CN112285074B - New application of 1,2, 4-triaminobenzene - Google Patents
New application of 1,2, 4-triaminobenzene Download PDFInfo
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- JSYBAZQQYCNZJE-UHFFFAOYSA-N benzene-1,2,4-triamine Chemical compound NC1=CC=C(N)C(N)=C1 JSYBAZQQYCNZJE-UHFFFAOYSA-N 0.000 title claims abstract description 111
- 238000001514 detection method Methods 0.000 claims abstract description 24
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 21
- 239000001963 growth medium Substances 0.000 claims abstract description 21
- 230000003834 intracellular effect Effects 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 230000008859 change Effects 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000010186 staining Methods 0.000 claims description 6
- 229910001868 water Inorganic materials 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 239000000975 dye Substances 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 4
- 229910002027 silica gel Inorganic materials 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
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- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
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- 206010058467 Lung neoplasm malignant Diseases 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 2
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- BUOYTFVLNZIELF-UHFFFAOYSA-N 2-phenyl-1h-indole-4,6-dicarboximidamide Chemical compound N1C2=CC(C(=N)N)=CC(C(N)=N)=C2C=C1C1=CC=CC=C1 BUOYTFVLNZIELF-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000004257 Potassium Channel Human genes 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
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- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01N21/6458—Fluorescence microscopy
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
- G01N21/80—Indicating pH value
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- G01N1/00—Sampling; Preparing specimens for investigation
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- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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Abstract
The application discloses new application of 1,2, 4-triaminobenzene, and specifically comprises application of 1,2, 4-triaminobenzene as a fluorescent probe in qualitative detection of pH value in cells or tissues and application of 1,2, 4-triaminobenzene as a fluorescent probe in qualitative detection of ammonia gas, wherein the qualitative detection of the intracellular pH value comprises the following steps: providing 1,2, 4-triaminobenzene, adding the 1,2, 4-triaminobenzene into a culture medium, uniformly mixing, incubating cells with the culture medium, and observing the cells under a fluorescence confocal microscope after incubating for a period of time. The preparation method of the fluorescent probe is simple and sensitive in reaction.
Description
Technical Field
The application relates to the technical field of analytical chemistry, in particular to a new application of 1,2, 4-triaminobenzene.
Background
The pH value in the cell is one of the important parameters of cell metabolism, and plays a key role in maintaining normal physiological processes of cell growth, proliferation, apoptosis, signal conduction and the like. At present, methods for detecting intracellular pH comprise a microelectrode method, an absorption spectrum method, a nuclear magnetic resonance method and the like, but have the defects of mechanical damage, high price and the like. The fluorescence detection method has the characteristics of high sensitivity, adjustable wavelength, capability of reflecting the change details of phenomena or objects along with time and space, namely high space-time resolution and the like, and is favorable for realizing in-situ observation and visual detection of pH and other information in cells and tissues.
The ammonia gas is colorless, toxic and inflammable gas with strong pungent odor, and easily damages the nervous system, internal organs and the like of a human body, so that the ammonia gas is sensitively, quickly and on-site detected in real time, and has great practical significance. The traditional method for detecting ammonia gas has the defects of a colorimetric method and an electrochemical method, more complex operation, difficult carrying and the like. The fluorescent probe sensing detection method is a novel detection method and has the advantages of intuition, rapidness, simple and convenient operation, high detection sensitivity, high accuracy and the like. At present, a paper-based fluorescent probe can rapidly detect ammonia gas, but the ammonia gas needs to be enriched in a solution before detection, so that a color reaction can be performed, and the detection of the ammonia gas in the environment has certain limitation.
Disclosure of Invention
The application aims at the problems of the prior art, and uses 1,2, 4-triaminobenzene as a simple and sensitive fluorescent probe for directly and quickly detecting the pH in cells and the ammonia in the environment.
The application provides an application of 1,2, 4-triaminobenzene as a fluorescent probe in qualitative detection of pH value in cells or tissues.
The 1,2, 4-triaminobenzene is a precursor of an effective opener for synthesizing a cell potassium ion channel, and the 1,2, 4-triaminobenzene derivative obtained through a complex derivatization process has high biological toxicity to cells, can be directly used as a fluorescent probe to detect the pH value in the cells because the 1,2, 4-triaminobenzene can also penetrate the cells, has low biological toxicity to the cells, and can effectively analyze and detect the pH value in tissues or the cells in real time, quickly and efficiently.
The cell refers to a cell cultured in vitro by a human or an animal, particularly a human cell, and can be a normal cell or a pathological cell, and optionally, the cell is an A549 lung cancer cell or a Hela human cervical cancer cell.
