CN113567409B - Method for detecting hypochlorous acid by using metal organic framework fluorescence ratio probe - Google Patents
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Abstract
The invention discloses a method for detecting hypochlorous acid by using a metal organic framework fluorescence ratio probe, which comprises the following steps: preparing a metal organic framework material into a suspension by using a phosphate buffer solution; adding sodium hypochlorite aqueous solutions with different concentrations into the suspension, and fully mixing and reacting; placing the mixed solution in a fluorescent cuvette; fixing the excitation wavelength to 370-410nm, detecting the mixed solution by using a fluorescence spectrometer, recording the emission peak intensities at the two emission wavelengths, and calculating the proportion; substituting the ratio into a linear equation to solve the concentration of the hypochlorous acid. The method has the advantages of good biological safety, low detection limit, wide linear range, strong specificity, quick and visual detection and strong universality, and provides an important technical support for hypochlorous acid detection of active oxygen.
Description
Technical Field
The invention relates to chemical analysis and detection, in particular to a method for detecting hypochlorous acid by using a metal organic framework fluorescence ratio probe.
Background
Hypochlorous acid (HClO) is produced by activated neutrophils, which release heme enzymes, myeloperoxidase, which catalyze H 2 O 2 With Cl - To produce HClO. Endogenous HClO is a powerful natural oxidant that reacts with proteins, DNA, RNA, fatty acids and cholesterol and is thought to play important physiological roles in living systems. On the one hand, HClO has microbicidal activity and is beneficial to the defense of the indigenous host against microbial invasion. On the other hand, oxidative stress caused by uncontrolled production of HClO in cells may adversely affect host physiology. It is known that HClO is closely related to the progression of many human diseases, and excessive HClO causes heart diseases, inflammatory diseases, nervous system injury, atherosclerosis, and the like.
The rapid and efficient detection method of HClO is not only useful in the field of analytical chemistry, but also useful in biological studies relating to its biological function, especially in the study of HClO production, distribution, concentration levels and metabolism. Many methods of detecting HClO have been reported over the past several decades, such as colorimetry, luminescence/fluorescence, electrochemistry and chromatography. Among them, the method using fluorescence sensing detection is one of the most effective methods, and has advantages of good selectivity, high sensitivity, and easy implementation. Compared with a probe with a single signal, the proportional fluorescent probe has higher sensitivity and selectivity because the proportional fluorescent probe can eliminate the influence of background or external factors.
Metal organic framework Materials (MOFs) have been widely studied and used in the synthesis of proportional probes in the field of fluorescence sensing due to their high specific surface area, high and permanent porosity, structure controllability, biocompatibility, and functionality. In fluorescence ratio probes based on Metal Organic Frameworks (MOFs), nanoparticles fabricated by physical trapping can leak fluorophores from the interior or inner region of the nanomaterial matrix. Problems such as this may lead to erroneous measurements based on the ratio between the emission intensities of the sensing fluorophore and the reference fluorophore. In addition, the organic fluorescent probe is limited in applications such as poor water solubility and aggregation-induced quenching, and these problems can be solved by combining organic molecules with MOFs. Therefore, it is still important to synthesize a new proportional type fluorescent probe capable of realizing accurate measurement.
Disclosure of Invention
The invention aims to: the invention aims to provide a method for detecting hypochlorous acid by using a metal organic framework fluorescence ratio probe, which has the advantages of good selectivity, high sensitivity, quick detection, good universality and the like.
The technical scheme is as follows: the invention provides a method for detecting hypochlorous acid by using a metal organic framework fluorescence ratio probe, which comprises the following steps:
(1) Preparing a metal organic framework material into a suspension by using a phosphate buffer solution;
(2) Adding sodium hypochlorite aqueous solutions with different concentrations into the suspension, and fully mixing and reacting;
(3) Placing the mixed solution in a fluorescent cuvette;
(4) Fixing the excitation wavelength to 370-410nm, detecting the mixed solution by using a fluorescence spectrometer, recording the emission peak intensities at the two emission wavelengths, and calculating the proportion;
(5) Substituting the ratio into a linear equation to solve the concentration of the hypochlorous acid.
Further, the metal organic framework material is an organic framework material taking 2-amino terephthalic acid as a ligand and zr (IV), al (III) and Fe (III) as metal centers, and is a first type probe.
