CN110161005B - Fluorescent carbon dot for detecting cell activity, preparation method and application thereof - Google Patents
Fluorescent carbon dot for detecting cell activity, preparation method and application thereof Download PDFInfo
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- CN110161005B CN110161005B CN201910438986.3A CN201910438986A CN110161005B CN 110161005 B CN110161005 B CN 110161005B CN 201910438986 A CN201910438986 A CN 201910438986A CN 110161005 B CN110161005 B CN 110161005B
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Abstract
The invention provides a fluorescent carbon dot for detecting cell activity, a preparation method and application thereof, 2, 4-dihydroxy benzaldehyde and 1, 2, 3, 3-tetramethyl-3H-iodoindole are dissolved in absolute ethyl alcohol, subjected to microwave reaction for 1H, cooled, purified by a filter membrane, subjected to rotary evaporation to obtain a dark red carbon dot solid, and dissolved in DMSO to serve as a stock solution. The carbon dots are very simple to synthesize and convenient to operate; the invention can detect the activity of cells; the invention can distinguish healthy, apoptotic and dead cells by monitoring the membrane potential of mitochondria with high sensitivity.
Description
Technical Field
The invention relates to the technical field of fluorescent carbon dots and biosensing, in particular to a fluorescent carbon dot for detecting cell activity, a preparation method and application thereof.
Background
Mitochondria are the power station of cells and play a key role in many important biological processes. Mitochondrial damage and dysfunction are associated with a range of diseases such as: cancer, diabetes, Alzheimer's disease and Parkinson's disease, myocardial ischemia-reperfusion injury, and aging degenerative disease. The decrease and disappearance of mitochondrial membrane potential represents mitochondrial damage and cell death. Therefore, methods for monitoring mitochondrial membrane potential changes are very important.
Carbon dots have been widely used in bioimaging due to the advantages of low toxicity, good biocompatibility, excellent photostability, ease of surface modification and low cost.
To date, although there are some reported carbon spots that can sense a decrease in mitochondrial membrane potential, there are no three different levels that can sensitively and selectively display mitochondrial membrane potential, i.e., normal, diminished and absent, and their use in mitochondrial-related biological processes.
The present invention is therefore intended to solve the problem of detecting mitochondrial membrane potential by targeting of carbon spots to organelles in different health states to reflect cell viability and to distinguish between healthy, apoptotic and dead cells.
Disclosure of Invention
The invention provides a fluorescent carbon dot for detecting cell activity, a preparation method and application thereof, wherein a benzaldehyde derivative can generate the carbon dot, 1, 2, 3, 3-tetramethyl-3H-indole has the capability of targeting mitochondria, the carbon dot is synthesized by condensation of the two, the membrane potential of the mitochondria can be monitored efficiently and sensitively, and healthy cells, apoptotic cells and dead cells can be distinguished by positioning in different organelles.
The technical scheme for realizing the invention is as follows:
a method for preparing fluorescent carbon dots for detecting cell activity comprises the steps of dissolving 2, 4-dihydroxybenzaldehyde and 1, 2, 3, 3-tetramethyl-3H-indole iodide in absolute ethyl alcohol, carrying out microwave reaction for 1H, cooling, purifying with a filter membrane, carrying out rotary evaporation to obtain a dark red carbon dot solid, and dissolving in DMSO to obtain a stock solution.
The synthesis route of the fluorescent carbon dots based on the detection of the cell activity is as follows:
the molar ratio of the 2, 4-dihydroxy benzaldehyde to the 1, 2, 3, 3-tetramethyl-3H-indole iodide is 3: 1.
The microwave reaction temperature is 130 ℃, and after cooling, the product is purified by a filter membrane of 0.22 mu M.
The fluorescent carbon dots realize monitoring of mitochondrial membrane potential and distinguishing healthy, apoptotic and dead cells through positioning in different organelles.
The application specifically comprises:
the targeting conditions of organelles of the carbon points in different health states are respectively tested, and the mitochondrial membrane potential is monitored so as to reflect the cell activity and distinguish healthy, apoptotic and dead cells. Has good prospect in biomedical research and diagnosis of related diseases.
The invention has the beneficial effects that: (1) the carbon point synthesis is very simple and convenient to operate; (2) the invention can detect the activity of cells; (3) the invention can distinguish healthy, apoptotic and dead cells by monitoring the membrane potential of mitochondria with high sensitivity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a TEM spectrum of a carbon dot of example 1.
FIG. 2 is an IR spectrum of a carbon dot of example 1.
FIG. 3 is an XPS spectrum of carbon dots of example 1.
