CN114573505A - Copper ion fluorescent probe containing naphthalene methoxyhydrazine pyridine structure and preparation method and application thereof - Google Patents
Copper ion fluorescent probe containing naphthalene methoxyhydrazine pyridine structure and preparation method and application thereof Download PDFInfo
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- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 title claims abstract description 59
- 229910001431 copper ion Inorganic materials 0.000 title claims abstract description 57
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- -1 naphthalene methoxyhydrazine pyridine structure Chemical group 0.000 title claims abstract description 19
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
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- C07D213/72—Nitrogen atoms
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Abstract
The invention discloses a copper ion fluorescent probe (NAMP) containing a naphthalene methoxyhydrazine pyridine structure, and a preparation method and application thereof. The structural general formula of the fluorescent probe NAMP is shown as (I); the fluorescent probe NAMP is prepared by heating reflux reaction of 6-methoxy-2-naphthaldehyde and 2-hydrazinopyridine; the fluorescent probe NAMP shows good copper ion selectivity and resistance to interference of other metal ions, shows high-sensitivity detection on copper ions in a water body, has a detection limit of 5nM, shows high-sensitivity detection on copper ions in living cells, and has good cell permeability, good stability, good solubility and biocompatibility.
Description
Technical Field
The invention belongs to the technical field of rapid fluorescence detection of copper ions, and particularly relates to a copper ion fluorescent probe containing a naphthalene methoxyhydrazine pyridine structure, and a preparation method and application thereof.
Background
Copper ions are a common environmental pollutant, and can cause harm to human health after being accumulated in a certain degree. The human body inhales excessive copper, which is manifested as Wilson's disease, a recessive disorder of chromosomes, and may be caused by excessive copper deposition in vital organs such as liver, kidney, and brain. Dermatitis and eczema can occur when the skin is exposed to copper compounds, and skin necrosis can occur when the skin is exposed to high concentrations of copper compounds. Conjunctivitis and eyelid edema may occur in the eye when exposed to copper salts, and haze and ulceration of the eye may occur in severe cases.
Copper is also highly toxic to aquatic organisms. Heavy metal ion Cu in seawater2+When the content exceeds a certain concentration, the amphioxus can be poisoned, so that the body of the amphioxus gradually bends to die. Cu in aqueous environments2+Can affect the ingestion rate of the macrobrachium nipponense and the alkaline phosphatase, the pepsin and the tryptase in the body. Cu2+Effect on alkaline phosphatase in hepatopancreas: adding low-concentration Cu in water environment2+The activity of pepsin and tryptase in the digestive tract can be activated when the concentration is (2-4 mu g/L), but the activity of pepsin and tryptase in the digestive tract can be inhibited when the concentration is high (8 mu g/L), and Cu2+Can make liver lysosome membrane phospholipid produce oxidation reaction, and can make lysosome membrane be broken, and the hydrolytic enzyme can be released in a large quantity so as to make the liver tissue be necrotized. After the heavy metal enters the water body, a series of influences are generated on the growth, development and physiological metabolic processes of aquatic animals. The accumulation of Zn, Cu, Mn, etc. has an influence on the sex, length, etc. of fish. In addition, heavy metals will also affect the genetic expression of aquatic animals. The mixed heavy metal ions of Cu, Zn, Pb, Cd and the like can inhibit the synthesis of crucian DNA.
Copper is found in many plants subject to toxicity to disorders associated with the absorption, transport and accumulation of essential nutrients. Plants with copper poisoning can develop iron deficiency and green loss. High concentrations of copper ions also cause peroxidation that damages the thylakoid membrane and also leads to green loss. In addition, copper can inhibit the activity of nitrite reductase, thereby influencing the absorption and utilization of nitrogen. Heavy metal stress inhibits photosynthesis of plants, and the reduction effect is correlated with the degree of heavy metal stress. The basal particle layer of chloroplast becomes sparse under low concentration, the level is reduced, the basal particle layer disappears along with the increase of the concentration, the chloroplast function is damaged, and the heavy metal generates obvious inhibition effect on leaf pigment.