Further, the metal organic framework material is UiO-66-NH 2 ，MIL-101-NH 2 (Al)，MIL-101-NH 2 (Fe)。
Further, the metal organic framework material is UiO-66-NH 2 And Eosin molecule Eosin Y, i.e. UiO-66-NH 2 @ EY, second type of probe.
Further, the two emission wavelengths are 432nm and 533nm, respectively.
Further, the linear equation is:
UiO-66-NH 2 ，I 533nm /I 432nm ＝0.00399c[ClO - ]+0.06127 at a concentration range of 0.1-150 μ M; MIL-101-NH 2 (Al)，I 533nm /I 432nm ＝0.00218c[ClO - ]+0.03584 at a concentration range of 5-150 μ M; MIL-101-NH 2 (Fe)，I 533nm /I 432nm ＝0.00226c[ClO - ]+0.03946 at a concentration range of 5-150 μ M; uiO-66-NH 2 @EY，I 533nm /I 432nm ＝0.00927c[ClO - ]+0.47346 at a concentration range of 0.1-200 μ M.
Further, the suspension and the sodium hypochlorite aqueous solution are sequentially added into a confocal culture dish to be incubated with cells, and the cells are excited by a laser confocal fluorescence microscope to perform fluorescence imaging.
Further, the cells are Hela cells and L02 cells.
Further, the suspension and an aqueous solution of sodium hypochlorite were injected into nude mice to perform in vivo fluorescence imaging.
The invention adopts the metal organic framework fluorescence ratio probe, the first class of probe does not need to additionally load other fluorophores as a second emission center, and the leakage of a loaded object and the error detection result caused by the leakage are avoided. The probe reacts with hypochlorite, secondary fluorescence emission can occur under the excitation of 370-410nm, a dual-emission platform is constructed together with the fluorescence emission of the probe, and the rapid detection of the hypochlorite can be realized by using the dual-emission fluorescence intensity ratio as a signal. The second type of probe is UiO-66-NH composed of the first type of probe 2 The probe effectively solves the problem of low selectivity of EY to hypochlorous acid by combining the nano-composite with an organic dye molecule EY.
The invention selects the metal organic framework material taking 2-aminobenzene dicarboxylic acid as ligand, because the ligand is provided with-NH 2 ，-NH 2 The sensitivity to the detection of environmental changes promotes the metal organic framework material to be used as a detection platform for hypochlorite. UiO-66-NH 2 ，MIL-101-NH 2 (Al)，MIL-101-NH 2 (Fe)，UiO-66-NH 2 The @ EY has good stability and high tolerance in an aqueous medium. The metal organic framework material is synthesized under mild conditions, and the concentration of the hypochlorous acid is accurately detected under the excitation of 370-410nm and within a certain concentration range based on the fluorescence dual-emission intensity ratio of the MOFs and the hypochlorous acid reaction. The probes of the method do not need to be loaded on MOFsOther fluorophores effectively avoid leakage of the loaded fluorophores and bring wrong detection results, and have wide application prospects. Another class of probes is a complex of MOFs and organic dye molecules EY, effectively improving the low selectivity of EY molecules to hypochlorous acid.
Has the advantages that: the method has the advantages of good selectivity, high sensitivity, quick detection, simplicity, feasibility, good universality and the like, and has potential application prospect in the detection of hypochlorous acid by using a proportional fluorescent probe based on MOFs.
Drawings
FIG. 1 shows (A) UiO-66-NH 2 Fluorescence response with 100 μ M hypochlorous acid at different excitation wavelengths; (B) UiO-66-NH 2 Reacting with hypochlorous acid in the concentration range of 0-150 μ M, and responding to fluorescence under excitation of 400 nm.
FIG. 2 shows a metal frame organic material UiO-66-NH 2 ，MIL-101-NH 2 (Al)，MIL-101-NH 2 (Fe) mechanism of reaction with hypochlorous acid;
FIG. 3 shows UiO-66-NH 2 Detecting hypochlorous acid in Hela cells for imaging, wherein A is a blank control group, B is a normal control group, and C is an experimental group;
FIG. 4 shows UiO-66-NH 2 And (3) detecting hypochlorous acid imaging in the nude mice, wherein the blank control group A, the normal control group B and the experiment group C are used.
