmental linear range of 0.05-10 U / L and 0.2-0.9 U / mL are 0.017 U / L and 0.067 U / mL, respectively.

2. The application of the low-dimensional Eu-BDC-NO2 / DPA nanomaterial as described in claim 1 in the determination of alkaline phosphatase activity, characterized in that, The Eu-BDC-NO2 / DPA material showed a segmented linear range of 0.05-10 U / L and 0.2-0.9 U / mL for alkaline phosphatase detection, with a correlation coefficient R0. 2 The values ​​are 0.9981 and 0.9943, respectively.

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