CN111333645A - Fluorescent probe for marking cytoskeleton - Google Patents

Abstract

The present invention provides a fluorescent probe for cytoskeleton labeling. The chemical structural features of the fluorescent probe are as follows: the fluorophore parent is a naphthalimide dye, and one end of the fluorescent probe is connected with a succinimide active group for labeling an antibody , and the specific structure of the fluorescent probe is shown in formula (1). Compared with other commonly used probes such as rhodamine, fluorescein, and BODIPY, the probe has better photostability and is more suitable for super-resolution microscopy imaging. The probe is labeled on the antibody corresponding to the cytoskeleton, which can label the cytoskeleton. and clear images.

Description

A Fluorescent Probe for Cytoskeleton Labeling

technical field

The invention belongs to the field of biological analysis and detection, in particular to a fluorescent probe used for cytoskeleton labeling.

Background technique

As the basic unit of biological activities, cells are the focus of attention in the field of biology. Using microscopic imaging technology to observe the internal components of cells in order to obtain a more complete understanding of the subcellular structure and function is a scientific research problem that scientists have been excavating for decades. Due to the limitation of light wave diffraction, traditional optical microscope imaging has reached the limit of resolution, and cannot observe and study nano-scale organelles and cellular components in cells. In recent years, with the emergence of new fluorescent molecules and the reform and innovation of digital imaging technology, laser scanning confocal fluorescence imaging technology came into being. This technology does not require fixation and inactivation of cells. The laser continuously scans different sections of fluorescently labeled cells layer by layer, and the obtained optical sections are reconstructed by a computer to reconstruct a three-dimensional model, which can accurately locate the components in living cells in real time. The three-dimensional spatial arrangement greatly improves the resolution of microscopic imaging, so it is widely used to explore the laws of various life activities in cells. The cytoskeleton with a diameter between 10 and 20 nanometers in the cell is the latest organelle discovered in the cell. Its diameter cannot be observed with a conventional optical microscope. With the help of the platform of laser scanning confocal fluorescence imaging, in recent years scientists The understanding of the structure and function of the cytoskeleton has taken a new level.

Cytoskeleton refers to a complex network system composed of proteins such as microfilaments, microtubules and intermediate fibers in the nucleus and the inner side of the cell membrane of eukaryotic cells. Its main function is to mechanically support the normal morphological structure of eukaryotic cells and protect organelles from cells. External injury, etc. In recent years, research evidence combined with confocal fluorescence imaging has shown that the cytoskeleton is also involved in the regulation of many important life activities in cells, such as pulling chromosomes during mitosis, providing directional tracks for intracellular material transport and signal transduction, and participating in cell apoptosis. Apoptosis, assisting the movement and migration of immune response cells in the body, and related to the tissue invasion and metastasis of tumor cells. At present, we do not have a complete understanding of the function of the cytoskeleton, so it is worth exploring further with the help of higher-resolution imaging methods. At this stage, the method to dynamically observe the cytoskeleton is mainly to label microfilament tubulin with specific fluorescent dyes, use excitation light of different wavelengths, and process the light to focus on a certain focal plane of the cell. The dye is excited to emit fluorescence at the corresponding wavelength, and then the optical signal is received, analyzed and fed back by the computer, thereby realizing the localization and imaging of the fluorescence in the cytoskeleton. In order to achieve the best signal-to-noise ratio and higher resolution for fluorescence imaging, increasing the intensity of the excitation light has become the first choice. However, the traditional fluorescent dyes are affected by the photobleaching effect. Under the continuous scanning of high-intensity excitation light in the confocal system, the light absorption tends to be saturated, and the molecules in the excited state in the fluorophore are irreversibly destroyed, so that the fluorescence signal increases with the irradiation time. Amplitude attenuation severely affects cytoskeleton localization imaging. Therefore, it is urgent to develop a new type of photostable fluorescent molecular probe targeting the cytoskeleton, reducing the photobleaching effect of fluorescent molecules during laser confocal imaging, and improving the high signal-to-noise ratio of cytoskeleton laser scanning confocal imaging. , high resolution, to enable continuous and dynamic exploration of more physiological functions of the cytoskeleton.

