A water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells, and its preparation method and application

Abstract

The invention discloses a water-soluble two-photon fluorescent probe for distinguishing live cells from dead cells, and a preparation method and application thereof. The probe is synthesized by using aldehyde disubstituted benzothiadiazole and 1-(3-trimethylaminopropyl)-4-methylpyridine dibromide as raw materials under the action of a catalyst. The water-soluble two-photon fluorescent probe can specifically stain the nuclei of dead cells to achieve the purpose of distinguishing live / dead cells, and has the advantages of high fluorescence brightness, large Stokes shift, good photostability, etc. The fluorescent probe provided by the invention is a universal dead cell nucleus staining agent, which is suitable for various types of cells such as normal cells, cancer cells, neuronal cells, and is suitable for paraformaldehyde-fixed or unfixed cells. The single-photon fluorescence imaging of fixed HeLa cells and the two-photon fluorescence imaging of neuronal cells SH-SY5Y are used as examples to demonstrate the application of the fluorescent probe in cell fluorescence imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells, and a preparation method and application thereof. Background Art

[0002] Apoptosis is a normal physiological process of cells that removes excess or damaged cells, especially during embryonic development, and is involved in the maintenance of tissue homeostasis. Distinguishing between live and dead cells is important for studying apoptosis.

[0003] A variety of methods have been developed to identify live / dead cells, including magnetic resonance imaging (MRI), X-ray computed tomography, optical microscopy, and electron microscopy. However, these methods are time-consuming and costly. Fluorescence imaging is the most widely used method for sensitive and rapid identification of live / dead cells. So far, various fluorescent materials have been developed to stain and distinguish live / dead cells. Due to the advantages of high sensitivity and spatial resolution, small molecule fluorescent probes have become a powerful tool for distinguishing live / dead cells.

[0004] The nucleus is a membrane-bound organelle in eukaryotic cells that maintains genetic integrity and controls cellular activities by regulating gene expression. The nucleus consists of a double-layered nuclear membrane containing protein and phospholipid molecules and a large number of nuclear pores, which are selectively hydrophilic channels that allow small molecules (such as ions and metabolites) to cross the membrane surface by simple diffusion. The nucleus is involved in transcription, gene replication, and the processing of transcripts. Now, imaging the nucleus can provide answers to many questions about nucleus-related events.

[0005] Although small molecule fluorescent probes have been synthesized in the prior art to distinguish live / dead cells by targeting the cell nucleus, most of these probes focus on specifically targeting the nuclei of live cells rather than targeting the nuclei of dead cells, and the strategy adopted is usually combined with the commercial nuclear dye Hoechst. Therefore, it is meaningful to develop small molecule fluorescent probes that specifically stain the nuclei of dead cells. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a water-soluble two-photon fluorescent probe for distinguishing live cells from dead cells, and its preparation method and application. The water-soluble two-photon fluorescent probe can specifically stain the nuclei of dead cells to achieve the purpose of distinguishing live / dead cells, and has the advantages of high fluorescence brightness, large Stokes shift, and good photostability. The fluorescent probe provided by the present invention is a universal dead cell nucleus stain, which is suitable for various types of cells such as normal cells, cancer cells, and neuronal cells, and is suitable for paraformaldehyde-fixed or unfixed cells. The application of the water-soluble two-photon fluorescent probe for distinguishing live / dead cells in the present invention takes single-photon fluorescence imaging of fixed HeLa cells and two-photon fluorescence imaging of neuronal cells SH-SY5Y as examples.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells, characterized in that the structural formula of the water-soluble two-photon fluorescent probe is:

[0009]

[0010] The present invention also provides a method for preparing the water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells, and the preparation method comprises the following steps:

[0011] (1) Synthesizing an aldehyde-disubstituted benzothiadiazole compound BTD-CHO, wherein the structural formula of the BTD-CHO is:

[0012] (2) Synthesizing 1-(3-trimethylaminopropyl)-4-methylpyridine dibromide Py-Br, wherein the structural formula of Py-Br is:

[0013] (3) Dissolve BTD-CHO and Py-Br in an organic solvent, then add a catalyst, and reflux under inert gas protection for 10 to 12 hours. After the reaction is completed, filter the reaction solution, wash, and dry to obtain the water-soluble two-photon fluorescent probe BTD-V for distinguishing live cells from dead cells.