Detailed Description
Example 1
A quick detection method of hypochlorous acid by a metal organic framework fluorescence ratio probe comprises the following specific steps:
the prepared metal organic framework material UiO-66-NH 2 A suspension of 50 μ g/mL was prepared by 0.15M phosphate buffer (pH = 7.4). 2mL of the suspension was added with an aqueous hypochlorous acid solution to a final concentration of 100. Mu.M, and mixed for reaction. Fixing the excitation wavelength at 400nm, detecting the mixed solution with a fluorescence spectrometer, recording fluorescence intensity at 432nm and 533nm, and calculating the ratio I 533nm /I 432nm . By linear equation I 533nm /I 432nm ＝0.00399c[ClO - ]+0.06127 the corresponding hypochlorous acid concentration was solved.
Preparing and taking 500 mumu.L of the suspension in g/mL is added into a confocal culture dish containing adherent Hela cells and L02 cells respectively, and the final concentration is 50 mu.g/mL. 0.002 mol.L of the product was prepared -1 Taking 50 mu L of sodium hypochlorite solution, respectively adding the sodium hypochlorite solution into the two confocal culture dishes, and incubating the sodium hypochlorite solution with cells for 0-6h. Fluorescence imaging was performed using 405nm and 488nm laser illumination. The imaging results were compared to a blank control and a normal control. Blue and green fluorescence respectively appeared in the blue channel and the green channel of the experimental group, which respectively correspond to 432nm and 533nm. The blank had no significant fluorescence. Only the blue channel of the normal control group exhibited blue fluorescence. The experimental phenomenon shows that UiO-66-NH 2 Hypochlorous acid in cells can be detected.
The blank control group did not have any treatment for Hela cells and L02 cells, and the normal control group added only the same volume of suspension as the experimental group at the same concentration.
100 mu L of 500 mu g/mL suspension is prepared and taken, and 100 mu L of 800 mu M sodium hypochlorite solution is prepared and taken, and the solution is sequentially injected to the same area of the abdomen of the nude mouse subcutaneously, and the imaging results are collected for 5min, 10min and 30 min. The imaging results were compared to a blank control and a normal control.
The nude mice of the blank control group were not treated and the normal control group was injected with the same volume of suspension as the experimental group.
The area of the red region displayed by the experimental group is the largest, the area of the red region displayed by the normal control group is small, and the red region is not displayed by the blank control group. The larger the red region, the higher the intensity of the fluorescence emission peak at 533nm. This indicates that UiO-66-NH 2 Can be used for detecting hypochlorous acid in vivo.
Example 2
A method for rapidly detecting hypochlorous acid by using a metal organic framework fluorescence ratio probe comprises the following specific steps:
the prepared metal organic framework material MIL-101-NH 2 (Al) prepared into a suspension of 50 μ g/mL by 0.15M phosphate buffer (pH = 7.4) by 0.15M phosphate buffer. 2mL of the suspension was added with aqueous hypochlorous acid,the final concentration was 100. Mu.M, and the reaction was mixed. Fixing excitation wavelength at 400nm, detecting the mixture with fluorescence spectrometer, recording fluorescence intensity at 432nm and 533nm, and calculating ratio I 533nm /I 432nm . By linear equation I 533nm /I 432nm ＝0.00218c[ClO - ]+0.03584 solves the corresponding hypochlorous acid concentration.
100 mu L of the suspension with the concentration of 500 mu g/mL is prepared and taken, and the suspension is respectively added into a confocal culture dish containing adherent Hela cells and L02 cells, and the final concentration is 50 mu g/mL. Preparation of 0.002 moL. L -1 Taking 50 mu L of sodium hypochlorite solution, respectively adding the sodium hypochlorite solution into the two confocal culture dishes, and incubating the sodium hypochlorite solution with cells for 0-6h. Fluorescence imaging was performed using 405nm and 488nm laser illumination. The imaging results were compared to a blank control and a normal control.
Blue and green fluorescence respectively appeared in the blue and green channels of the experimental group, corresponding to 432nm and 533nm, respectively. The blank had no significant fluorescence. Only the blue channel of the normal control group showed blue fluorescence. The experimental phenomenon shows that MIL-101-NH 2 (Al) hypochlorous acid in cells can be detected.
The blank control group did not have any treatment for Hela cells and L02 cells, and the normal control group added only the same volume of suspension as the experimental group at the same concentration.
