Development and application of high-signal-to-noise-ratio fluorescent probe for detecting in-vivo and in-vitro mitochondrial viscosity change of organism

Abstract

The invention discloses development and application of a high-signal-to-noise-ratio fluorescent probe for detecting in-vivo and in-vitro mitochondrial viscosity changes of a living body, and relates to the technical field of fluorescent probes. The chemical name of the fluorescent probe is (E)-5, 6-dichloro-1, 3-diethyl-2-(8-hydroxyjulolidine-9-vinyl) benzimidazole-3-onium iodide, and the chemical name of the fluorescent probe is (E)-5, 6-dichloro-1, 3-diethyl-2-(8-hydroxyjulolidine-9-vinyl) benzimidazole-3-onium iodide. The fluorescent probe has the characteristics of low staining concentration, short staining time, high biocompatibility, specific targeting and high signal-to-noise ratio, can be used for monitoring the viscosity of mitochondria in chromosome in-vitro living cells and the viscosity of mitochondria in in-vitro living cells, and can be used for preparing products for staining mitochondria in organisms and products for monitoring the viscosity change of mitochondria in organisms. The fluorescent probe JL-JC is a powerful tool for real-time and in-situ tracking of mitochondrial dynamic change and monitoring of mitochondrial viscosity change, is beneficial to mitochondrial dynamics and potential application thereof in drug development, and provides important reference for deep understanding of mitochondrial related diseases.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probes, and in particular to the development and application of a fluorescent probe for detecting changes in mitochondrial viscosity in vivo and in vitro with a high signal-to-noise ratio. Background Art

[0002] The metabolic homeostasis of mitochondria is essential for eukaryotic cells to maintain normal function. When cells expand and eventually divide, they require more "fuel" and therefore more mitochondria. As an indispensable energy generator for maintaining tissue homeostasis and a pathway for programmed cell apoptosis, the core function of mitochondria also requires strict control of their quality and quantity. Among the many indicators used to evaluate mitochondria, viscosity becomes a key parameter, which is an indicator of the diffusion rate of various species within mitochondria. In addition, the biological dynamics of a variety of active macromolecules, such as protein folding, signal transduction, and enzyme catalysis, usually maintain stable viscosity changes under normal conditions. Therefore, changes in mitochondrial viscosity can provide clues to understand the state and function of these organelles. On the contrary, abnormal changes in mitochondrial viscosity may disrupt cell function and lead to the occurrence of various diseases. Therefore, real-time tracking and monitoring of mitochondrial viscosity changes are necessary for studying and diagnosing diseases related to mitochondrial viscosity. However, the lack of mature technologies and means in this process is a continuous challenge.

[0003] Fluorescence microscopy of fluorescent probes is considered one of the most attractive methods in biosensing, which can display information in real time at the cellular and biological levels. Traditional detection methods, such as chemiluminescence, electroanalysis, and Raman spectroscopy, are limited by complex sample structures, expensive instruments, and difficulties in real-time signal analysis. In contrast, fluorescence microscopy has the characteristics of simple operation, high sensitivity, high temporal and spatial resolution, and strong real-time in situ imaging capabilities. In cooperation with fluorescence lifetime microscopy, super-resolution fluorescence microscopy, and two-photon imaging microscopy, it can more conveniently perform non-destructive detection of related molecules in organisms, and has good application prospects in the biomedical field.

[0004] Changes in mitochondrial viscosity are often closely related to tumors, atherosclerosis and other diseases. In recent years, many fluorescent probes have been developed to detect disease-related intracellular mitochondrial viscosity. Although some progress has been made in the probes to feedback changes in intracellular mitochondrial viscosity, the viscosity changes in the complex environment of the body have not been studied in detail.