SUMMARY OF THE INVENTION

The invention provides a fluorescent probe for cytoskeleton labeling; the stability of the probe is stronger than that of traditional rhodamine, fluorescein, BODIPY and other dyes. Imaging under a microscope yields a clear structure.

The present invention is a fluorescent probe for cytoskeleton labeling, and the molecular structural formula of the probe is as follows:

A method for synthesizing a fluorescent probe for cytoskeleton labeling, the synthesizing steps are as follows:

(1) Synthesis of intermediate BCOMe-NBr:

Dissolve 4-bromo-5-nitro-1,8-naphthalene anhydride, 4-aminobutyric acid ethyl ester hydrochloride, and triethylamine in absolute ethanol. The reaction solution was heated to 40-90 °C and stirred for 1-24 h. After cooling the reaction solution to room temperature, after removing the solvent under reduced pressure, the silica gel column is separated, and the volume ratio is 1:0.25～6 of dichloromethane and petroleum ether or the volume ratio of 1:0～0.01 of dichloromethane and methanol As eluent, the solvent was removed under reduced pressure to obtain off-white solid BCOMe-NBr;

(2) Synthesis of probe BCOOH-DAC:

BCOMe-NBr was dissolved in ethylene glycol methyl ether, and cyclohexanediamine was added thereto. The reaction solution was slowly heated to 100-140° C. and reacted under nitrogen protection for 10-24 h. The solvent was removed under reduced pressure, separated on a silica gel column, and the solvent was removed by using dichloromethane and methanol with a volume ratio of 50 to 400:1 as the eluent to obtain a brown-yellow solid BCOMe-DAC;

BCOMe-DAC methanol, and 2M sodium hydroxide solution was added dropwise to the reaction solution. After reacting at room temperature for 1-3 hours, methanol was distilled off under reduced pressure, filtered, washed with water, and the filter cake was dried to obtain BCOOH-DAC;

(3) Synthesis of fluorescent dyes with NHS active groups

After dissolving BCOOH-DAC and DCC in dry N,N-dimethylformamide, stir at room temperature for 10-40min. N-hydroxysuccinimide was dissolved in 1 mL of dry N,N-dimethylformamide and added to the reaction solution. After 2-5 hours, the solvent was removed under reduced pressure, and the silica gel column was used for separation, and dichloromethane and ethyl acetate in a volume ratio of 4-20:1 were used as the eluent to remove the solvent to obtain the fluorescent dye NHSB-DAC with NHS active group;

In step (1), the mass ratio of 4-bromo-5-nitro-1,8-naphthalene anhydride, 4-aminobutyric acid ethyl ester hydrochloride, and triethylamine is 1:1-3:1-3; The mass ratio of 4-bromo-5-nitro-1,8-naphthalene anhydride to the volume of ethanol is 1:20-80g/mL;

In step (2), wherein, the mass ratio of BCOMe-NBr to cyclohexanediamine is 1:1-3; the mass ratio of BCOMe-NBr to ethylene glycol methyl ether is 10-50:1 g/mL;

The mass ratio of BCOMe-DAC to methanol is 10-20:1 g/mL; the mass ratio of BCOMe-DAC to 2M sodium hydroxide solution is 10-20:1 g/mL; the mass of BCOMe-DAC is 10-20:1 g/mL; The volume ratio of water is 10-20:1g/mL;

In step (3), the mass ratio of BCOOH-DAC, DCC and NHS is 1:1-5:1-10; the mass ratio of BCOOH-DAC to N,N-dimethylformamide is 10-20:1 (g:mL).

The fluorescent probe used for cytoskeleton labeling is characterized in that the probe is used for cytoskeleton labeling, and the labeling method is as follows: (1) a monoclonal antibody corresponding to a cytoskeleton protein is used to label cells; (2) the The fluorescent probe is labeled with the corresponding polyclonal antibody; (3) the fluorescently labeled polyclonal antibody is labeled with the monoclonal antibody, and then the corresponding cytoskeletal protein is labeled.