[0014] In step (1), the synthesis method of BTD-CHO comprises the following steps: adding 4,7-dibromo-2,1,3-benzothiadiazole, 4-formylphenylboronic acid pinacol ester, tetrabutylammonium bromide, tetrakis(triphenylphosphine)palladium and potassium carbonate to a mixed solution of toluene, ethanol and deionized water; stirring and reacting at 75 to 80° C. for 20 to 24 hours under the protection of inert gas nitrogen; after the reaction is completed, extracting, concentrating, purifying and drying the reaction solution to obtain the BTD-CHO.

[0015] In step (2), the synthesis method of Py-Br comprises the following steps: adding 4-methylpyridine and (3-bromopropyl)trimethylammonium bromide to anhydrous N,N-dimethylformamide, stirring and reacting at 95-105° C. for 1.5-2.5 hours, and then filtering, washing and drying the reaction solution to obtain the Py-Br.

[0016] Furthermore, the molar ratio of 4-methylpyridine to (3-bromopropyl)trimethylammonium bromide is 1:1 to 1.1.

[0017] The concentration of 4-methylpyridine in anhydrous N,N-dimethylformamide is 0.5-0.6M.

[0018] In step (3), the organic solvent is anhydrous ethanol; the catalyst is piperidine; and the inert gas is nitrogen.

[0019] In step (3), the molar ratio of BTD-CHO, Py-Br and piperidine is 1:3.5-4.0:0.01-0.02.

[0020] In step (3), the concentration of BTD-CHO in anhydrous ethanol is 0.01 to 0.02 M.

[0021] The present invention also provides the use of the water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells in imaging for distinguishing living cells from dead cells.

[0022] The present invention also provides application of the water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells in cell fluorescence imaging.

[0023] In the preparation method of the water-soluble two-photon fluorescent probe provided by the present invention, benzothiadiazole and 1-(3-trimethylaminopropane)-4-methylpyridine dibromide, which are disubstituted with aldehyde groups, are used as raw materials. Under the action of a catalyst, a water-soluble two-photon fluorescent probe BTD-V for distinguishing living cells from dead cells is simply and conveniently synthesized. The structural characteristics are that benzothiadiazole is a fluorophore, 1-(3-trimethylaminopropane)-4-methylpyridine bromide is a water-soluble group, and the two are connected by a double bond. There is a rotatable single bond between BTD-V molecules, and the molecule will be distorted when this single bond rotates. In addition, there are donors and acceptors in this molecular configuration, and charge transfer will occur between them. The distortion between BTD-V molecules and the occurrence of intramolecular charge transfer (TICT) lead to the intensification of non-radiative transitions and the weakening of fluorescence intensity. However, when BTD-V binds to DNA in the cell nucleus, the probe BTD-V enters the minor groove of the cell nucleus DNA, the rotation of the BTD-V single bond is inhibited, and the fluorescence is enhanced. The results of the co-localization experiment showed that BTD-V can specifically target the nuclei of dead cells. This is because the permeability of the cell membrane changes after cell death, and BTD-V can penetrate the membrane of dead cells to reach the nucleus and stain the nucleus; while due to the integrity of the cell membrane of living cells, BTD-V cannot penetrate the cell membrane, so it cannot stain living cells. In order to further confirm that BTD-V can specifically identify the nuclei of dead cells, the commercial dye pyridinium iodide (PI) and BTD-V, which specifically stain dead cells, were used for cell co-staining imaging. The results of the two-photon absorption spectrum experiment showed that BTD-V has two-photon performance, with a maximum two-photon absorption wavelength of 750nm. After binding to DNA in the cell nucleus, the two-photon performance is enhanced, and the maximum two-photon absorption wavelength is red-shifted to 790nm, with an absorption cross section of 78GM. Secondly, BTD-V has good optical properties, such as high fluorescence quantum in aqueous solution, large molar absorption coefficient, large Stokes shift, and good biocompatibility. More importantly, it can perform two-photon fluorescence imaging of the nuclei of dead cells. Therefore, BTD-V has good application prospects in cell nucleus-related research and monitoring.

