CN111349070B - Near-infrared fluorescent molecular probe for detecting biological cell viscosity and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of fluorescent probe detection, in particular to a near-infrared fluorescent molecular probe for detecting biological cell viscosity and a preparation method and application thereof. The molecular probe is preferably 9- (2-carboxyphenyl) -6 (diethylamino) -4- [4- (methylpiperazine) -1 benzylidene ] -1,2,3, 4-tetrahydroanthracene, wherein 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-methylthiobenzylidene) -1,2,3, 4-tetrahydroanthracene is used as a fluorescent group, and an activated double bond is used as a reactive group. The molecular probe prepared by the invention can be applied to biological sample detection, and has the advantages of small background fluorescence, long emission wavelength, near-infrared luminescence and excellent light sensitivity in biological sample imaging by twisting an intramolecular charge transfer mechanism.
Description
Technical Field
The invention relates to the technical field of fluorescent probe detection, in particular to a near-infrared fluorescent molecular probe for detecting biological cell viscosity and a preparation method and application thereof.
Background
The stability of intracellular fluids is the basis for normal activities of living cells, and abnormal changes are associated with many abnormalities in cellular skills, and the viscosity of intracellular fluids plays a crucial role, including signaling and transport, particularly in regulating diffusion-mediated cellular processes. However, conventional viscometers, although suitable for macroscopic liquid media, cannot be used to detect the viscosity of living cells. The small-molecule fluorescent probe has the advantages of high sensitivity, real-time detection, rapid nondestructive analysis and the like, and is widely applied to the field of fluorescence imaging, so that the small-molecule fluorescent probe can be used for ideally visualizing and monitoring the dynamic change of the viscosity in living cells.
Disclosure of Invention
Aiming at the defects in the prior art, the technical problem to be solved by the invention is to provide a near-infrared fluorescent molecular probe for detecting the viscosity of biological cells and a preparation method and application thereof, the prepared molecular probe can be applied to biological cell sample detection, the molecular probe has small background fluorescence, long emission wavelength, near-infrared luminescence and excellent light sensitivity in biological sample imaging by twisting an intramolecular charge transfer mechanism, and the molecular probe is suitable for biological cell imaging due to the reduction of the background fluorescence, the small light damage to the biological sample and the increase of the penetration depth.
The technical scheme adopted by the invention for realizing the purpose is as follows: a near-infrared fluorescence molecular probe for detecting the viscosity of biological cells, which takes 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-methylthio-benzylidene) -1,2,3, 4-tetrahydro-anthracene as a fluorescent group and takes an activated double bond as a reactive group, is preferably 9- (2-carboxyphenyl) -6 (diethylamino) -4- [4- (methylpiperazine) -1 benzylidene ] -1,2,3, 4-tetrahydro-anthracene, and has the chemical structural formula:
the invention also comprises a method for preparing the near-infrared fluorescent molecular probe for detecting the viscosity of the biological cells, which comprises the following steps:
(1) adding concentrated sulfuric acid into a flask, adding cyclohexanone and 2- [ (4-diethylamino) -2-hydroxybenzoyl]Benzoic acid, reacting at 70-90 ℃, after the reaction is finished, cooling the solution to room temperature, putting the solution into an ice water bath, uniformly stirring, then dropwise adding perchloric acid into the mixed liquid, and separating out a solid to obtain an intermediate product 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroanthraceneIs named as F 376 ；
(2) Intermediate product F obtained in step (1) 376 Dissolving with anhydrous ethanol, adding 4- (4-methylpiperazino) benzaldehyde, stirring and refluxing at 70-90 deg.C, cooling to room temperature, spin-drying the filtrate, and separating with chromatography column to obtain 9- (2-carboxyphenyl) -6 (diethylamino) -4- [4- (methylpiperazino) -1-benzylidene]-1,2,3, 4-tetrahydroanthracene, designated F 562 。
Further, in the step (1),
the molar ratio of 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid to cyclohexanone is preferably 1:2,
the reaction time is preferably 2 hours,
the preferred sequence of addition is to add concentrated sulfuric acid, stir in an ice-water bath for 5 minutes, then add cyclohexanone, stir at 0 ℃ for 5 minutes, and finally add cyclohexanone and 2- [ (4-diethylamino) -2-hydroxybenzoyl) ] benzoic acid.
