CN110156687B - Fluorescent probe for detecting divalent copper ions and preparation method and application thereof - Google Patents
Fluorescent probe for detecting divalent copper ions and preparation method and application thereof Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 40
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 title claims abstract description 17
- 229910001431 copper ion Inorganic materials 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 239000010949 copper Substances 0.000 claims abstract description 47
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000126 substance Substances 0.000 claims abstract description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 19
- 238000001514 detection method Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 11
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- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 6
- SIOXPEMLGUPBBT-UHFFFAOYSA-N picolinic acid Chemical compound OC(=O)C1=CC=CC=N1 SIOXPEMLGUPBBT-UHFFFAOYSA-N 0.000 claims description 6
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- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
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- 125000004185 ester group Chemical group 0.000 description 2
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- 238000004252 FT/ICR mass spectrometry Methods 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
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- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/06—Ring systems of three rings
- C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention discloses a fluorescent probe for detecting bivalent copper ions and a preparation method and application thereof, wherein the chemical formula of the fluorescent probe is C 31 H 27 N 3 O 5 The structure is as follows:
the fluorescent probe has simple preparation conditions, and can treat Cu under both ultraviolet and fluorescent conditions 2+ Responsive and capable of detecting Cu in pure water and cells 2+ Has great application potential in the aspect of biological medicine.
Description
Technical Field
The invention belongs to the technical field of metal ion detection, and particularly relates to a fluorescent probe for detecting divalent copper ions, and a preparation method and application thereof.
Background
With the improvement of living standard, people pay more attention to the change of physical quality, and the attention to trace elements in the body system is far higher than the past. The content of common trace elements such as iron, copper, magnesium and the like in a human body in a living body is very important for the health of a life system, so that the invention of the method for efficiently detecting the trace elements in the cells is very important.
Copper is an essential trace element and nutrient substance in a life system, plays an extremely important role in the life system, for example, the copper ions cannot be separated in the hematopoiesis process in organisms, participate in the synthesis of various enzymes in the heart, and once the copper ions are deficient, the possibility of coronary heart disease is increased. In addition, if the copper ions are not in a certain range in the life process, the possibility of suffering from the Alzheimer disease and the familial muscular atrophy is increased, so that the research on the detection of the content of the copper ions in a living body has an inevitable effect on maintaining the normal operation of a living system. Meanwhile, compared with normal cells of human body, the content of copper ions in tumor cells is obviously greater than that of normal cells, mainly because the copper ions are easier to enrich in tumor cells.
The general monitoring methods for the copper ion content comprise atomic absorption spectrometry, inductively coupled plasma-mass spectrometry, wind-solar photometry, cyclic voltammetry and the like, but the methods have the defects of complicated detection process, insensitive detection result, incapability of large-scale positioning detection and real-time monitoring and the like. In comparison, the fluorescent probe overcomes the limitations of the method, can quickly, sensitively and specifically detect copper ions, and can cause minimal damage to cells without influencing the detection result simply by modifying the structure of the probe. Currently, methods for detecting cells using fluorescent probes are widely used. The synthesis of the efficient and stable fluorescent probe capable of detecting the copper ions in the cells in real time has important significance.
Disclosure of Invention
The invention aims to provide a fluorescent probe for detecting divalent copper ions and a preparation method and application thereof, and aims to solve the technical problem that Cu can be detected and identified through molecular design synthesis 2+ The fluorescent probe of (1). The invention is based on Cu 2+ Catalyzing hydrolysis principle of ester group, and fluorescent probe is on Cu 2+ Under the action of Cu, the ester group is covered by 2+ Hydrolyzing the hydroxyl group to generate fluorescence quenching, and designing and synthesizing a novel fluorescent probe compound.
The invention relates to a fluorescent probe for detecting bivalent copper ions, which has a chemical formula of C 31 H 27 N 3 O 5 The structural formula is as follows:
the preparation method of the fluorescent probe for detecting the divalent copper ions comprises the following steps:
adding the compound 1 (100 mg) into anhydrous DMSO (5 mL), heating to 70 ℃, and cooling to room temperature to obtain a solution A; adding 30mg of 2-picolinic acid), EDC (90.8 mg) and DMAP (27.0 mg) into anhydrous DMSO (5 mL), and dissolving to obtain a solution B; mixing the solution A with the solution B, N 2 Protecting, and reacting for 24 hours at normal temperature; and after the reaction is finished, dropwise adding the product into about 200mL of distilled water, separating out a yellow product, distilling under reduced pressure to obtain a crude product, recrystallizing with absolute ethyl alcohol, and then carrying out suction filtration under reduced pressure to obtain a target product, wherein the final fluorescent probe is obtained without separation and purification by a silica gel column chromatography.
The synthetic process of the invention is as follows:
the application of the fluorescent probe of the invention is in detecting Cu 2+ The method (2) is used as a detection reagent.