[0024] Compared with the prior art, the water-soluble two-photon fluorescent probe BTD-V for distinguishing living cells from dead cells provided by the present invention has the advantages of good biocompatibility, strong specificity, high fluorescence brightness, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the structural formula of probe BTD-V;

[0026] Figure 2 The synthetic route of the probe BTD-V is shown in Figure 2.

[0027] Figure 3 is the H NMR spectrum of BTD-CHO;

[0028] Figure 4 is the NMR carbon spectrum of BTD-CHO;

[0029] Figure 5 This is the high-resolution mass spectrum of BTD-CHO;

[0030] Figure 6 is the H NMR spectrum of Py-Br;

[0031] Figure 7 is the NMR carbon spectrum of Py-Br;

[0032] Figure 8 This is the high-resolution mass spectrum of Py-Br;

[0033] Fig. 9 is the H NMR spectrum of BTD-V;

[0034] Fig.10 is the NMR carbon spectrum of BTD-V;

[0035] Fig.11 This is the high-resolution mass spectrum of BTD-V;

[0036] Fig.12 The UV-visible absorption spectrum (A) and fluorescence emission spectrum (B) of the probe BTD-V in aqueous solution in Example 2;

[0037] Fig.13 is a curve showing the change of the fluorescence emission intensity of the probe BTD-V as a function of pH in Example 2, wherein the pH value ranges from 3 to 11;

[0038] Fig.14 This is a graph showing the relationship between the fluorescence emission intensity of the probe BTD-V in Example 2 and time under continuous irradiation with 415 nm excitation light for 1 h;

[0039] Fig.15 is the two-photon absorption spectrum of the probe BTD-V;

[0040] Fig.16 The UV absorption spectrum (A) and fluorescence emission spectrum (B) of the interaction between probe BTD-V and cell nuclear DNA, and the circular dichroism spectrum (C) of the interaction between probe BTD-V and cell nuclear DNA;

[0041] Fig.17 It is the HeLa cell cytotoxicity graph of probe BTD-V;

[0042] Fig.18 SH-SY5Y cell cytotoxicity graph of probe BTD-V;

[0043] Fig.19The cell co-localization image of the probe BTD-V (A); and the co-staining image of the probe BTD-V and the commercial dye PI (B);

[0044] Fig. 20 This is the fluorescence imaging of the probe BTD-V on HeLa cells fixed with paraformaldehyde;

[0045] Fig.21 Fluorescence imaging of the probe BTD-V to distinguish between live and dead cells;

[0046] Fig. 22 This is the two-photon fluorescence imaging of the probe BTD-V on the neuronal cell SH-SY5Y. DETAILED DESCRIPTION

[0047] The present invention is described in detail below with reference to the embodiments.

[0048] Example 1

[0049] A water-soluble two-photon fluorescent probe BTD-V for distinguishing living cells from dead cells, the structural formula of which is:

[0050]

[0051] The synthetic route for the fluorescent probe is as follows: Figure 2 As shown, the synthesis method comprises the following steps:

[0052] (1) Synthesis of compound BTD-CHO: 4,7-dibromo-2,1,3-benzothiadiazole (0.58 g, 1.97 mmol), 4-formylphenylboronic acid pinacol ester (0.61 g, 2.65 mmol), tetrabutylammonium bromide (0.12 g, 0.36 mmol), tetrakis(triphenylphosphine)palladium (0.06 g, 0.05 mmol) and anhydrous potassium carbonate (0.81 g, 5.84 mmol) were added to a round-bottom flask, and 9 mL of toluene, 6 mL of anhydrous ethanol and 3 mL of deionized water were added. The air in the round-bottom flask was evacuated and refluxed at 75 °C for 24 h under nitrogen atmosphere. After the reaction was completed, the mixture was naturally cooled to room temperature and quickly poured into 100 mL of water to quench the reaction. Then, the mixture was extracted three times with 50 mL of dichloromethane, washed three times with 50 mL of water and then washed three times with 50 mL of saturated brine. After drying over anhydrous magnesium sulfate, the dichloromethane was removed by rotary evaporation and purified by silica gel column using ethyl acetate:petroleum ether in a volume ratio of 1:19 as eluent to obtain compound BTD-CHO after vacuum drying.