Compared with normal cells, the physiological and biochemical functions of pathological cells are changed, the metabolic activity is greatly changed, and the pH value in the cells is changed.
The qualitative detection of the intracellular pH value refers to the detection of the change of the intracellular pH value or the pH value difference among different cells, but not the detection of the specific value.
Optionally, the qualitative detection of intracellular pH comprises the following steps:
providing 1,2, 4-triaminobenzene, adding the 1,2, 4-triaminobenzene into a culture medium, uniformly mixing, incubating cells with the culture medium, and observing the cells under a fluorescence confocal microscope after incubating for a period of time.
Incubating cells through a culture medium, activating and passaging, wherein 1,2, 4-triaminobenzene in the culture medium penetrates through a cell membrane and enters cytoplasm in the cell incubation process; the change of the pH value of the environment outside the cell can cause the change of the pH value of cytoplasm, the 1,2, 4-triaminobenzene in intercellular substance can change color under the condition of different pH values, and the fluorescence intensity of the 1,2, 4-triaminobenzene can be detected through a fluorescence confocal microscope to know the change of the pH value in the cell. The incubation carrier is generally a culture dish, and before the incubation with the probe solution, the incubation needs to be carried out by using a common culture medium to allow cells to adhere to the wall, and then the culture medium containing the fluorescent probe is used for replacing the incubation.
The incubation time is generally based on sufficient access of the 1,2, 4-triaminobenzene to the interior of the cells, and is generally from 1 to 5 hours.
Optionally, the cells are fixed after incubation; then, staining the cells by using a cell nucleus dye; finally, the cells were observed under a fluorescent microscope.
The 1,2, 4-triaminobenzene can penetrate through a cell membrane to enter cytoplasm and even enter cell nucleus, and the cell nucleus is dyed by using a cell nucleus dye, so that the fluorescent probe accurately enters the cell, the visual effect of the cell under a confocal fluorescent microscope is better, and the sensitive capture of the slight change of the pH value in the cell is facilitated.
The tissue refers to isolated tissue of human or animal, which is a cell group with similar shape, structure and function, the cells are closely arranged, intercellular substance exists among the cells, the intercellular substance is the liquid environment for the cell life, and the intercellular substance can provide substances required by the metabolism for the cells and receive the metabolites of the cells or unused substances. Differences in the pH of the intercellular matrix are caused by the different metabolic activities of different cell populations.
The qualitative detection of the tissue pH value refers to the change of the tissue intercellular substance pH value or the difference of the different tissue intercellular substance pH values, and does not refer to the specific value of the tissue intercellular substance pH value.
Optionally, the process of detecting the pH value in the tissue includes the following steps:
preparing 1,2, 4-triaminobenzene into an aqueous solution, immersing a tissue slice in the aqueous solution for staining for a period of time, taking out and cleaning the tissue slice, and then observing the tissue slice under a fluorescence microscope.
The pH value of the intercellular substance can reflect the metabolic condition and change of cells, 1,2, 4-triaminobenzene enters the intercellular substance, the fluorescence intensity change of the tissue slice is detected by a fluorescence microscope, and the change of the pH value of the intercellular substance is sensitively reflected. In addition, the 1,2, 4-triaminobenzene has good sensitivity to different tissue sections as a fluorescence probe, and has wide application range. Optionally, the tissue is derived from lung, heart, kidney, liver or spleen.
Optionally, the 1,2, 4-triaminobenzene is purified by a silica gel column.
The cell culture conditions are severe, impurities contained in a commercially available 1,2, 4-triaminobenzene sample can influence the metabolism in cells, the purity of the 1,2, 4-triaminobenzene can be improved through silica gel column purification, the influence of the impurities on the cell culture is reduced, and the detection accuracy is improved.
Optionally, the concentration of the 1,2, 4-triaminobenzene in the culture medium is 50 μ g/mL to 250 μ g/mL.
Optionally, the concentration of the 1,2, 4-triaminobenzene in the culture medium is 100 μ g/mL to 200 μ g/mL. The concentration range can ensure the effect of cell staining, and the imaging effect of the probe can be influenced when the concentration of the 1,2, 4-triaminobenzene is lower than 50 mu g/mL or higher than 250 mu g/mL.
The application also provides application of the 1,2, 4-triaminobenzene as a fluorescent probe in qualitative detection of ammonia gas.
The environment is air and is mainly used for qualitatively detecting the ammonia concentration in the air and observing the change of the ammonia concentration and the difference of the ammonia concentration in different environments.