Further, the fluorescent probe can specifically recognize Cu in a hydrolysis mode 2 Can detect Cu by ultraviolet or fluorescence 2+ All have specific recognition. Fluorescence intensity and Cu in fluorescence spectrum 2+ Concentration is inversely proportional with Cu 2+ The concentration increases and decreases.
The fluorescent probe can quantitatively detect Cu in pure water 2+ It can also be applied to Cu in living cells 2+ Detection of (3).
The preparation condition of the invention is simple, and the fluorescent probe can treat Cu under both ultraviolet and fluorescent conditions 2+ Responsive and capable of detecting Cu in pure water and cells 2+ Has great application potential in the aspect of biological medicine.
Drawings
FIG. 1 shows the fluorescent probe of the present invention in DMSO-d6 as a solvent 1 H-NMR(400MHz)。
FIG. 2 is a mass spectrum of the fluorescent probe of the present invention.
FIG. 3 is an infrared spectrum of the fluorescent probe of the present invention.
FIG. 4 shows the UV-Vis absorbances of the fluorescent probe of the present invention after adding different metal ions to the aqueous solvent.
FIG. 5 is a graph showing the addition of Cu of various concentrations ranging from 0 to 30. Mu. Mol/L to a probe solution 2+ The UV-Vis absorbance after the reaction.
FIG. 6 is a fluorescence spectrum of a probe solution to which different metal ions were added.
FIG. 7 shows the addition of Cu to the probe solution at different concentrations ranging from 0 to 30. Mu. Mol/L 2+ Fluorescence quenching pattern after the reaction.
FIG. 8 is a graph showing the addition of Cu of various concentrations ranging from 0 to 10. Mu. Mol/L to a probe solution 2+ Subsequent linear fit, coefficient of linearity R 2 ＝0.96892。
FIG. 9 is the addition of 10. Mu.L, 10 to HeLa cells -3 Adding 2 times of equivalent Cu into the cells cultured by mol/L probe solution for half an hour and the cells cultured for half an hour 2+ Two-photon confocal images of cells after ionization. Wherein, λ ex =355nm, a is a confocal fluorescence image after adding a fluorescent probe into a HeLa cell, B is a bright field image, and C is a combined image of A, B. D is to add a fluorescent probe into the HeLa cells, culture for half an hour, and then add 2 times of equivalent Cu 2+ The subsequent confocal fluorescence image, E is a bright field image, and F is a combined image of D, E.
Detailed Description
Example 1: synthesis of fluorescent probes
Adding the compound 1 (100 mg) into anhydrous DMSO (5 mL), heating to 70 ℃, and cooling to room temperature to obtain a solution A; adding 30mg of 2-picolinic acid), EDC (90.8 mg) and DMAP (27.0 mg) into anhydrous DMSO (5 mL), and dissolving to obtain a solution B; mixing the solution A with the solution B, N 2 Protecting, and reacting for 24 hours at normal temperature; and after the reaction is finished, adding the product into 200mL of distilled water, precipitating yellow solid, distilling under reduced pressure to obtain a crude product, recrystallizing twice by using absolute ethyl alcohol, and finally carrying out suction filtration under reduced pressure to obtain the final fluorescent probe.
1H NMR (400mhz, dmso) δ 8.90-8.81 (m, 1H), 8.58 (d, J =7.4hz, 2h), 8.35-8.27 (m, 2H), 8.12 (t, J =8.1hz, 1h), 7.90 (dd, J =13.0,7.7hz, 2h), 7.81-7.74 (m, 1H), 7.70 (d, J =8.2hz, 2h), 7.57 (d, J = 8.8 hz, 2h), 4.15 (t, J =6.8hz, 2h), 3.44 (s, 4H), 3.31 (s, 1H), 2.41 (s, 2H), 2.33 (s, 3H), 1.84 (t, J =6.6hz, 2h), FTMS: m/z =522.2, theoretical value: 521.52.
example 2:
1. to examine whether or not the synthesized probe can be bound to Cu in pure water 2+ Has ultraviolet spectrum selectivity, 10 -5 4mL of the probe was pipetted using a pipette tip and 5-fold equivalent of Ag was added to each of the pipettes + ，Al 3+ ，Ca 2+ ，Cd 2+ ，Co 2+ ，Cu 2+ ，Fe 2+ ，Hg 2+ ，K + ，Li + ，Mg 2+ ，Na + ，Ni 2+ ，Pb 2+ The added solution is compared in an ultraviolet spectrum, and Cu is added 2+ The probe of (2) is obviously red-shifted, the position of the maximum absorption peak is 374nm, and the red-shift is 10nm. This indicates that the probe can be used for Cu 2+ Has selective ultraviolet spectrum response.