FIG. 4 shows the carbon dots (5. mu.g mL) of example 1-1) Uv and fluorescence spectra in PBS (10mM pH 7.4).
FIG. 5 is a fluorescence lifetime map of the carbon dots of example 1.
FIG. 6 is a graph of the photostability of the carbon dots of example 1.
FIG. 7 is a fluorescence spectrum showing the change with time of fluorescence intensity at 546nm after reaction at 5. mu.g/mL carbon spots with 200. mu.M different amino acids, 200. mu.M different ROS, 1mM anion and cation, respectively, in a PBS-buffered (10mM, pH 7.4) system.
FIG. 8 is a co-localization map of example 1 carbon spots in healthy and dead cells. (A) Healthy cells were incubated with carbon spots for 30 minutes and imaged after incubation with commercial mitochondrial dye for 30 minutes; (B) cells were dead, fixed with 4% paraformaldehyde for 30min, incubated for 30min with carbon spots, and imaged after incubation with commercial nuclear dye for 30 min.
FIG. 9 is a graph of the co-localization of carbon spots in apoptotic cells from example 1. (A) Panels were incubated with carbon spots and commercial lysosomal probes prior to addition of CCCP (20 μ M) for apoptosis; (B) group was incubated with carbon dots and commercial lysosomal probes prior to addition of H2O2(8mM) apoptosis; (C) panels were incubated with carbon spots and commercial lysosomal probes, followed by HBSS apoptosis.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The carbon dots are synthesized by the following steps:
mixing 2, 4-dihydroxy benzaldehyde and 1, 2, 3, 3-tetramethyl-3H-indole iodide in a molar ratio of 3:1 in absolute ethyl alcohol, reacting at 130 ℃ for 1 hour by microwave, cooling, purifying by a 0.22 mu M filter membrane, performing rotary evaporation to obtain a dark red carbon dot solid, and dissolving in DMSO to obtain a stock solution.
FIG. 1 is a TEM image of carbon dots, approximately spherical in structure, with a statistical size of 1.7nm, corresponding to a 0.23nm lattice, indicating that carbon dots have been formed.
FIG. 2 is an IR spectrum of carbon dots, 3428.11cm-1Stretching vibration corresponding to-OH, 3156.79cm-1Stretching vibration corresponding to-N-H, 1620.50, 1578.45, 1503.36, 1477.81cm-1Corresponding to aromatic Hydrogen, 1620.50cm-1Corresponding to C/C-N stretching vibration, 1316.79cm-1Stretching vibration corresponding to C-N, 1263.19cm-1Stretching vibration corresponding to C-O, 854.33, 705.97cm-1Corresponding to the bending vibration of C-H, -OH. Indicating that the two raw materials have been combined to form carbon dots.
FIG. 3 is an XPS spectrum of carbon dots, 284.8, 400.92 and 532.36eV, corresponding to C1s, N1s and O1s, respectively, at 77.02%, 1.09% and 21.89% respectively, further illustrating the formation of carbon dots.
FIG. 4 is a carbon dot (5. mu.g mL)-1) Uv and fluorescence spectra in PBS (10mM pH 7.4) with maximum absorption and emission of 525nm and 546nm, respectively.
FIG. 5 is a graph of the fluorescence lifetime of carbon dots in PBS (10mM pH 7.4) at 546nm, the fluorescence lifetime being 1.0337 ns.
Fig. 6 is a graph of the light stability of carbon dots in PBS (10mM pH 7.4), and the fluorescence intensity of the carbon dots does not change significantly when the carbon dots are continuously irradiated under a 250W xenon lamp for 30min, which shows that the carbon dots can resist photobleaching and are suitable for biological imaging.
Example 2
Reaction of 5. mu.g/mL carbon dots with 200. mu.M different amino acids, 200. mu.M different ROS, 1mM cation and anion
To a 2mL PBS-buffered (10mM, pH 7.4) system containing 5 μ g/mL carbon dots, 20 μ L of 20mM analyte: 1: carbon points; 2: threonine; 3: arginine; 4: methionine; 5: (ii) proline; 6: glycine; 7: lysine; 8: leucine; 9: tryptophan; 10: isoleucine; 11: alanine; 12: (ii) histidine; 13: glutamic acid; 14: aspartic acid; 15: valine; 16: serine; 17: (ii) cysteine; 18: homocysteine; 19: glutathione; 20: h2O2；21：NaNO2；22：t-BuOOH；23：.OH；24：KCl；25：CaCl2；26：NaCl；27：MgCl2； 28：AlCl3；29：ZnSO4；30：FeCl3；31：FeCl2；32：CuSO4；33：NiCl2；34：CrCl3；35： K2CrO7；36：MnCl2；37：NaF；38：NaCl；39：NaBr；40：KI；41：Na2SO4；42：Mg(NO3)2Fluorescence spectrometry was performed. The fluorescence intensity at 546nm of the different analytes was also compared. The experimental data of fig. 7 show that the carbon dots do not react with these analytes.