Further, in the step (2),
intermediate product F 376 The molar ratio to 4- (4-methylpiperazinyl) benzaldehyde is preferably 1:2,
the reflux reaction time is preferably 30 min.
The invention also comprises the application of the near-infrared fluorescent molecular probe for detecting the viscosity of the biological cells, wherein the fluorescent molecular probe has the structure or is prepared by the preparation method, and is applied to detecting the internal viscosity of the biological cells and the viscosity change of living cells and fluorescence imaging.
Further, a molecular probe F was established 562 The step of titration linear curve for viscosity includes,
(1) preparing a PBS buffer salt solution with the pH value of 7.40 and the concentration of 20 mM; molecular Probe F at a concentration of 1mM 562 A DMSO solution of (1);
(2) the proportion of methanol to glycerol is respectively as follows: 10 parts of 10:0, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10 solutions are mixed in a test tube uniformly, and then 10 mu L of molecular probe F with the concentration of 1mM is added 562 Mixing the DMSO solution uniformly, and performing fluorescence colorimetryCompleting sample measurement in a dish;
(3) testing the fluorescence intensity by a fluorescence spectrophotometer to obtain a fluorescence intensity ratio, wherein the excitation wavelength of the fluorescence intensity for detecting the cell viscosity is 605 nm;
(4) and respectively taking the content of the glycerol as an abscissa and the fluorescence intensity ratio as an ordinate to obtain a linear equation about the viscosity and the fluorescence intensity ratio.
Further, detecting molecular probe F 562 The uv absorption step at the minimum and maximum viscosities includes,
the proportion of methanol to glycerol is respectively as follows: 2 portions of 10:0 and 0:10 solutions are mixed in a test tube, 10 mul of molecular probe F with the concentration of 1mM is added 562 And (3) uniformly mixing the DMSO solution, and completing sample measurement in a cuvette to obtain ultraviolet absorption spectra of two samples.
Further, molecular probe F 562 The step of imaging the macrophage includes the step of,
(1) preparing a DMSO standard solution of a molecular probe with the concentration of 1mM and a monensin solution with the concentration of 1 mu g/ml;
(2) cell culture: culturing recovered macrophage cell, wherein the culture medium contains 10% bovine embryo serum, 1% double antibody, 89% DMEM, and 5% CO at 37 deg.C 2 Culturing for 24h in the environment to obtain cells with good growth vigor for later use;
(3) culturing macrophage in culture medium, and culturing 3 groups with inoculum size of 2 × 10 7 ～ 9×10 7 Culturing for 24h, dividing into three groups A, B and C, and adding 10 μ M molecular probe F into macrophage of group A 562 Incubating for 30 min; group B macrophages were incubated with 15. mu.M monensin for 30min and then with 10. mu.M molecular probe F 562 Incubating for 30 min; group C cells were incubated with 2. mu.M nystatin for 30min and then with 10. mu.M molecular probe F 562 Incubating for 30min, and performing confocal laser fluorescence imaging on the macrophages to obtain a confocal map of the three groups of cells and obtain an intensity map of a fluorescence image of the three groups of cells.
Further, molecular probe F 562 For cellsThe step of survival rate influencing comprises the step of,
molecular probes F were added to the cell culture medium at concentrations of 0M,5M,10M,20M,30M and 50M, respectively 562 At 37 ℃ 5% CO 2 The culture chamber of (1) was used for 24 hours, 25. mu.L of 5mg/mL of 4-methylthiazolyltetrazole MTT was added to the cell culture solution and cultured for 4 hours, and the cell viability was evaluated by the MTT cuvette method.