2. To examine whether or not the synthesized probe can be bound to Cu in pure water 2+ Has fluorescence selectivity, 10 is prepared - 5 The mol/L probe was pipetted with a pipette to obtain 4mL of the probe, and 5-fold equivalent of Ag was added thereto + ，Al 3+ ，Ca 2+ ，Cd 2+ ，Co 2+ ，Cu 2+ ，Fe 2+ ，Hg 2 + ，K + ，Li + ，Mg 2+ ，Na + ，Ni 2+ ，Pb 2+ Metal ions, detecting the response in the fluorescence spectrum. As can be seen from the fluorescence spectrogram, other ions do not respond to the fluorescent probe, and Cu is added 2+ Then, fluorescence is obviously quenched, and the maximum emission peak is at 475nm, thus proving that the fluorescent probe can be used for detecting Cu in a fluorescence spectrum 2+ And also selectively responds.
3. To examine whether the synthesized probe can be used for Cu in water 2+ Quantitatively detecting the prepared 10 -5 4mL of the solution transfer gun for mol/L probe was transferred to4mL centrifuge tube, then respectively adding 1-30 mu mol/L Cu 2+ Comparison in the UV spectrum shows that with the addition of Cu 2+ The probe gradually red-shifted with increasing concentration, indicating Cu at different concentrations 2+ The occurrence of the ultraviolet absorption maximum can be red-shifted. 10 -5 Adding 1 mu mol/L,2 mu mol/L,3 mu mol/L,4 mu mol/L,5 mu mol/L,6 mu mol/L,7 mu mol/L,8 mu mol/L,9 mu mol/L and 10 mu mol/L Cu into the mol/L probe respectively 2+ Fluorescence spectra in the concentration range can be seen with Cu 2+ The fluorescence gradually quenches when the concentration is continuously increased, and the linear coefficient R is 2 =0.96892. This indicates that the fluorescent probe can quantitatively detect Cu in the fluorescence spectrum 2+ 。
4. To determine whether the synthesized probe can be used for detecting Cu in cells 2+ With good response, 10 μ L of 10 -3 Adding mol/L probe into HeLa cells cultured in a 1mL culture dish, culturing for half an hour, detecting the fluorescence intensity in the cells under a two-photon confocal microscope, and adding 2 times of equivalent Cu into the cells cultured for half an hour by using the fluorescent probe 2+ The cells were cultured for another half hour and the fluorescence intensity of the cells was photographed under a two-photon confocal microscope. As can be seen, in the cells, cu was added 2+ Then adding Cu 2+ The previous fluorescence was significantly reduced. This indicates that the probe is paired with Cu in the cell 2+ Also has good response, which is to detect Cu in cells 2+ A very good detection method is provided.
Claims (6)
1. A fluorescent probe for detecting divalent copper ions, which is characterized in that:
the chemical formula of the fluorescent probe is C 31 H 27 N 3 O 5 The structural formula is as follows:
2. a method for preparing the fluorescent probe of claim 1, characterized by comprising the steps of:
adding 100mg of compound 1 into anhydrous DMSO, heating to 70 ℃, and cooling to room temperature to obtain a solution A; adding 30mg of 2-picolinic acid, 90.8mg of EDC and 27.0mg of DMAP into anhydrous DMSO, and dissolving to obtain a solution B; mixing the solution A with the solution B, N 2 Protecting, and reacting for 24 hours at normal temperature; after the reaction is finished, dropwise adding the product into distilled water, separating out a yellow product, then carrying out reduced pressure distillation to obtain a crude product, recrystallizing with absolute ethyl alcohol, and carrying out reduced pressure suction filtration to obtain a target product;
the structural formula of the compound 1 is as follows:
3. use of a fluorescent probe according to claim 1, characterized in that:
the fluorescent probe is used for preparing and detecting Cu 2+ The detection reagent of (1).
4. Use according to claim 3, characterized in that:
the fluorescent probe can specifically recognize Cu in a hydrolysis mode 2+ Can be detected by ultraviolet or fluorescence, and can be used for detecting Cu 2+ All have specific recognition.
5. Use according to claim 3, characterized in that:
the fluorescent probe is used for preparing and detecting Cu in pure water or living cells 2+ The detection reagent of (4).
6. Use according to claim 5, characterized in that:
the fluorescent probe is used for preparing the Cu in the quantitative detection pure water 2+ A detection reagent of concentration.
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| CN108250188A (en) * | 2018-01-16 | 2018-07-06 | 商丘师范学院 | A kind of long-wavelength fluorescent probe for detecting copper ion and its synthetic method and application |
| CN109320535A (en) * | 2018-09-29 | 2019-02-12 | 江汉大学 | A kind of detection Cu2+Ratiometric fluorescent probe, preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| A highly sensitive naphthalimide-based fluorescent probe for detection of Cu2+ via selective hydrolysis reaction and its application in practical samples;Xueyun Li等;《J Chin Chem Soc.》;20191212;1070-1077 * |
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