The existing technology for detecting copper ions lacks sensitivity and needs complex equipment. The method can be used for on-site detection, is simple to operate, is rapid in detection, is high in sensitivity, is mild in detection condition, has good selectivity, and aims to provide more technical support for the fluorescence detection technology of the copper ions.
Disclosure of Invention
The invention provides a copper ion fluorescent probe containing a naphthalene methoxyhydrazine pyridine structure, a preparation method and application thereof, aiming at overcoming the defects of the prior art.
The technical scheme adopted by the invention is as follows:
a copper ion fluorescent probe containing a naphthalene methoxyhydrazine pyridine structure has a structural general formula shown as (I):
the preparation method of the copper ion fluorescent probe containing the naphthalene methoxyhydrazine pyridine structure comprises the following steps: adding 6-methoxy-2-naphthaldehyde and 2-hydrazine pyridine into 20mL of ethanol, heating and refluxing for reaction for 30min, adding 10mL of ethanol, continuously heating and refluxing for reaction for 1h, then adding 10mL of methanol every 20min, continuously heating and refluxing for reaction until yellow floccule is generated, finishing the reaction, cooling to room temperature, performing rotary evaporation until 1mL of residual liquid is obtained, performing suction filtration, washing a filter cake with ethanol, and drying the filter cake to obtain light yellow solid powder, namely the copper ion fluorescent probe containing the naphthalene methoxyhydrazine pyridine structure.
Further, in the preparation method, the molar ratio of the 6-methoxy-2-naphthaldehyde to the 2-hydrazinopyridine is 1: 1.
Further, in the above preparation method, the temperature of the heating reflux reaction is 80 ℃.
Further, in the above preparation method, the drying temperature is 60 ℃.
The copper ion fluorescent probe containing the naphthalene methoxyhydrazine pyridine structure is applied to detecting copper ions in environmental water.
The copper ion fluorescent probe containing the naphthalene methoxyhydrazine pyridine structure is applied to detection of copper ions in living cells.
Further, in the above application, the living cell is a Hela cell line, an MCF-7 cell line or a RAW264.7 cell line.
The invention has the beneficial effects that:
1. the fluorescent probe can rapidly detect the copper ions in the water body or in the living cells with high sensitivity. The fluorescent probe shows better copper ion selectivity and resistance to interference of other metal ions; the fluorescence spectrum is not influenced by pH in the range of pH 7-12; the fluorescent probe shows high sensitivity detection on copper ions in a water body, and the detection limit reaches 5 nM; the complex constant of copper ions and the probe is 3.2 multiplied by 106M, indicating that the probe has stronger binding capacity with copper ions, and the binding of the probe with the copper ions is reversible. In addition, the fluorescent probe shows obvious color change in the detection process of copper ions in the water body, and when 6.0 multiplied by 10 is added-7After mol/L of copper ions, the color of the probe solution changes from colorless to light yellow, and the solution color continuously deepens along with the increase of the concentration of the copper ions and finally changes into light yellow.
2. The fluorescent probe can enter living cells, copper ions respond quickly, the sensitivity is high, and the copper ions in the fluorescent probe can be detected on line in real time; microscopic imaging experiments also show that the probe has better cell permeability, stability, solubility and biocompatibility, and can carry out microscopic imaging on the distribution of copper ions in cells.
Drawings
FIG. 1 is a graph showing the trend of absorbance of a fluorescent probe NAMP at 366nm as a function of pH.
FIG. 2 is a graph showing the change of fluorescence spectra of the fluorescent probe NAMP for different metal ions.
FIG. 3 is a graph showing the trend of fluorescence spectra of the fluorescent probe NAMP at different concentrations of copper ions.
FIG. 4 is a photograph of the detection of copper ions in a water body by a fluorescent probe NAMP.