The near-infrared fluorescent molecular probe for detecting the viscosity of the biological cells, the preparation method and the application thereof have the beneficial effects that:
(1) the near-infrared fluorescence labeling reagent of the invention uses 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroxanthene as a fluorescent group, uses an activated double bond as a reaction site, twists a near-infrared fluorescence probe of an intramolecular charge transfer mechanism, and leads the probe to have obvious fluorescence signal reading for viscosity.
(2) The near-infrared fluorescence labeling reagent disclosed by the invention is sensitive in response, and the response time to viscosity is within seconds.
(3) The near-infrared fluorescence labeling reagent has low detection limit, and compared with a commercialized fluorescence labeling reagent, the detection limit of the viscosity is 1.5nm/L, so that the disease can be timely found and early diagnosed conveniently.
(4) The invention is applied to the detection of living cells, and further promotes the research of the effect of the biological micromolecules in the living body.
(5) The change of the ultraviolet color before and after the reaction is obvious, so that the ultraviolet color can be detected by naked eyes, and the method is convenient and quick.
(6) Since its long wavelength is in the infrared part, it is less harmful to biological cells.
Drawings
FIG. 1 shows a molecular probe F according to example 1 of the present invention 562 Synthetic roadmaps of (a);
FIG. 2 shows a molecular probe F according to example 1 of the present invention 562 Mass spectrogram of (1);
FIG. 3 shows a molecular probe F according to example 1 of the present invention 562 Nuclear magnetic H spectrum of (1);
FIG. 4 shows a molecular probe F according to example 1 of the present invention 562 Nuclear magnetic spectrum C of (1);
FIG. 5 shows a molecular probe F according to example 2 of the present invention 562 Fluorescence titration plot of in vitro viscosity;
FIG. 6 shows a molecular probe F according to example 2 of the present invention 562 A linear equation for the fluorescence intensity of the in vitro viscosity;
FIG. 7 shows a molecular probe F according to example 3 of the present invention 562 Uv absorption profile in both pure ethanol and glycerol, i.e. the viscosity at the maximum and minimum;
FIG. 8 shows a molecular probe F according to example 4 of the present invention 562 Confocal mapping of macrophages;
FIG. 9 shows a molecular probe F according to example 4 of the present invention 562 Intensity profile of confocal spectra on macrophages.
Detailed Description
The invention is further explained in detail with reference to the drawings and the specific embodiments;
example 1:
as shown in fig. 1-4, a near-infrared fluorescent molecular probe for detecting biological cell viscosity, which uses 9- (2-carboxyphenyl) -6 (diethylamino) -4 (4-methylthiobenzylidene) -1,2,3, 4-tetrahydroanthracene as a fluorescent group and uses an activated double bond as a reactive group, is 9- (2-carboxyphenyl) -6 (diethylamino) -4- [4- (methylpiperazine) -1 benzylidene ] -1,2,3, 4-tetrahydroanthracene, and has a chemical structural formula:
the invention also comprises a method for preparing the near-infrared fluorescent molecular probe for detecting the viscosity of the biological cells, which comprises the following steps:
(1) adding concentrated sulfuric acid into a flask, adding cyclohexanone and 2- [ (4-diethylamino) -2-hydroxybenzoyl]Benzoic acid reacts at 70-90 ℃, after the reaction is finished, the solution is cooled to room temperature, the solution is placed into ice water bath and is evenly stirred, perchloric acid is dripped into the mixed liquid to separate out solid, and an intermediate product 9- (2-carboxyphenyl) -6 (diethylamino) -1,2,3, 4-tetrahydroanthracene, named as F, is obtained 376 ；
(2) Intermediate product F obtained in step (1) 376 Dissolving with anhydrous ethanol, adding 4- (4-methylpiperazino) benzaldehyde, stirring and refluxing at 70-90 deg.C, cooling to room temperature, spin-drying the filtrate, and separating with chromatography column to obtain 9- (2-carboxyphenyl) -6 (diethylamino) -4- [4- (methylpiperazino) -1-benzylidene]-1,2,3, 4-tetrahydroanthracene, designated F 562 。
In the above-mentioned step (1),
the mol ratio of the 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid to the cyclohexanone is 1:2,
the reaction time is 2 hours,
the sample was added in the order of concentrated sulfuric acid, stirred in an ice-water bath for 5 minutes, then cyclohexanone was added, stirred at 0 ℃ for 5 minutes, and finally cyclohexanone and 2- [ (4-diethylamino) -2-hydroxybenzoyl) ] benzoic acid were added.