[0053] Its H NMR spectrum is Figure 3 As shown, 1H NMR (400MHz, Chloroform-d) δ 10.14 (s, 2H), 8.18 (d, J = 6.6 Hz, 4H), 8.08 (d, J = 8.4 Hz, 4H), 7.91 (s, 2H).

[0054] Its NMR carbon spectrum is Figure 4 As shown, 13 C NMR (100MHz, Chloroform-d) δ191.67,153.58,142.76,135.86,132.82,129.76,128.52.

[0055] Its high-resolution mass spectrometry Figure 5 As shown, [M+H] + The theoretical value is 345.0692 and the measured value is 345.0697.

[0056] (2) Synthesis of compound Py-Br: Weigh 4-methylpyridine (0.52 g, 5.64 mmol) and (3-bromopropyl)trimethylammonium bromide (1.41 g, 5.40 mmol) and add them to 10 mL of anhydrous N,N-dimethylimide, stir at 100 °C for 2 h, cool to room temperature, filter, wash with dichloromethane, and vacuum dry to obtain 1-(3-trimethylaminopropyl)-4-methylpyridine dibromide Py-Br.

[0057] Its H NMR spectrum is Figure 6 As shown, 1 H NMR(400MHz, Deuterium Oxide)δ8.69(d,J=6.8Hz,2H),7.90(d,J=6.4Hz,2H),4.64(t,J=7.7Hz,2H),3.53–3.47(m,2H),3.16(s,9H),2.65(s,3H),2.61–2.51(m,2H).

[0058] Its NMR carbon spectrum is Figure 7 As shown, 13 C NMR (100MHz, Deuterium Oxide) δ161.04,143.21,129.08,62.49,57.09,53.19,24.55,21.50.

[0059] Its high-resolution mass spectrometry Figure 8 As shown, the theoretical value of [M-Br] is 273.0961 and the measured value is 273.0961.

[0060] (3) Synthesis of fluorescent probe BTD-V: Weigh BTD-CHO (0.05 g, 0.14 mmol) and Py-Br (0.18 g, 0.52 mmol) and add them to 10 mL of anhydrous ethanol. Stir evenly and add 200 μL of piperidine. Reflux for 12 h under nitrogen protection. After the reaction is completed, cool to room temperature, filter, wash with dichloromethane, and dry to obtain the fluorescent probe BTD-V.

[0061] Its H NMR spectrum is Fig. 9 As shown, 1 H NMR (400MHz, Methanol-d4) δ8.93(d,J=6.9Hz,4H),8.31(d,J=6.9Hz,4H),8.23(d,J=8.4Hz,4H),8.11(d,J=16.2Hz,2H),8.05(s ,2H),7.97(d,J=8.4Hz,4H),7.60(d,J=16.2Hz,2H),4.68(t,J=7.7Hz,4H),3.63–3.56(m,4H),3.23(s,18H),2.66–2.57(m,4H).

[0062] Its NMR carbon spectrum is Fig.10 As shown, 13 C NMR(100MHz,DMSO-d6)δ152.88,152.76,144.20,140.14,138.14,134.82,13 1.36,129.45,128.27,128.09,123.66,123.50,61.35,56.27,52.07,23.74.

[0063] Its high-resolution mass spectrometry Fig.11 As shown, [M] 4+ Theoretical value is 174.0988, measured value is 174.0980, [M+2Br] 2+ The theoretical value is 428.1155 and the measured value is 428.1106.

[0064] Example 2

[0065] Optical properties of the probe BTD-V

[0066] Take 8 μL of a 1 mM methanol solution of the probe BTD-V and add it to a centrifuge tube. After the solvent is completely evaporated to dryness, add 1 mL of deionized water to obtain an aqueous solution with a concentration of 8 μM.

[0067] The ultraviolet absorption spectrum and fluorescence emission spectrum of the probe aqueous solution were tested respectively.