Example 3
Co-localization of carbon spots in cells of different health states
Healthy cells: carbon spots (10. mu.g/mL) were added to 1mL of cell culture medium, incubated for 30min, washed three times with PBS, incubated for 30min with the Mito Tracker Deep Red (100nM) commercial mitochondrial probe, washed three times with PBS, and very good overlap of carbon spots with commercial mitochondrial probes was observed in confocal microscopy, indicating that carbon spots can target mitochondria.
Apoptotic cells: carbon spots (10. mu.g/mL) were added to 1mL of cell culture medium, incubated for 30min, washed three times with PBS, added with the commercial lysosomal probe Lyso Tracker Deep Red (100nM), incubated for 30min, washed three times with PBS, added to 1mL of cell culture medium, and poor overlap of carbon spots with the commercial lysosomal probe was observed in a confocal microscope. Then, carbonyl cyano 3-chlorophenylhydrazone (CCCP), a substance capable of reducing the membrane potential on both sides of the mitochondrial inner membrane and promoting apoptosis, was added at 20 μ M), which was observed in a confocal microscope, and the overlap was better after addition of CCCP, indicating that the carbon point is reduced in the mitochondrial membrane potential and can target lysosome during apoptosis.
Carbon dots (10. mu.g/mL) were added to 1mL of cell culture medium, incubated for 30min, washed three times with PBS, added with the commercial lysosomal probe Lyso Tracker Deep Red (100nM), incubated for 30min, washed three times with PBS, added to 1mL of cell culture medium, and the carbon dots were observed to overlap poorly with the commercial lysosomal probe in a confocal microscope. Addition of H2O2(8mM, a substance that inhibits oxidative respiratory chain, induces cell damage) observed in a confocal microscope, to which H was added2O2The overlap was better later, suggesting that the carbon spot could target lysosomes during apoptosis.
Carbon dots (10. mu.g/mL) were added to 1mL of cell culture medium, incubated for 30min, washed three times with PBS, added with the commercial lysosomal probe Lyso Tracker Deep Red (100nM), incubated for 30min, washed three times with PBS, added to 1mL of cell culture medium, and the carbon dots were observed to overlap poorly with the commercial lysosomal probe in a confocal microscope. The replacement with 1mL starvation medium, hanks' balanced salt solution (HBSS, a substance that induces autophagy by starvation), overlapped better and better within 180 minutes, indicating that carbon spots can target lysosomes during apoptosis.
Dead cells: adding 1mL of 4% paraformaldehyde into a cell culture dish to fix cells for 30min, washing with PBS three times, adding a carbon dot (10 mu g/mL) into 1mL of cell culture medium, culturing for 30min, washing with PBS three times, adding a commercial nuclear probe DAPI (1 mu g/mL), culturing for 30min, washing with PBS three times, adding 1mL of cell culture medium, and observing that the carbon dot is well overlapped with the commercial nuclear probe in a confocal microscope, which indicates that the carbon dot can target the nucleus in dead cells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (4)
1. A preparation method of a fluorescent carbon dot for detecting cell activity is characterized by comprising the following steps: dissolving 2, 4-dihydroxy benzaldehyde and 1, 2, 3, 3-tetramethyl-3H-indole iodide in absolute ethyl alcohol, performing microwave reaction for 1H, cooling, purifying with a filter membrane, and performing rotary evaporation to obtain a dark red carbon dot solid.
2. The method for preparing a fluorescent carbon dot for detecting cell activity according to claim 1, wherein: the molar ratio of the 2, 4-dihydroxy benzaldehyde to the 1, 2, 3, 3-tetramethyl-3H-indole iodide is 3: 1.
3. The method for preparing a fluorescent carbon dot for detecting cell activity according to claim 2, wherein: the microwave reaction temperature is 130 ℃, and after cooling, the product is purified by a filter membrane of 0.22 mu M.
4. A fluorescent carbon dot produced by the production method according to any one of claims 1 to 3, characterized in that: the fluorescent carbon dots are reagents for detecting cell activity.
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| CN104694116A (en) * | 2013-12-06 | 2015-06-10 | 中国科学院大连化学物理研究所 | A carbon-base fluorescence probe for a targeting living cell mitochondrion and preparation thereof |
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