The method for labeling a cytoskeleton with a fluorescent probe is characterized in that: the cytoskeleton marked by the method is microtubules, microfilaments and intermediate fibers.

The method for labeling a cytoskeleton with a fluorescent probe is characterized in that: the cytoskeleton marked by the method can be used for imaging with a confocal microscope or a super-resolution microscope.

The advantages and beneficial effects of the present invention are:

The stability of the probe is stronger than that of traditional dyes such as rhodamine, fluorescein, and BODIPY. It can be used to label cytoskeletal proteins and image them under a confocal microscope or super-resolution microscope to obtain clear structural images.

Description of drawings

The hydrogen spectrum of the fluorescent probe NHSB-DAC prepared in Example 3 of Fig. 1;

Figure 2 High-resolution mass spectrum of the fluorescent probe NHSB-DAC prepared in Example 3;

FIG. 3 is the photostability detection pattern of the fluorescent probe NHSB-DAC prepared in Example 3 described in Example 4. FIG.

FIG. 4 is an SDS-PAGE electrophoresis image after labeling the polyclonal antibody goat anti-mouse IgG with the fluorescent probe NHSB-DAC prepared in Example 3 described in Example 5. FIG.

FIG. 5 is a fluorescent confocal imaging of the NHSB-DAC-labeled polyclonal antibody goat anti-mouse IgG-labeled filaggrin prepared in Example 5 described in Example 6. FIG.

FIG. 6 is a fluorescent confocal imaging of NHSB-DAC-labeled polyclonal antibody goat anti-mouse IgG-labeled tubulin prepared in Example 5 described in Example 7. FIG.

Detailed ways

The following examples will further illustrate the present invention, but do not limit the present invention accordingly.

Example 1

Synthesis of Intermediate 6-(N-(4-Bromo-5-nitro-1,8-naphthalimide))aminobutyric Acid Ethyl Ester (BCOMe-NBr)

4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 80 mL of ethanol, and to it was added ethyl 4-aminobutyrate hydrochloride (1.00 g, 6.00 mmol) ) and 1.00 g of triethylamine. After reacting at 90° C. for 1 h, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:petroleum ether=3:1, V/V) to obtain 608 mg of a white solid with a yield of 45%.

Synthesis of BCOOH-DAC

BCOMe-NBr (200 mg, 0.46 mmol) was dissolved in 20 mL of ethylene glycol methyl ether, and 600 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 100 °C and reacted for 24 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=80:1, V/V) to obtain 103 mg of a dark yellow solid with a yield of 53%.

BCOMe-DAC (80 mg, 0.19 mmol) was dissolved in 40 mL of methanol, and 8 mL of 2M sodium hydroxide solution was slowly added dropwise to the reaction solution. After the dropwise addition, the reaction solution was reacted at room temperature for 1 h, methanol was distilled off under reduced pressure, the turbid solution was filtered, washed with 8 mL of water, and the filter cake was dried to obtain 65 mg of BCOOH-DAC with a yield of 87%.

Synthesis of NHSB-DAC

BCOOH-DAC (50 mg, 0.12 mmol) and dicyclohexylcarbene (DCC) (50 mg, 0.24 mmol) were dissolved in 2.5 mL of N,N-dimethylformamide and stirred at room temperature for 40 min. N-hydroxysuccinimide (50 mg, 0.44 mmol) was dissolved in 1 mL of N,N-dimethylformamide, and then added dropwise to the reaction solution. After 5 h, the solvent was removed under reduced pressure, and the mixture was separated on a silica gel column. Dichloromethane:ethyl acetate=5:1 was used as the eluent, and the solvent was removed to obtain 55 mg of a khaki solid with a yield of 89%.

After detection, its structure is shown in the above formula NHSB-DAC, the absorption wavelength of the dye in water is 481nm, the fluorescence emission wavelength is 489nm, and the fluorescence quantum yield is 0.78.