[0068] like Fig.12As shown in the figure, it can be seen from the UV absorption spectrum that the maximum UV absorption peak of the probe BTD-V in aqueous solution is 414nm. It can be seen from the fluorescence emission spectrum that the maximum emission wavelength of the probe BTD-V under this condition is 590nm. The fluorescence test conditions are E X slit=10nm,E m slit=10nm,PMT=300V.

[0069] The fluorescence emission intensity of the probe BTD-V changes in the pH range of 3 to 11. Fig.13 As shown in the figure, it can be seen that the probe BTD-V has excellent stability in the pH range investigated; in addition, the results further illustrate the possibility of the probe's application in biological systems.

[0070] The change trend of the fluorescence emission intensity of the probe BTD-V under continuous irradiation of excitation light with a wavelength of 415 nm for 1 h is as follows: Fig.14 As shown in the figure, it can be seen that the fluorescence intensity of the probe BTD-V is basically stable over time, which indicates that the probe BTD-V has good resistance to photobleaching and has the potential for long-term imaging in cell experiments. X slit=10nm,E m slit=10nm,PMT=300V.

[0071] In order to evaluate the lipid-water partition coefficient Log P of the BTD-V probe o / w , 80 μM probe solution was distributed in a mixture containing 1.5 mL of n-octanol and 1.5 mL of water. The mixture was first vortexed and then centrifuged at 2000 rpm for 5 minutes. When these procedures were completed, the n-octanol layer and the aqueous layer were separated. The absorbance of the n-octanol and aqueous layers was measured respectively, and the Log P was calculated to be -2.35 by the logarithmic ratio of the BTD-V concentration in the organic phase and the aqueous phase, indicating that the probe BTD-V has the potential for nucleus targeting.

[0072] Example 3

[0073] Two-photon performance of the probe BTD-V

[0074] The final concentration of the probe BTD-V in the solution in Example 2 was 8 μM. At the same time, 2 μM fluorescein at pH = 11 was used as a reference, and a Leica TCS SP8 confocal scanning microscope and a multiphoton femtosecond laser were used as the excitation light source to record the fluorescence intensity at a wavelength of 700 to 880 nm. Fig.15 As shown, the two-photon maximum absorption wavelength of the probe BTD-V is 750 nm.

[0075] Example 4

[0076] Effects of probe BTD-V and nuclear DNA

[0077] First, nuclear DNA was synthesized, and its sequence was 5'-CGCGAATTCGCGTTTTCGCGAATTCGCG-3'. After annealing, the DNA was prepared into a 100 μM master solution with TE buffer. In the experiment, the concentration of the final probe BTD-V was kept at 8 μM, and the concentration of DNA was kept at 2 μM. The ultraviolet absorption spectra of BTD-V, DNA, BTD-V and DNA, and the fluorescence emission spectra of BTD-V, BTD-V and DNA were measured respectively. Fig.16 As shown in Figures A and B, after the probe binds to DNA, the ultraviolet absorption wavelength moves toward the long wavelength direction and the fluorescence intensity increases by nearly four times.

[0078] In order to further determine the binding mode of BTD-V to DNA, circular dichroism (CD) studies were performed on DNA, BTD-V, and BTD-V and DNA. Fig.16 As shown in Figure C, the results show that the probe BTD-V without a chiral center has no CD optical activity. Nuclear DNA (2μM) shows a positive peak at 285nm and a negative peak at 255nm in the CD spectrum. After the interaction between probe BTD-V and nuclear DNA, the positive peak of the CD signal around 285nm and the negative peak near 255nm did not change significantly, indicating that the DNA helical structure did not change. However, after the nuclear DNA was exposed to 8μM and 16μM probe BTD-V, a new induced positive CD signal appeared near 470nm, indicating that BTD-V binds to the minor groove of nuclear DNA, so the fluorescence intensity is enhanced after the probe BTD-V interacts with DNA.

[0079] Example 5

[0080] Cytotoxicity of probe BTD-V

[0081] Cell culture: HeLa cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 1% 100U / mL penicillin and 100μg / mL streptomycin. Neuronal SH-SY5Y cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% 100U / mL penicillin and 100μg / mL streptomycin. The culture conditions of the biological incubator were 5% CO2 and 37°C.