In the step (2) described above, the step (c),
intermediate product F 376 The mol ratio of the 4- (4-methylpiperazino) benzaldehyde to the N-methyl-N-ethyl-N-methyl-N-ethyl-N-methyl-phenyl-formaldehyde is 1:2,
the reflux reaction time was 30 min.
The invention also comprises the application of the near-infrared fluorescent molecular probe for detecting the viscosity of the biological cells, wherein the fluorescent molecular probe has the structure or is prepared by the preparation method, and is applied to detecting the internal viscosity of the biological cells and the viscosity change of living cells and fluorescence imaging.
Example 2:
as shown in FIGS. 5 and 6, a molecular probe F was established 562 The step of titration linear curve for viscosity includes,
(1) preparing a PBS buffer salt solution with the pH value of 7.40 and the concentration of 20 mM; molecular Probe F at a concentration of 1mM 562 A DMSO solution of (1);
(2) the proportion of methanol to glycerol is respectively as follows: 10 parts of 10:0, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10 solutions are mixed in a test tube uniformly, and then 10 mu L of molecular probe F with the concentration of 1mM is added 562 Uniformly mixing the DMSO solution, and completing sample measurement in a fluorescent cuvette;
(3) testing the fluorescence intensity by a fluorescence spectrophotometer to obtain a fluorescence intensity ratio, wherein the excitation wavelength of the fluorescence intensity for detecting the cell viscosity is 605 nm;
(4) and respectively taking the content of the glycerol as an abscissa and the fluorescence intensity ratio as an ordinate to obtain a linear equation about the viscosity and the fluorescence intensity ratio.
Example 3:
as shown in FIG. 7, the detecting molecular probe F 562 The uv absorption step at the minimum and maximum viscosities includes,
the proportion of methanol to glycerol is respectively as follows: 2 portions of 10:0 and 0:10 solutions are mixed in a test tube, 10 mul of molecular probe F with the concentration of 1mM is added 562 And (3) uniformly mixing the DMSO solution, and completing sample measurement in a cuvette to obtain ultraviolet absorption spectra of two samples.
Example 4:
as shown in FIGS. 8 and 9, the molecular probe F 562 The step of imaging the macrophage includes the step of,
(1) preparing a DMSO standard solution of a molecular probe with the concentration of 1mM and a monensin solution with the concentration of 1 mu g/ml;
(2) cell culture: culturing recovered macrophage cell, wherein the culture medium contains 10% bovine embryo serum, 1% double antibody, 89% DMEM, and 5% CO at 37 deg.C 2 Culturing for 24h in the environment to obtain cells with good growth vigor for later use;
(3) culturing macrophage in culture medium, and culturing 3 groups with inoculum size of 2 × 10 7 ～ 9×10 7 Culturing for 24h, dividing into three groups A, B and C, and adding 10 μ M molecular probe F into macrophage of group A 562 Incubating for 30 min; group B macrophages were incubated with 15. mu.M monensin for 30min and then with 10. mu.M molecular probe F 562 Incubating for 30 min; group C cells were incubated with 2. mu.M nystatin for 30min, followed by 10. mu.M molecular probe F 562 Incubating for 30min, and performing confocal laser fluorescence imaging on the macrophages to obtain a confocal map of the three groups of cells and obtain an intensity map of a fluorescence image of the three groups of cells.