[0005] Therefore, in order to solve the above problems, it is necessary to develop a high signal-to-noise ratio fluorescent probe to observe the changes in mitochondrial viscosity in cells and organisms under different conditions, and to observe the dynamic process of mitochondria in situ. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a development and application of a fluorescent probe for detecting changes in mitochondrial viscosity in vivo and in vitro with a high signal-to-noise ratio. The chemical name of the mitochondrial viscosity-responsive fluorescent probe with a high signal-to-noise ratio prepared by the present invention is (E)-5.6-dichloro-1,3-diethyl-2-(2-(8-hydroxyl-9-vinyl)-benzimidazole-3-iodide, referred to as JL-JC. It can detect mitochondria in extrachromosomal living cells, and can monitor the breakage and fusion of mitochondria in situ under the premise of excellent photostability, and can sensitively feedback changes in mitochondrial viscosity in living cells caused by drug induction and environmental changes. At the same time, the fluorescent probe is also used to prepare products for mitochondrial staining in vivo and products for monitoring changes in mitochondrial viscosity in vivo. Affected by the molecular structure, the fluorescent probe has a sensitive response to the environmental viscosity in vivo and in vitro, and has a signal-to-noise ratio that is superior to that of commercial mitochondrial probes.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect of the present invention, a mitochondrial viscosity-responsive fluorescent probe with a high signal-to-noise ratio is provided, wherein the chemical structure of the fluorescent probe is shown in formula (I):

[0009]

[0010] The chemical name of the fluorescent probe is: (E)-5.6-dichloro-1,3-diethyl-2-(2-(8-hydroxyl-9-vinyl)-benzimidazole-3-iodide, abbreviated as JL-JC.

[0011] Preferably, the maximum absorption wavelength of the fluorescent probe is in the range of 470-500 nm, and the maximum emission wavelength is in the range of 560-580 nm.

[0012] The fluorescent probe shows a sensitive response to the environmental viscosity, and the fluorescence emission intensity increases by dozens of times in the solution test. The fluorescent probe molecule has a cationic salt structure and can quickly target mitochondria in living cells through electrostatic interactions. Compared with commercial mitochondrial probes, the fluorescent probe molecule has a higher signal-to-noise ratio and can image mitochondria in living cells more clearly. In addition, the fluorescent probe can correctly feedback the changes in mitochondrial viscosity in living cells in vitro and in vivo, and with the support of excellent photostability, the dynamic changes of mitochondria in living cells can be observed in real time through the probe.

[0013] The second aspect of the present invention provides a method for preparing the above-mentioned mitochondrial viscosity-responsive fluorescent probe with a high signal-to-noise ratio, comprising the following steps:

[0014] (1) mixing 5,6-dichloro-2-methylbenzimidazole and iodoethane, dissolving the mixture in acetonitrile, reacting the mixture, and washing the reaction product to obtain compound C1;

[0015] (2) Compound C1 and 8-hydroxyljulidine-9-carboxaldehyde are mixed and dissolved in ethanol to obtain a mixture, and piperidine is added to react. After the reaction is completed, the ethanol is removed and the mixture is purified to obtain a fluorescent probe.

[0016] Preferably, in step (1), the ratio of the added amounts of 5,6-dichloro-2-methylbenzimidazole, iodoethane and acetonitrile is (1.8-2.2) mmol: (2.0-2.4) mmol: (8-12) mL.

[0017] Preferably, in step (1), the reaction temperature is 80-90° C. and the reaction time is 10-14 h.

[0018] Preferably, in step (2), the added amount ratio of the compound C1, 8-hydroxyljulidine-9-carboxaldehyde and piperidine is (0.8-1.2) mmol: (1.0-2.0) mmol: (90-110) μL.

[0019] Preferably, in step (2), the reaction temperature is 80-90° C. and the reaction time is 10-14 h.

[0020] The chemical reaction formula involved in the above preparation method is as follows:

[0021]

[0022] The third aspect of the present invention provides the use of the above-mentioned mitochondrial viscosity-responsive fluorescent probe with a high signal-to-noise ratio in staining mitochondria in living cells.

[0023] Preferably, the living cells are rat embryonic cardiomyocytes (H9c2).

[0024] Preferably, the excitation wavelength of the fluorescent probe JL-JC is 488 nm, and it emits bright fluorescence in the wavelength range of 550-650 nm, making the mitochondria show a green fluorescent pseudo-color.

[0025] Preferably, the fluorescent probe JL-JC has a lower cell staining concentration and a very fast staining time during the staining of mitochondria in living cells.

[0026] More preferably, the cell staining concentration is 1 μM and the staining time is 10 min.