Example 2

Synthesis of Intermediate 6-(N-(4-Bromo-5-nitro-1,8-naphthalimide))aminobutyric Acid Ethyl Ester (BCOMe-NBr)

4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 20 mL of ethanol, and thereto was added ethyl 4-aminobutyrate hydrochloride (3.00 g, 18.0 mmol) ) and 3.00 g of triethylamine. After reacting at 40° C. for 24 hours, the solvent was distilled off under reduced pressure, and the residue was separated by silica gel column (dichloromethane:petroleum ether=3:1, V/V) to obtain 500 mg of white solid with a yield of 37%. Its nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.71 (d, J=7.8 Hz, 1H), 8.52 (d, J=7.9 Hz, 1H), 8.22 (d, J=7.9 Hz, 1H), 7.93 (d ,J=7.8Hz,1H),4.25(t,J=7.1Hz,2H),4.10(q,J=7.1Hz,2H),2.44(t,J=7.4Hz,2H),2.09(p,J = 7.3Hz, 2H), 1.24 (t, J = 7.1Hz, 3H). ¹³ C NMR (101MHz, CDCl ₃ )δ172.72, 162.85, 162.09, 151.33, 136.00, 132.40, 131.30, 130.57, 125.65, 124.24, 123.56, 122.36, 121.24, 60.53, 40.11, 31.82, 23.20, 14.23.

Its high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₁₈ H ₁₆ BrN ₂ O ₆ [M+H] ⁺ 435.0192, actual value 435.0193.

Synthesis of BCOOH-DAC

BCOMe-NBr (200 mg, 0.46 mmol) was dissolved in 4 mL of ethylene glycol methyl ether, and 200 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 140 °C and reacted for 10 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=80:1, V/V) to obtain 86 mg of a dark yellow solid with a yield of 44%. Its nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.04 (d, J=8.6 Hz, 2H), 7.51 (s, 2H), 6.82 (d, J=8.7 Hz, 2H), 4.00 (dt, J= 14.1, 5.3Hz, 4H), 3.14 (d, J=8.8Hz, 2H), 2.30 (t, J=7.5Hz, 2H), 2.19 (d, J=11.7Hz, 2H), 1.89–1.80 (m, 2H), 1.73 (d, J=6.8Hz, 2H), 1.31 (dt, J=30.1, 15.8Hz, 4H), 1.14 (t, J=7.1Hz, 3H). ¹³ C NMR (101MHz, DMSO-d6 )δ172.88,163.49,154.56,134.79,133.35,110.58,107.74,106.44,60.18,59.48,38.55,32.07,31.80,23.75,23.63,14.53.

BCOMe-DAC (200 mg, 0.48 mmol) was dissolved in 10 mL of methanol, and 10 mL of 2M sodium hydroxide solution was slowly added dropwise to the reaction solution. After the dropwise addition, the reaction solution was reacted at room temperature for 3 hours, methanol was distilled off under reduced pressure, the turbid solution was filtered, washed with 10 mL of water, and the filter cake was dried to obtain 65 mg of BCOOH-DAC with a yield of 87%. Its nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ 12.01(s, 1H), 8.04(d, J=8.6Hz, 2H), 7.51(s, 2H), 6.82(d, J=8.7Hz, 2H) ,3.99(dd,J＝9.2,4.6Hz,2H),3.15(d,J＝9.1Hz,2H),2.21(dd,J＝16.7,9.3Hz,4H),1.88–1.76(m,2H), 1.72(d, J=8.0Hz, 2H), 1.42-1.19(m, 4H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ174.48, 163.50, 154.57, 134.79, 133.36, 110.58, 107.76, 106.47, 59.50, 47.97,33.82,32.08,31.90,25.79,24.93,23.86,23.63.