Example 5:
molecular probe F 562 The step of influencing the viability of the cells comprises,
molecular probes F were added to the cell culture medium at concentrations of 0M,5M,10M,20M,30M and 50M, respectively 562 At 37 ℃ 5% CO 2 The culture chamber of (1) was used for 24 hours, 25. mu.L of 5mg/mL of 4-methylthiazolyltetrazole MTT was added to the cell culture solution and cultured for 4 hours, and the cell viability was evaluated by the MTT cuvette method.
The macrophages in the examples of the present invention are: wuhanponosic life science and technology ltd, mouse kidney macrophage, product number: CP-M187; high performance liquid-mass spectrometry was performed using an Agilent 1100 mass spectrometry system (Agilent, USA) equipped with a degasser, quaternary pump, autosampler, and high performance liquid chromatography was performed with a Hypersil GOLD C18 column (2.1mm x 50mm,1.8 μmi.d., Agilent, USA). The fluorescence detection is carried out by using a Hitachi F-4600 fluorescence spectrometer for ONOO - The detection excitation wavelength is 500nm, the excitation and emission slit widths are both 10.0nm, the voltage is 400V, and the scanning speed is 2400 nm/min. Fluorescence imaging observations were performed by Olympus Fluo View FV1000 (Japan) confocal, with 40-fold objective. The separation and purification of the compound are realized by adopting a thin-layer chromatography silica gel column, wherein the filler is 300-400 meshes.
The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention accordingly, and not to limit the protection scope of the present invention accordingly. All equivalent changes or modifications made in accordance with the spirit of the present disclosure are intended to be covered by the scope of the present disclosure.
Claims (9)
1. A near-infrared fluorescent molecular probe for detecting the viscosity of biological cells is characterized in that: the activated double bond is taken as a reactive group, and the chemical structural formula is as follows:
2. a method for preparing a near-infrared fluorescent molecular probe for detecting the viscosity of biological cells, wherein the fluorescent molecular probe has a chemical structural formula as defined in claim 1, and the method comprises the following steps:
(1) adding concentrated sulfuric acid into a flask, adding cyclohexanone and 2- [ (4-diethylamino) -2-hydroxybenzoyl]Benzoic acid, reacting at 70-90 ℃, after the reaction is finished, cooling the solution to room temperature, putting the solution into an ice water bath, uniformly stirring, then dropwise adding perchloric acid into the mixed liquid, separating out a solid, and obtaining an intermediate product F 376 The structural formula is as follows:
(2) intermediate product F obtained in step (1) 376 Dissolving with anhydrous ethanol, adding 4- (4-methylpiperazinyl) benzaldehyde, stirring and refluxing at 70-90 deg.C, cooling to room temperature, spin drying the filtrate, and purifying with chromatographic column to obtain the final product.
3. The method for preparing near-infrared fluorescent molecular probe for detecting the viscosity of biological cells as claimed in claim 2, wherein:
in the step (1), the step (c),
the mol ratio of the 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid to the cyclohexanone is 1:2,
the reaction time is 2 hours,
the sample is added in sequence, concentrated sulfuric acid is added firstly, the mixture is stirred for 5 minutes in ice-water bath, then cyclohexanone is added, the mixture is stirred for 5 minutes at the temperature of 0 ℃, and finally cyclohexanone and 2- [ (4-diethylamino) -2-hydroxybenzoyl ] benzoic acid are added.
4. The method for preparing near-infrared fluorescent molecular probe for detecting the viscosity of biological cells as claimed in claim 2, wherein:
in the step (2), the step (c),
intermediate product F 376 The mol ratio of the 4- (4-methylpiperazino) benzaldehyde to the N-methyl-N-ethyl-N-methyl-N-ethyl-N-methyl-phenyl-formaldehyde is 1:2,
the reflux reaction time was 30 min.