Optionally, the application includes the following steps:
dissolving 1,2, 4-triaminobenzene in water to prepare a solution, immersing the carrier in the solution, taking out the carrier, and then drying the carrier in the air to prepare a sensor;
and placing the sensor in an environment to be detected, and observing the color change of the sensor under visible light or ultraviolet light.
Optionally, the fluorescence intensity is highest under 365nm ultraviolet light, and the color change of the sensor can be better observed.
Optionally, the carrier is filter paper, the filter paper is paper with good filtering performance, the paper is loose, and the filter paper has strong liquid absorption performance.
The application also provides a sensor for detecting ammonia gas, which comprises a carrier and the 1,2, 4-triaminobenzene loaded on the carrier. The pH value is increased along with the increase of the ammonia concentration, the color of the sensor is changed from light purple red to light yellow and then to brown yellow by observing under visible light through naked eyes, the color is changed from red to brown and then to orange by placing under 356nm ultraviolet rays, and the orange is gradually deepened when the pH value is continuously increased.
The application discloses a new application of 1,2, 4-triaminobenzene, which can be used as a simple and sensitive fluorescent probe to be applied to the detection of pH value in cells and the qualitative detection of ammonia gas.
Drawings
FIG. 1 is a graph showing the response of purified 1,2, 4-triaminobenzene to different pH values in visible light (above) and under 365nm UV light (below);
FIG. 2 is a confocal image of pH measurements taken with 1,2, 4-triaminobenzene as a fluorescent probe in cells of different organs, wherein (a) is an overlay of (b) and (c), (b) is a confocal microscope of A549 cells incubated with the fluorescent probe at different pH values, and (c) is a nuclear DAPI staining of A549 lung cancer cells;
FIG. 3 is a confocal microscope of 1,2, 4-triaminobenzene as a fluorescent probe for detecting mouse tissue sections (a) is a fluorescence image and (b) is a bright field image;
FIG. 4 shows the test paper under visible light (top) and under 365nm UV lamp (bottom) for different NH concentrations3·H2O response graph.
Detailed Description
The technical solutions described in the present application will be further described with reference to the following embodiments, but the present application is not limited thereto.
Example 1
After the column was packed with silica gel, commercially available 1,2, 4-triaminobenzene powder was purified by a solvent (dichloromethane to methanol volume ratio of 4: 1). And drying the material after column chromatography purification by using a rotary evaporator, and dissolving the material in absolute ethyl alcohol for storage. Before use, the liquid is transferred to a test tube by a liquid transfer gun, and the drying can be quickly finished by a vacuum drying oven.
Preparing citric acid-sodium citrate buffer solutions with pH values of 3.0,4.0,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2,6.4,6.6,6.8,7.0,7.2,8.0,9.0 and 10.0 respectively; dissolving the purified 1,2, 4-triaminobenzene in water to prepare a 1,2, 4-triaminobenzene solution with the concentration of 100 mu g/mL; mixing 180 μ L of 1,2, 4-triaminobenzene solution with 20 μ L of citric acid-sodium citrate buffer solution with different pH values, and observing color change of the solution under visible light and 365nm ultraviolet lamp.
As shown in fig. 1, the color of the aqueous solution changed from light purple to light yellow and then to brown-yellow under visible light with increasing pH; under 365nm ultraviolet rays, the color changes from red to brown and then to orange, and the orange is gradually deepened when the pH value is continuously increased.
Example 2
Dissolving 1,2, 4-triaminobenzene in water to obtain aqueous solution, adding the aqueous solution when preparing culture medium to make concentration of 1,2, 4-triaminobenzene in the culture medium 100 μ g/mL, and adjusting pH of the culture medium to 4.8, 5.5, 6.3, 7.3 and 8.5 respectively.
The concentration is 8x104A549/mL lung cancer cells were plated on a 20mm diameter glass-bottom plate at 37 deg.C with 5% CO2Incubate under conditions for 24h to allow for adherence. Pouring the original culture medium before confocal imaging, adding the culture medium with different pH values, placing the culture medium in an incubator for incubation for two hours, washing the culture medium with Phosphate Buffered Saline (PBS) for three times, then adding 1mL of 4% paraformaldehyde, fixing the cells, pouring the culture medium after half an hour, washing the cells with PBS for 3 times, adding 1mL of 1 mu g/mL DAPI (4, 6-diamidino-2-phenylindole) as a nuclear dye, reacting for 15min, dyeing the nuclei, washing the nuclei for 3 times with PBS, pouring the nuclei, and imaging the nuclei under a confocal microscope.