[0082] Cytotoxicity experiment: Cytotoxicity was determined using the MTT method of a cell counting kit. HeLa cells and SH-SY5Y cells in logarithmic phase growth were cultured in 96-well plates. After adhesion, the cells were incubated with a series of concentrations of BTD-V for 24 hours. Then, 10 μL of MTT solution (5 mg / mL) was added to each well and incubated for 4 hours. After the incubation, formazan solution was added and incubated for another 4 hours. Finally, the absorbance at a wavelength of 570 nm was measured using a microplate reader, and the cell survival rate was calculated based on the measured absorbance results. Fig.17 As shown in Figure 2, when the concentration of probe BTD-V was 12 μM, the survival rate of HeLa cells was still as high as over 80%. Fig.18 As shown in the figure, when the concentration of probe BTD-V is 24 μM, the survival rate of SH-SY5Y cells is still as high as over 80%. These results indicate that the probe BTD-V has good biocompatibility and can be used for fluorescence imaging of biological systems such as cells.

[0083] Example 6

[0084] Probe BTD-V cell co-localization experiment

[0085] HeLa cells were used in the experiment to explore the localization ability of the probe BTD-V to subcellular organelles. The commercial nuclear fluorescent probe DAPI was selected to verify the nuclear targeting ability of the probe BTD-V. In the experiment, HeLa cells were pretreated with 4% paraformaldehyde fixative for 15 minutes. Then, the fixed HeLa cells were incubated with 8μM BTD-V solution and 10μM DAPI staining solution for 15 minutes. After the incubation, PBS was washed 2 to 3 times. The co-localization results are shown in Figure 2. Fig.19 As shown in Figure (A), the experimental results show that the fluorescence of the probe BTD-V and the commercial cell nucleus localization dye DAPI has a good overlap, and the Pearson colocalization coefficient can reach 0.91. This also shows that the probe BTD-V has excellent targeting ability to the cell nucleus.

[0086] To further demonstrate that the probe BTD-V can specifically bind to dead cells, the dead cell commercial dye pyridinium iodide (PI) was used for co-staining with BTD-V. Fig.19 As shown in Figure (B), BTD-V can specifically stain the nuclei of dead cells, thereby achieving the purpose of distinguishing living cells from dead cells.

[0087] Example 7

[0088] Fluorescence imaging of fixed HeLa cell nuclei using probe BTD-V

[0089] HeLa cells were selected to study the fluorescence imaging ability of the probe BTD-V on fixed cell nuclei. In the fluorescence imaging experiment, HeLa cells growing in logarithmic phase were cultured in a 4-well cell culture dish. After adhesion, each well was first washed 2-3 times with PBS, and then 400 μL of paraformaldehyde fixative was added to each well for 15 minutes. After fixation, the four wells were washed with PBS, and then 700 μL of probe BTD-V at concentrations of 0.1 μM, 0.5 μM, 2 μM, and 8 μM were added to each well and incubated for 5 minutes. Before imaging, the cells were washed three times with PBS solution. Fluorescence imaging of the cells was obtained in the red channel (570 nm to 620 nm) excited at 405 nm by laser confocal microscopy. Fig. 20 The results showed that the probe BTD-V could complete the staining process of fixed HeLa cell nuclei at 0.5 μM and within 5 min, and had the advantages of being sensitive and rapid.

[0090] Example 8

[0091] Fluorescence imaging of unfixed HeLa cell nuclei using probe BTD-V

[0092] HeLa cells were selected to study the ability of the probe BTD-V to image the fluorescence of unfixed nuclei. For the fluorescence imaging experiment of unfixed nuclei, HeLa cells growing in logarithmic phase were cultured in a 4-well cell culture dish. After adhesion, each well was first washed 2-3 times with PBS, and then 8 μM of the probe BTD-V was added to three of the wells and incubated for 10 min, 20 min, and 35 min, respectively. Fluorescence imaging of the cells was obtained in the red channel (570 nm-620 nm) excited at 405 nm by laser confocal microscopy. Fig.21 The results showed that the probe BTD-V can complete the staining of unfixed HeLa cell nuclei in 10 minutes, and the imaging results showed that the probe BTD-V can specifically identify dead cells, so it can quickly and sensitively distinguish between live cells and dead cells.