100 mu L of 500 mu g/mL suspension is prepared and taken, and 100 mu L of 800 mu M sodium hypochlorite solution is prepared and taken, and the solution is sequentially injected to the same area of the abdomen of the nude mouse subcutaneously, and the imaging results are collected for 5min, 10min and 30 min. The imaging results were compared to a blank control and a normal control.
The nude mice of the blank control group were not treated and the normal control group was injected with the same volume of suspension as the experimental group.
The area of the red region displayed by the experimental group is the largest, the area of the red region displayed by the normal control group is small, and the red region is not displayed by the blank control group. The larger the red region, the higher the intensity of the fluorescence emission peak at 533nm. Thus indicating MIL-101-NH 2 (Al) can be used for detecting hypochlorous acid in vivo.
Example 3
A method for rapidly detecting hypochlorous acid by using a metal organic framework fluorescence ratio probe comprises the following specific steps:
the prepared metal organic framework material MIL-101-NH 2 (Fe), formulated into a suspension of 50 μ g/mL by 0.15M phosphate buffer (pH = 7.4). 2mL of the suspension was added with an aqueous hypochlorous acid solution to a final concentration of 100. Mu.M, and the reaction mixture was mixed. Fixing the excitation wavelength at 400nm, detecting the mixed solution with a fluorescence spectrometer, recording fluorescence intensity at 432nm and 533nm, and calculating the ratio I 533nm /I 432nm . By linear equation I 533nm /I 432nm ＝0.00226c[ClO - ]+0.03946 the corresponding hypochlorous acid concentration was solved.
100 mu L of the suspension with the concentration of 500 mu g/mL is prepared and taken, and the suspension is respectively added into a confocal culture dish containing adherent Hela cells and L02 cells, and the final concentration is 50 mu g/mL. 0.002 mol.L of the product was prepared -1 Taking 50 mu L of sodium hypochlorite solution, respectively adding the sodium hypochlorite solution into the two confocal culture dishes, and incubating the sodium hypochlorite solution with cells for 0-6h. Fluorescence imaging was performed using 405nm and 488nm laser irradiation. The imaging results were compared to a blank control and a normal control.
Blue and green fluorescence respectively appeared in the blue channel and the green channel of the experimental group, which respectively correspond to 432nm and 533nm. The blank had no significant fluorescence. Only the blue channel of the normal control group exhibited blue fluorescence. The experimental phenomenon shows that MIL-101-NH 2 (Fe) hypochlorous acid in cells can be detected.
The blank control group did not have any treatment for Hela cells and L02 cells, and the normal control group only added the same volume of suspension as the experimental group.
100 mu L of 500 mu g/mL suspension is prepared and taken, and 100 mu L of 800 mu M sodium hypochlorite solution is prepared and taken, and the solution is sequentially injected to the same area of the abdomen of the nude mouse subcutaneously, and the imaging results are collected for 5min, 10min and 30 min. The imaging results were compared to a blank control and a normal control.
The nude mice of the blank control group were not treated and the normal control group was injected with the same volume of suspension as the experimental group.
The area of the red region displayed by the experimental group is the largest, the area of the red region displayed by the normal control group is small, and the red region is not displayed by the blank control group. The larger the red region, the higher the intensity of the fluorescence emission peak at 533nm. Thus indicating MIL-101-NH 2 (Fe) can be used for detecting hypochlorous acid in vivo.
Example 4
A method for rapidly detecting hypochlorous acid by using a metal organic framework fluorescence ratio probe comprises the following specific steps:
the prepared metal organic framework material UiO-66-NH 2 @ EY, formulated into a suspension of 50 μ g/mL by 0.15M phosphate buffer (pH = 7.4). 2mL of the suspension was added with an aqueous hypochlorous acid solution to a final concentration of 100. Mu.M, and mixed for reaction. Fixing the excitation wavelength at 400nm, detecting the mixed solution with a fluorescence spectrometer, recording fluorescence intensity at 432nm and 533nm, and calculating the ratio I 533nm /I 432nm . By linear equation I 533nm /I 432nm ＝0.00927c[ClO - ]+0.47346, solving the corresponding hypochlorous acid concentration.