Synthesis of NHSB-DAC

BCOOH-DAC (20 mg, 0.05 mmol) and dicyclohexylcarbene (DCC) (100 mg, 0.48 mmol) were dissolved in 2 mL of N,N-dimethylformamide and stirred at room temperature for 10 min. N-hydroxysuccinimide (200 mg, 1.74 mmol) was dissolved in 1 mL of N,N-dimethylformamide, and then added dropwise to the reaction solution. After 2 h, the solvent was removed under reduced pressure, and the mixture was separated on a silica gel column. Dichloromethane:ethyl acetate=5:1 was used as the eluent, and the solvent was removed to obtain 19 mg of a khaki solid with a yield of 77%. Its nuclear magnetic spectrum hydrogen spectrum is shown in Figure 1, and the nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.19-7.93(m, 2H), 7.53(s, 2H), 6.83(d, J=8.7Hz, 2H), 4.05(t, J=6.5Hz, 2H), 3.15(d, J＝9.2Hz, 1H), 2.80(s, 4H), 2.72(t, J＝7.7Hz, 2H), 2.19(d, J＝11.4Hz, 2H), 1.97–1.88( m, 2H), 1.73 (d, J=7.2Hz, 2H), 1.31 (dt, J=28.8, 15.2Hz, 4H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ170.66, 169.11, 163.47, 154.65, 134.87,133.42,110.63,107.66,106.43,59.48,38.35,32.07,28.69,25.90,23.73,23.63.

The high-resolution mass spectrometry is shown in Figure 2, and the specific data are as follows: The theoretical value of high-resolution mass spectrometry C ₂₆ H ₂₇ N ₄ O ₆ [M+H] ⁺ 491.1931, the actual value 491.1981.

After detection, its structure is shown in the above formula NHSB-DAC, the absorption wavelength of the dye in water is 481nm, the fluorescence emission wavelength is 489nm, and the fluorescence quantum yield is 0.78.

Example 3

Synthesis of Intermediate 6-(N-(4-Bromo-5-nitro-1,8-naphthalimide))aminobutyric Acid Ethyl Ester (BCOMe-NBr)

4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 20 mL of ethanol, and thereto was added ethyl 4-aminobutyrate hydrochloride (3.00 g, 18.0 mmol) ) and 3.00 g of triethylamine. After reacting at 60°C for 18 hours, the solvent was distilled off under reduced pressure, and the residue was separated by silica gel column (dichloromethane:petroleum ether=3:1, V/V) to obtain 743 mg of white solid with a yield of 55%. Its nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.71 (d, J=7.8 Hz, 1H), 8.52 (d, J=7.9 Hz, 1H), 8.22 (d, J=7.9 Hz, 1H), 7.93 (d ,J=7.8Hz,1H),4.25(t,J=7.1Hz,2H),4.10(q,J=7.1Hz,2H),2.44(t,J=7.4Hz,2H),2.09(p,J = 7.3Hz, 2H), 1.24 (t, J = 7.1Hz, 3H). ¹³ C NMR (101MHz, CDCl ₃ )δ172.72, 162.85, 162.09, 151.33, 136.00, 132.40, 131.30, 130.57, 125.65, 124.24, 123.56, 122.36, 121.24, 60.53, 40.11, 31.82, 23.20, 14.23.

Its high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₁₈ H ₁₆ BrN ₂ O ₆ [M+H] ⁺ 435.0192, actual value 435.0193.

Synthesis of BCOOH-DAC

BCOMe-NBr (200 mg, 0.46 mmol) was dissolved in 4 mL of ethylene glycol methyl ether, and 200 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 140 °C and reacted for 8 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=80:1, V/V) to obtain 90 mg of a dark yellow solid with a yield of 46%. Its nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.04 (d, J=8.6 Hz, 2H), 7.51 (s, 2H), 6.82 (d, J=8.7 Hz, 2H), 4.00 (dt, J= 14.1, 5.3Hz, 4H), 3.14 (d, J=8.8Hz, 2H), 2.30 (t, J=7.5Hz, 2H), 2.19 (d, J=11.7Hz, 2H), 1.89–1.80 (m, 2H), 1.73 (d, J=6.8Hz, 2H), 1.31 (dt, J=30.1, 15.8Hz, 4H), 1.14 (t, J=7.1Hz, 3H). ¹³ C NMR (101MHz, DMSO-d6 )δ172.88,163.49,154.56,134.79,133.35,110.58,107.74,106.44,60.18,59.48,38.55,32.07,31.80,23.75,23.63,14.53.