As shown in fig. 2, wherein (a) is an overlay of (b) and (c), (b) is a confocal microscope of a549 cells incubated with 1,2, 4-triaminobenzene as a fluorescent probe at different pH values, and (c) is a nuclear DAPI staining pattern of a549 cells. When the pH was increased from 4.8 to 5.5, the orange intensity of the fluorescent probe increased, as shown in panel (b); the graph (c) is a graph of the staining effect of the cell nucleus of the A549 lung cancer cell under different pH conditions, and the fluorescence intensity is kept unchanged; combining the two channels (b) and (c), the result is shown in (a), the purple intensity displayed by the fluorescent probe is clearer than that of the graph (c), and the 1,2, 4-triaminobenzene as the fluorescent probe can sensitively respond to the slight change of the intracellular pH value.
Example 3
After being sliced, the diabetic mouse tissue is placed in a petri dish filled with 15mL of 0.1mg/mL 1,2, 4-triaminobenzene solution (the solution is over the tissue slice), the microtome is placed on a shaker for shaking for 4h, then the microtome is taken out, and the microtome is repeatedly rinsed with ultrapure water for five times to remove the solution remained on the surface of the section, and then the section is imaged under a confocal microscope. As shown in fig. 3(a), each tissue section exhibited orange fluorescence of different intensity, and each exhibited characteristics of each organ, in which the fluorescence intensity of lung, heart, kidney, liver, and spleen was sequentially increased. The result shows that 1,2, 4-triaminobenzene has good dyeing effect as a probe, and the tissue sections of different organs have different fluorescence intensities due to different pH values.
Example 4
The preparation process comprises the following steps: the filter paper was placed in a petri dish and 15mL of 1,2, 4-triaminobenzene solution was added to immerse the filter paper. After shaking for 15 minutes, the filter paper was transferred to another clean petri dish and allowed to air dry at room temperature. Prior to use, the test strips were cut into small discs with a punch.
And (3) detection process: 150 μ L of NH at different concentrations3·H2O and 50. mu.L of 20M NaOH solution were added to the small round piece, and the seal was stuck with a test strip and reacted for 35 minutes
FIG. 4 shows the results with NH3·H2The color of the test paper changed from light purple to light yellow and then to brown yellow with the naked eye as the O concentration increased. Under 365nm ultraviolet rays, the color changes from red to brown and then to orange, and the orange is gradually deepened when the pH value is continuously increased.
The above description of the embodiments is provided to facilitate understanding and use of the invention by those skilled in the art, and appropriate changes and modifications may be made by those skilled in the art to which the present invention pertains. Therefore, the present application is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present application.
Claims (10)
1.1,2, 4-triaminobenzene is used as a fluorescent probe in qualitative detection of pH value in cells or tissues.
2. The use according to claim 1, wherein said qualitative detection of the intracellular pH value comprises the following steps:
providing 1,2, 4-triaminobenzene, adding the 1,2, 4-triaminobenzene into a culture medium, uniformly mixing, incubating cells with the culture medium, and observing the cells under a fluorescence confocal microscope after incubating for a period of time.
3. Use according to claim 2, wherein the cells are incubated and subsequently fixed, followed by staining of the cells with a nuclear dye and finally by observation of the cells under a fluorescent microscope.
4. The use according to claim 2, wherein the concentration of 1,2, 4-triaminobenzene in the culture medium is from 50 μ g/mL to 250 μ g/mL.
5. The use according to claim 1, wherein said qualitative detection of the pH value in the tissue comprises the following steps:
preparing 1,2, 4-triaminobenzene into an aqueous solution, immersing a tissue slice in the aqueous solution for staining for a period of time, taking out and cleaning the tissue slice, and then observing the tissue slice under a fluorescence confocal microscope.
6. Use according to claim 1, characterized in that the 1,2, 4-triaminobenzene is purified by means of a silica gel column.
Application of 1,2, 4-triaminobenzene as a fluorescent probe in qualitative detection of ammonia gas.
8. Use according to claim 7, characterized in that it comprises the following steps:
dissolving 1,2, 4-triaminobenzene in water to prepare a solution, immersing the carrier in the solution, taking out the carrier, and then drying the carrier in the air to prepare a sensor;
and placing the sensor in an environment to be detected, and observing the color change of the sensor under visible light or ultraviolet light.
9. Use according to claim 8, wherein the support is filter paper.
10. A sensor for detecting ammonia gas, which is characterized by comprising a carrier and 1,2, 4-triaminobenzene loaded on the carrier.
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