5. The application of a near-infrared fluorescent molecular probe for detecting the viscosity of biological cells is characterized in that: the fluorescent molecular probe has the chemical structural formula as described in claim 1, or is prepared by the preparation method as described in any one of claims 2 to 4, and is applied to detection of the viscosity in biological cells, detection of the viscosity change of living cells and fluorescence imaging.
6. The use of the near-infrared fluorescent molecular probe for detecting the viscosity of biological cells as claimed in claim 5, wherein: the step of establishing a titration linear curve of the molecular probe for viscosity comprises,
(1) preparing a PBS buffer salt solution with the pH value of 7.40 and the concentration of 20 mM; a DMSO solution of molecular probes at a concentration of 1 mM;
(2) the proportion of methanol to glycerol is respectively as follows: 10 parts of solutions of 10:0, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10 are mixed uniformly in a test tube, then 10 mu L of DMSO solutions of molecular probes with the concentration of 1mM are respectively added, the mixture is mixed uniformly, and the sample measurement is finished in a fluorescence cuvette;
(3) testing the fluorescence intensity by a fluorescence spectrophotometer to obtain a fluorescence intensity ratio, wherein the excitation wavelength of the fluorescence intensity for detecting the cell viscosity is 605 nm;
(4) and respectively taking the content of the glycerol as an abscissa and the fluorescence intensity ratio as an ordinate to obtain a linear equation about the viscosity and the fluorescence intensity ratio.
7. The use of the near-infrared fluorescent molecular probe for detecting the viscosity of biological cells as claimed in claim 5, wherein: the step of detecting the uv absorption of the molecular probe at the minimum and maximum viscosities comprises,
the proportion of methanol to glycerol is respectively as follows: 2 parts of 10:0 solution and 2 parts of 0:10 solution are respectively and uniformly mixed in a test tube, then 10 mu L of DMSO solution of the molecular probe with the concentration of 1mM is respectively added and uniformly mixed, and the sample measurement is completed in a cuvette, so that the ultraviolet absorption spectra of two samples are obtained.
8. The use of the near-infrared fluorescent molecular probe for detecting the viscosity of biological cells as claimed in claim 5, wherein: the step of imaging the macrophage with the molecular probe includes,
(1) preparing a DMSO standard solution of a molecular probe with the concentration of 1mM and a monensin solution with the concentration of 1 mu g/ml;
(2) cell culture: culturing recovered macrophage cell, wherein the culture medium contains 10% bovine embryo serum, 1% double antibody, 89% DMEM, and 5% CO at 37 deg.C 2 Culturing for 24h in the environment to obtain cells with good growth vigor for later use;
(3) culturing macrophage in culture medium, and culturing 3 groups with inoculum size of 2 × 10 7 ～9×10 7 Culturing for 24h per mL, dividing into three groups A, B and C, and only adding 10 μ M molecular probe into macrophage of group A and incubating for 30 min; group B macrophages are firstly cultured for 30min by using 15 mu M monensin, and then are incubated for 30min by using 10 mu M molecular probes; and (3) incubating the cells in the group C for 30min by using 2 mu M nystatin, then incubating for 30min by using 10 mu M molecular probes, and performing confocal laser fluorescence imaging on macrophages to obtain a confocal map of the cells in the three groups and obtain an intensity map of a fluorescence image of the cells in the three groups.
9. The use of the near-infrared fluorescent molecular probe for detecting the viscosity of biological cells as set forth in claim 5, wherein: the step of influencing cell viability by the molecular probe comprises,
adding molecular probes with concentration of 0M,5M,10M,20M,30M and 50M to the cell culture solution, and adding 5% CO at 37 deg.C 2 The culture chamber of (1) was used for 24 hours, 25. mu.L of 5mg/mL of 4-methylthiazolyltetrazole MTT was added to the cell culture solution and cultured for 4 hours, and the cell viability was evaluated by the MTT cuvette method.
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