BCOMe-DAC (200 mg, 0.48 mmol) was dissolved in 10 mL of methanol, and 10 mL of 2M sodium hydroxide solution was slowly added dropwise to the reaction solution. After the dropwise addition, the reaction solution was reacted at room temperature for 2 h, methanol was distilled off under reduced pressure, the turbid solution was filtered, washed with 10 mL of water, and the filter cake was dried to obtain 62 mg of BCOOH-DAC with a yield of 83%. Its nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ 12.01(s, 1H), 8.04(d, J=8.6Hz, 2H), 7.51(s, 2H), 6.82(d, J=8.7Hz, 2H) ,3.99(dd,J＝9.2,4.6Hz,2H),3.15(d,J＝9.1Hz,2H),2.21(dd,J＝16.7,9.3Hz,4H),1.88–1.76(m,2H), 1.72(d, J=8.0Hz, 2H), 1.42-1.19(m, 4H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ174.48, 163.50, 154.57, 134.79, 133.36, 110.58, 107.76, 106.47, 59.50, 47.97,33.82,32.08,31.90,25.79,24.93,23.86,23.63.

Synthesis of NHSB-DAC

BCOOH-DAC (20 mg, 0.05 mmol) and dicyclohexylcarbene (DCC) (80 mg, 0.38 mmol) were dissolved in 2 mL of N,N-dimethylformamide and stirred at room temperature for 10 min. N-hydroxysuccinimide (150 mg, 1.31 mmol) was dissolved in 1 mL of N,N-dimethylformamide, and then added dropwise to the reaction solution. After 2 h, the solvent was removed under reduced pressure, and the mixture was separated on a silica gel column. Dichloromethane:ethyl acetate=5:1 was used as the eluent, and the solvent was removed to obtain 20 mg of a khaki solid with a yield of 81%. Its nuclear magnetic spectrum hydrogen spectrum is shown in Figure 1, and the nuclear magnetic spectrum hydrogen spectrum and carbon spectrum data are as follows:

¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.19-7.93(m, 2H), 7.53(s, 2H), 6.83(d, J=8.7Hz, 2H), 4.05(t, J=6.5Hz, 2H), 3.15(d, J＝9.2Hz, 1H), 2.80(s, 4H), 2.72(t, J＝7.7Hz, 2H), 2.19(d, J＝11.4Hz, 2H), 1.97–1.88( m, 2H), 1.73 (d, J=7.2Hz, 2H), 1.31 (dt, J=28.8, 15.2Hz, 4H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ170.66, 169.11, 163.47, 154.65, 134.87,133.42,110.63,107.66,106.43,59.48,38.35,32.07,28.69,25.90,23.73,23.63.

After detection, its structure is shown in the above formula NHSB-DAC, the absorption wavelength of the dye in water is 481nm, the fluorescence emission wavelength is 489nm, and the fluorescence quantum yield is 0.78.

Example 4

In vitro stability detection of the fluorescent probe NHSB-DAC prepared in Example 3

NHSB-DAC, Rhodamine 123, Fluorescein, and BODIPY were dissolved in PBS solution to a final concentration of 10 μM. Irradiated under strong light, and detected its fluorescence intensity at 0, 0.5, 1.5, 2, 3, 4, 6, and 8 hours, respectively. Taking time as the abscissa and the normalized value of the maximum emission intensity as the ordinate, Figure 3 is obtained.

The stability test chart of NHSB-DAC, rhodamine 123, fluorescein, and BODIPY is shown in Figure 3: the emission intensity of rhodamine 123, fluorescein, and BODIPY decreased significantly with time, while the amplitude of the decrease in the emission intensity of NHSB-DAC was obvious. lower than the other three fluorescent dyes, proving that it is more photostable.

Example 5

The fluorescent probe NHSB-DAC prepared in Example 3 was labeled with polyclonal antibody and purified

NHSB-DAC was dissolved in DMSO solution to prepare a 1 mM stock solution for later use. 10 μL of NHSB-DAC stock solution was added to 100 μL solution containing polyclonal antibody goat anti-mouse IgG (0.5 mg/mL), left standing for 1 h at room temperature, and excess fluorescent small molecules were removed by Sephadex G-25. The polyclonal antibody labeled NHSB-DAC was run on 12% SDS-PAGE, firstly imaged with UV irradiation, and then with Coomassie brilliant blue staining to obtain Figure 4

The fluorescence imaging of the polyclonal antibody labeled with the fluorescent probe NHSB-DAC is shown in Figure 4: the first line in Figure 4 is the protein marker; the second line is the polyclonal antibody labeled with NHSB-DAC. Coomassie brilliant blue staining was consistent with the band under UV irradiation, which proved that the fluorescent probe had been labeled on the polyclonal antibody.