[0093] Example 9

[0094] Two-photon fluorescence imaging of fixed SH-SY5Y neuronal cell nuclei using probe BTD-V

[0095] SH-SY5Y cells were used as an example to study the two-photon fluorescence imaging ability of the probe BTD-V on fixed cell nuclei. In the fluorescence imaging experiment, SH-SY5Y cells growing in the logarithmic phase were cultured in a single-well cell culture dish. After adhesion, they were first washed 2 to 3 times with PBS, and then 700 μL of paraformaldehyde fixative was added for 15 minutes. After fixation, they were washed with PBS and then 700 μL of 8 μM probe BTD-V was added and incubated for 5 minutes. Before imaging, they were washed 2 to 3 times with PBS solution. Fluorescence imaging of the cells was obtained in the red channel (570 nm to 620 nm) excited at 790 nm by laser confocal microscopy. Fig. 22 This indicates that the probe BTD-V can perform two-photon fluorescence imaging on the fixed SH-SY5Y cell nucleus, and also indicates that the probe BTD-V is a two-photon fluorescence probe.

[0096] The detailed description of a water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells, its preparation method and application with reference to the above-mentioned embodiments is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells, characterized in that: The structural formula of the water-soluble two-photon fluorescent probe is:

2. The method for preparing a water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Synthesizing an aldehyde-disubstituted benzothiadiazole compound BTD-CHO, wherein the structural formula of the BTD-CHO is: (2) Synthesizing 1-(3-trimethylaminopropyl)-4-methylpyridine dibromide Py-Br, wherein the structural formula of Py-Br is: (3) BTD-CHO and Py-Br are dissolved in an organic solvent, and then a catalyst is added, and reflux reaction is carried out under the protection of an inert gas for 10 to 12 hours. After the reaction is completed, the reaction solution is filtered, washed, and dried to obtain the water-soluble two-photon fluorescent probe BTD-V for distinguishing between live cells and dead cells.

3. The preparation method according to claim 2, characterized in that: In step (1), the synthesis method of BTD-CHO comprises the following steps: adding 4,7-dibromo-2,1,3-benzothiadiazole, 4-formylphenylboronic acid pinacol ester, tetrabutylammonium bromide, tetrakis(triphenylphosphine)palladium and potassium carbonate to a mixed solution of toluene, ethanol and deionized water; stirring and reacting at 75 to 80° C. for 20 to 24 hours under the protection of inert gas nitrogen; after the reaction is completed, extracting, concentrating, purifying and drying the reaction solution to obtain the BTD-CHO.

4. The preparation method according to claim 2, characterized in that: In step (2), the synthesis method of Py-Br comprises the following steps: adding 4-methylpyridine and (3-bromopropyl)trimethylammonium bromide to anhydrous N,N-dimethylformamide, stirring and reacting at 95-105° C. for 1.5-2.5 hours, and then filtering, washing and drying the reaction solution to obtain the Py-Br.

5. The preparation method according to claim 4, characterized in that: The molar ratio of 4-methylpyridine to (3-bromopropyl)trimethylammonium bromide is 1:1 to 1.

1.

6. The preparation method according to claim 2, characterized in that: In step (3), the organic solvent is anhydrous ethanol; the catalyst is piperidine; and the inert gas is nitrogen.

7. The preparation method according to claim 6, characterized in that: In step (3), the molar ratio of BTD-CHO, Py-Br and piperidine is 1:3.5-4.0:0.01-0.

02.

8. The preparation method according to claim 6, characterized in that: In step (3), the concentration of BTD-CHO in anhydrous ethanol is 0.01 to 0.02 M.

9. Use of the water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells as claimed in claim 1 in imaging for distinguishing living cells from dead cells.

10. Use of the water-soluble two-photon fluorescent probe for distinguishing living cells from dead cells as claimed in claim 1 in cell fluorescence imaging.

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