FIG. 5 is a photograph showing the detection of copper ions in Hela cells by the fluorescent probe NAMP.
Detailed Description
Example 1 copper ion fluorescent Probe (NAMP) containing a Naphthyl-methoxyhydrazinopyridine Structure
The preparation reaction formula is as follows:
853.15mg of 6-methoxy-2-naphthaldehyde (4.5817 mmol) and 500mg of 2-hydrazinopyridine (4.5817 mmol) are weighed into a 250mL single-mouth bottle, 20mL of ethanol is added, the mixture is heated and refluxed for reaction at 80 ℃ for 30min, the solubility is poor, 10mL of ethanol is added, the heating and refluxing reaction at 80 ℃ is continued for 1h, the color change and the solubility are still poor, 10mL of methanol is added every 20min, the heating and refluxing reaction at 80 ℃ is continued, the color and state change are observed until yellow floccules are generated, TLC detection is carried out, the reaction is completed, the reaction is cooled to room temperature, the liquid is rotated and evaporated to the residual 1mL, suction filtration is carried out, the filter cake is washed by ethanol, the filter cake is transferred to a small medicine bottle, the filter cake is dried at 60 ℃, and then weighing is carried out, and 1.3815g of light yellow solid powder is obtained, namely the copper ion fluorescent probe (NAMP) containing the naphthalene methoxyhydrazine pyridine structure.
Example 2 application of copper ion fluorescent probe containing naphthalene methoxyhydrazine pyridine structure
The application test is carried out by using the copper ion fluorescent probe (NAMP) containing the naphthalene methoxyhydrazine pyridine structure prepared in the example 1.
1. And (4) measuring an absorption spectrum. The concentration of prepared fluorescent probe NAMP is 2 multiplied by 10-5Adjusting the pH of a probe aqueous solution containing 60% acetonitrile by using sodium hydroxide and hydrochloric acid respectively according to mol/L of the probe aqueous solution, preparing the probe aqueous solution with the pH range of 2-12 and different pH values respectively, testing absorption spectra respectively, and selecting a maximum absorption intensity to map the pH value, as shown in figure 1, wherein each point in figure 1 represents the absorbance of a fluorescent probe NAMP aqueous solution with different pH values at a position of 366nm, and the result shows that the absorbance is continuously enhanced along with the reduction of the pH value, and the fluorescent probe NAMP is insensitive when the pH value is in the range of 7-12.
2. And (4) selectively measuring metal ions. The concentration of prepared fluorescent probe NAMP is 2 multiplied by 10-5mol/L probe water solution containing 60% acetonitrile, pH 7. Different kinds of metal salts (the addition amount is 100 times of the molar mass of the fluorescent probe NAMP) are respectively added into the probe aqueous solution for fluorescence spectrum measurement. As shown in the results of FIG. 2, other metal ions besides copper ions have no influence on the fluorescence spectrum of the fluorescent probe NAMP, i.e., the probe shows better copper ion selectivity.
3. And (4) carrying out fluorescence titration on the copper ions. The fluorescent probe NAMP is added with different amounts of metal copper ions in buffer solution with pH value of 7 respectively, and fluorescence spectrum determination is carried out respectively. FIG. 3 shows that when 0.3equiv of copper ion was added, the concentration was 6X 10-7At mol/L, the fluorescence intensity of the probe is obviously enhanced, and the probe shows higher sensitivity. The detection limit of the probe reaches 5nM by calculation, and the complexing constant of the probe and copper ions is 3.2 multiplied by 106M。
4. And (4) detecting copper ions in the water body. The fluorescent probes NAMP were added to aqueous solutions containing different metal ions, respectively, and it can be seen from fig. 4 that the aqueous solution in which the absorption appeared yellowish and the fluorescence appeared bluish green strong fluorescence contained copper ions.