Example 6

The NHSB-DAC-labeled polyclonal antibody prepared in Example 5 was used in the filaggrin fluorescence imaging experiment. The NHSB-DAC-labeled polyclonal antibody was dissolved in an aqueous solution to prepare a 0.5 mg/mL stock solution for later use. HeLa cells (proliferating epidermal cancer cells) were plated in a petri dish containing 1 mL of DMED high-glucose medium containing 10% fetal bovine serum, and cultured at 37°C and 5% carbon dioxide to a cell density of about 70%. The cells were gently washed twice with PBS buffer, fixed with 4% paraformaldehyde for 30 min, discarded the fixative and washed three times with PBS, then permeabilized with 0.2% TritonX-100 for 20 min and washed three times with PBS, 5 min each time , and then blocked with 5% BSA blocking solution for 20 minutes and washed 3 times with PBS. 200 μL of PBS solution containing anti-β-filaggrin monoclonal antibody (about 10 μg/mL) was added and incubated overnight at 4°C. The next day, after washing three times with PBS, 200 μL of PBS solution containing NHSB-DAC-labeled polyclonal antibody (about 10 μg/mL) was added, and incubated at 37° C. for 3 hours. Finally, after washing three times with PBS, the images were imaged under a fluorescence confocal microscope to obtain Figure 5.

Figure 5 shows the fluorescence imaging of filaggrin by NHSB-DAC-labeled polyclonal antibody: Figure 5-a is the imaging of filaggrin, Figure 5-b is the imaging of nuclei labeled with commercial nuclear dyes, Figure 5-c The superimposed images of Fig. 5-a and Fig. 5-b, and the bright-field image of Fig. 5-d.

Example 7

Fluorescence imaging experiment of tubulin by the NHSB-DAC-labeled polyclonal antibody prepared in Example 5 The NHSB-DAC-labeled polyclonal antibody was dissolved in an aqueous solution to prepare a 0.5 mg/mL stock solution for later use. HeLa cells (proliferating epidermal cancer cells) were plated in a petri dish containing 1 mL of DMED high-glucose medium containing 10% fetal bovine serum, and cultured at 37°C and 5% carbon dioxide to a cell density of about 70%. The cells were gently washed twice with PBS buffer, fixed with 4% paraformaldehyde for 30 min, discarded the fixative and washed three times with PBS, then permeabilized with 0.2% TritonX-100 for 20 min and washed three times with PBS, 5 min each time , and then blocked with 5% BSA blocking solution for 20 minutes and washed 3 times with PBS. 200 μL of PBS solution containing anti-α-tubulin monoclonal antibody (about 10 μg/mL) was added and incubated overnight at 4°C. The next day, after washing three times with PBS, 200 μL of PBS solution containing NHSB-DAC-labeled polyclonal antibody (about 10 μg/mL) was added, and incubated at 37° C. for 3 hours. Finally, after washing three times with PBS, the images were imaged under a fluorescence confocal microscope, as shown in Figure 6.

Figure 6 shows the fluorescence imaging of tubulin by NHSB-DAC-labeled polyclonal antibody: Figure 6-a is the imaging of tubulin, Figure 6-b is the imaging of nuclei labeled with commercial nuclear dyes, Figure 6- c is the superimposed image of Fig. 6-a and Fig. 6-b, and Fig. 6-d is the bright-field image.