100 mu L of the suspension with the concentration of 500 mu g/mL is prepared and taken, and the suspension is respectively added into a confocal culture dish containing adherent Hela cells and L02 cells, and the final concentration is 50 mu g/mL. 0.002 mol.L of the product was prepared -1 Taking 50 mu L of sodium hypochlorite solution, respectively adding the sodium hypochlorite solution into the two confocal culture dishes, and incubating the sodium hypochlorite solution with cells for 0-6h. Fluorescence imaging was performed using 405nm and 488nm laser irradiation. The imaging results were compared to a blank control and a normal control.
The blank control group did not have any treatment for Hela cells and L02 cells, and the normal control group only added the same volume of suspension as the experimental group.
Blue and green fluorescence respectively appeared in the blue and green channels of the experimental group, corresponding to 432nm and 533nm, respectively. The blank had no significant fluorescence. The normal control group also exhibited fluorescence in both the blue and green channels, but the green channel fluorescence was weaker than that in the experimental group. Experiment is nowElephant indicates UiO-66-NH 2 @ EY can detect hypochlorous acid in cells.
100 mu L of 500 mu g/mL suspension is prepared and used, and 100 mu L of 800 mu M sodium hypochlorite solution is prepared and used, and the solution is sequentially injected to the same area of the abdomen of the nude mouse subcutaneously, and the imaging results are collected for 5min, 10min and 30 min. The imaging results were compared to a blank control group and a normal control group.
The nude mice of the blank control group were not treated and the normal control group was injected with the same volume of suspension as the experimental group.
The area of the red area displayed by the experimental group is the largest, the area of the red area displayed by the normal control group is small, and the red area is not displayed by the blank control group. The larger the red region, the greater the intensity of the fluorescence emission peak representing 533nm. This indicates that UiO-66-NH 2 @ EY can be used for detecting hypochlorous acid in vivo.
Claims (5)
1. A method for detecting hypochlorous acid by using a metal organic framework fluorescence ratio probe is characterized by comprising the following steps: the method comprises the following steps:
(1) Preparing a metal organic framework material into a suspension by using a phosphate buffer solution;
(2) Adding a sodium hypochlorite aqueous solution into the suspension, and fully mixing and reacting;
(3) Placing the mixed solution in a fluorescent cuvette;
(4) Fixing the excitation wavelength to 370-410nm, detecting the mixed solution by using a fluorescence spectrometer, recording the emission peak intensities at the two emission wavelengths, and calculating the proportion;
(5) Substituting the ratio into a linear equation to solve the concentration of the hypochlorous acid,
the metal organic framework material takes 2-amino terephthalic acid as a ligand and takes Zr (IV), al (III) and Fe (III) as metal centers, and specifically is UiO-66-NH 2 、MIL-101-NH 2 (Al)、 MIL-101-NH 2 (Fe)、UiO-66-NH 2 @ EY, said UiO-66-NH 2 @ EY being UiO-66-NH 2 And Eosin molecule Eosin Y,
the two emission wavelengths are 432nm and 533nm, respectively.
2. The method for detecting hypochlorous acid by using the metal-organic framework fluorescence ratio probe according to claim 1, wherein the method comprises the following steps: the linear equation is:
UiO-66-NH 2 ，I 533nm /I 432nm =0.00399c[ClO - ]+0.06127 at a concentration range of 0.1-150 μ M; MIL-101-NH 2 (Al)，I 533nm /I 432nm =0.00218c[ClO - ]+0.03584 at a concentration range of 5-150 μ M; MIL-101-NH 2 (Fe)，I 533nm /I 432nm =0.00226c[ClO - ]+0.03946 at a concentration range of 5-150 μ M; uiO-66-NH 2 @EY，I 533nm /I 432nm =0.00927c[ClO - ]+0.47346 at a concentration range of 0.1-200 μ M.
3. The method for detecting hypochlorous acid by using the metal-organic framework fluorescence ratio probe according to claim 1, wherein the method comprises the following steps: and sequentially adding the suspension and the sodium hypochlorite aqueous solution into a confocal culture dish to co-incubate with cells, and exciting by using a laser confocal fluorescence microscope to perform fluorescence imaging.
4. The method for detecting hypochlorous acid by using the metal-organic framework fluorescence ratio probe according to claim 3, wherein the method comprises the following steps: the cells are adherent Hela cells and L02 cells.
5. The method for detecting hypochlorous acid by using the metal-organic framework fluorescence ratio probe according to claim 1, wherein the method comprises the following steps: the suspension and sodium hypochlorite aqueous solution are injected into a nude mouse body to carry out in vivo fluorescence imaging.
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