5. And (4) fluorescence microscopic imaging. To a culture dish containing a live cell line, 2X 10 cells were added- 5mixing mol/L dimethyl sulfoxide solution of fluorescent probe NAMP with cell culture solution, staining for 5min, and performing with phosphate buffer solution with pH of 7.4Washing three times, adding 2X 10-5And (3) a copper ion solution in mol/L, and finally placing the culture dish under a confocal microscope for observation. The experimental result shows that as shown in fig. 5, the Hela cells stained with fluorescent probe NAMP exhibit obvious fluorescence, and the experimental result shows that NAMP has better cell permeability, stability, solubility and biocompatibility, and can carry out microscopic imaging on the distribution of copper ions in the cells.
Claims (8)
1. A copper ion fluorescent probe containing a naphthalene methoxyhydrazine pyridine structure is characterized in that the copper ion fluorescent probe containing the naphthalene methoxyhydrazine pyridine structure has a structural general formula shown as (I):
2. the preparation method of the copper ion fluorescent probe containing the naphthalene methoxyhydrazine pyridine structure, which is described in claim 1, is characterized by comprising the following steps: adding 6-methoxy-2-naphthaldehyde and 2-hydrazine pyridine into 20mL of ethanol, heating and refluxing for reaction for 30min, adding 10mL of ethanol, continuously heating and refluxing for reaction for 1h, then adding 10mL of methanol every 20min, continuously heating and refluxing for reaction until yellow floccule is generated, finishing the reaction, cooling to room temperature, performing rotary evaporation until 1mL of residual liquid is obtained, performing suction filtration, washing a filter cake with ethanol, and drying the filter cake to obtain light yellow solid powder, namely the copper ion fluorescent probe containing the naphthalene methoxyhydrazine pyridine structure.
3. The method according to claim 2, wherein the molar ratio of 6-methoxy-2-naphthaldehyde to 2-hydrazinopyridine is 1: 1.
4. The method according to claim 2, wherein the temperature of the heating reflux reaction is 80 ℃.
5. The method of claim 2, wherein the drying temperature is 60 ℃.
6. The application of the copper ion fluorescent probe containing the naphthalene methoxyhydrazine pyridine structure in the detection of copper ions in environmental water body according to claim 1.
7. The application of the copper ion fluorescent probe containing the naphthalene methoxyhydrazine pyridine structure in detecting copper ions in living cells, as claimed in claim 1.
8. The use of claim 7, wherein the living cell is a Hela cell line, an MCF-7 cell line, or a RAW264.7 cell line.
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| CN107417675A (en) * | 2017-08-15 | 2017-12-01 | 新乡医学院 | A kind of novel coumarin class Schiff base derivatives, preparation method and its application in copper ion fluorescence and cell imaging detection |
| CN108383823A (en) * | 2018-03-23 | 2018-08-10 | 河南理工大学 | Aphthopyrans kappa hydazone derivative and its preparation method and application |
| CN109593078A (en) * | 2018-12-29 | 2019-04-09 | 太原师范学院 | N- butyl -4- hydroxyl -1,8- naphthalimide -3- formaldehyde-(2- pyridine) hydrazone and application |
| CN110981856A (en) * | 2019-11-22 | 2020-04-10 | 河南理工大学 | Pyrrole-naphthalimide derivative fluorescent probe and preparation method and application thereof |
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| CN107417675A (en) * | 2017-08-15 | 2017-12-01 | 新乡医学院 | A kind of novel coumarin class Schiff base derivatives, preparation method and its application in copper ion fluorescence and cell imaging detection |
| CN108383823A (en) * | 2018-03-23 | 2018-08-10 | 河南理工大学 | Aphthopyrans kappa hydazone derivative and its preparation method and application |
| CN109593078A (en) * | 2018-12-29 | 2019-04-09 | 太原师范学院 | N- butyl -4- hydroxyl -1,8- naphthalimide -3- formaldehyde-(2- pyridine) hydrazone and application |
| CN110981856A (en) * | 2019-11-22 | 2020-04-10 | 河南理工大学 | Pyrrole-naphthalimide derivative fluorescent probe and preparation method and application thereof |
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