Claims (8)

1. A fluorescent probe for cytoskeleton labeling, characterized in that the molecular structural formula of the probe is as follows:

2. the method of claim 1, wherein the method comprises the following steps:

(1) synthesis of intermediate BCOMe-NBr:

dissolving 4-bromo-5-nitro-1, 8-naphthalic anhydride, 4-ethyl aminobutyric acid hydrochloride and triethylamine in absolute ethyl alcohol, heating the reaction solution to 40-90 ℃, stirring for 1-24h, cooling the reaction solution to room temperature, removing the solvent under reduced pressure, separating by using a silica gel column, and mixing dichloromethane and petroleum ether in a volume ratio of 1: 0.25-6, or in a volume ratio of: taking dichloromethane and methanol of 0-0.01 as an eluent, and removing the solvent under reduced pressure to obtain an off-white solid BCOMe-NBr;

(2) synthesizing a probe BCOOH-DAC:

dissolving BCOMe-NBr in ethylene glycol monomethyl ether, and adding cyclohexanediamine; slowly heating the reaction liquid to 140 ℃ at 100 ℃, and reacting for 10-24h under the protection of nitrogen; removing the solvent under reduced pressure, separating by using a silica gel column, and removing the solvent by using dichloromethane and methanol in a volume ratio of 50-400: 1 as an eluent to obtain a brown yellow solid BCOMe-DAC;

BCOMe-DAC was dissolved in methanol, and 2M sodium hydroxide solution was added dropwise to the reaction solution. Reacting for 1-3h at room temperature, distilling under reduced pressure to remove methanol, filtering, washing with water, and drying to obtain BCOOH-DAC;

(3) synthesis of fluorescent dye with NHS active group

Dissolving BCOOH-DAC and DCC in dry N, N-dimethylformamide, and stirring at room temperature for 10-40 min; dissolving N-hydroxysuccinimide in 1mL of dry N, N-dimethylformamide, and adding the solution into the reaction solution; and (3) decompressing after 2-5h, removing the solvent, separating by using a silica gel column, and removing the solvent by using dichloromethane and ethyl acetate in a volume ratio of 4-20: 1 as an eluent to obtain the NHSB-DAC (NHS-reactive group).

3. The method for synthesizing a fluorescent probe for cytoskeleton labeling according to claim 1, characterized in that in the step (1), the mass ratio of 4-bromo-5-nitro-1, 8-naphthalic anhydride, ethyl 4-aminobutyric acid hydrochloride and triethylamine is 1:1-3: 1-3; the volume ratio of the mass of the 4-bromo-5-nitro-1, 8-naphthalic anhydride to the volume of the ethanol is 1:20-80 g/mL.

4. The method for synthesizing a fluorescent probe for cytoskeleton labeling according to claim 1, wherein in the step (2), the mass ratio of BCOMe-NBr to cyclohexanediamine is 1: 1-3; the volume ratio of the mass of BCOMe-NBr to ethylene glycol monomethyl ether is 10-50:1 g/mL; the volume ratio of the mass of the BCOMe-DAC to the methanol is 10-20:1 g/mL;

the volume ratio of the mass of the BCOMe-DAC to the 2M sodium hydroxide solution is 10-20:1 g/mL; the ratio of the mass of the BCOMe-DAC to the volume of the water is 10-20:1 g/mL.

5. The method for synthesizing a fluorescent probe for cytoskeleton labeling according to claim 1, wherein in the step (3), the mass ratio of BCOOH-DAC, DCC and NHS is 1:1-5: 1-10; the volume ratio of the mass of BCOOH-DAC to the N, N-dimethylformamide is 10-20:1 g/mL.

6. The use of a fluorescent probe for cytoskeleton labeling according to claim 1, wherein the probe is used for cytoskeleton labeling by the following method:

(1) labeling cells with monoclonal antibodies corresponding to cytoskeletal proteins;

(2) labeling fluorescent probes to corresponding polyclonal antibodies;

(3) the fluorescently-labeled polyclonal antibody labels the monoclonal antibody, and then labels the corresponding cytoskeletal protein.

7. Use of a fluorescent probe for cytoskeletal labeling according to claim 6, wherein: the cytoskeleton marked by the method is a microtubule, a microfilament and a middle fiber.

8. Use of a fluorescent probe for cytoskeletal labeling according to claim 6, wherein: the cytoskeleton marked by the method is used for imaging by a confocal microscope or a super-resolution microscope.

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