[0027] When the cell staining concentration is 1 μM and the staining time is 10 min, the filamentous mitochondrial structure in the cell can be clearly observed. Compared with the commercial mitochondrial probes MTDR and MTG, the fluorescent probe JL-JC of the present invention has a higher imaging signal-to-noise ratio.

[0028] Specifically, the commercial mitochondrial probes MTDR and MTG have a high background signal during confocal imaging, and the signal-to-noise ratio of the fluorescence signal in the labeled area is 4.3 times and 23.3 times, respectively, while the signal-to-noise ratio of the background signal of the fluorescent probe JL-JC of the present invention is 37 times. It can be seen that the imaging signal-to-noise ratio capability of probe JL-JC is significantly higher than that of probes MTDR and MTG, and the excellent signal-to-noise ratio imaging capability of probe JL-JC provides the possibility for long-range tracking of dynamic changes of mitochondria under a fixed field of view. In addition, probe JL-JC has good biocompatibility.

[0029] A fourth aspect of the present invention provides the use of the above-mentioned mitochondrial viscosity-responsive fluorescent probe with a high signal-to-noise ratio in in situ monitoring of dynamic changes of mitochondria in living cells.

[0030] Preferably, the living cells are rat embryonic cardiomyocytes, and the dynamic changes of mitochondria include mitochondrial fragmentation and fusion.

[0031] Specifically, after staining the mitochondria in living cells with the fluorescent probe JL-JC and observing them under a fixed field of view of a microscope for 600 seconds, the mitochondrial breakage in the living cells can be clearly observed. At the same time, the fusion of mitochondria in the living cells can also be clearly observed.

[0032] The fifth aspect of the present invention provides the use of the above-mentioned mitochondrial viscosity-responsive fluorescent probe with high signal-to-noise ratio in viscosity response.

[0033] Preferably, the fluorescent probe responds sensitively to viscosity changes caused by living cell pathological models and / or environmental changes.

[0034] Specifically, in vitro spectral tests have shown that in solvents with different water-glycerol ratios, the fluorescence emission intensity of the fluorescent probe JL-JC of the present invention shows a significant enhancement as the proportion of glycerol increases. This shows that it has a sensitive response to environmental viscosity. When cells are treated with different concentrations of nystatin to induce an increase in mitochondrial viscosity, it is found that compared with the fluorescence emission intensity of the control group (without nystatin), the fluorescence brightness of the probe in cells treated with nystatin is significantly enhanced, and is positively correlated with the treatment concentration of nystatin. In addition, the same batch of cells were subjected to low-temperature treatment, and the enhanced fluorescence intensity caused by the increase in mitochondrial viscosity was also observed.

[0035] The sixth aspect of the present invention provides the use of the above-mentioned mitochondrial viscosity-responsive fluorescent probe with a high signal-to-noise ratio in the preparation of in vivo mitochondrial staining products and products for monitoring changes in mitochondrial viscosity in an organism.

[0036] Preferably, the organism is zebrafish.

[0037] Specifically, after 3-day-old zebrafish were treated with different concentrations of nystatin, the fluorescence emission intensity in the zebrafish was significantly enhanced compared with the control group (zebrafish were not treated with nystatin), and the intensity change showed a significant positive correlation with the drug concentration. This shows that probe JL-JC can be used to prepare mitochondrial staining products in vivo and products for monitoring changes in mitochondrial viscosity in vivo.

[0038] Beneficial effects of the present invention:

[0039] The present invention discloses a mitochondrial long-range tracking fluorescent probe JL-JC with high signal-to-noise ratio and viscosity response. The fluorescent probe JL-JC uses the property of the inner membrane of the mitochondria having a potential of -180mV to target the mitochondria through electrostatic action, and has a better signal-to-noise ratio than the commercial probes MTDR and MTG. The fluorescent probe JL-JC of the present invention has a low cell staining concentration, a fast staining time, excellent biocompatibility, and is sensitively responsive only to mitochondrial viscosity in a complex environment.

[0040] The fluorescent probe JL-JC of the present invention can detect mitochondria in extrachromosomal living cells, and under the premise of excellent light stability, it can monitor the dynamic changes of mitochondrial breakage and fusion in living cells in situ. It is worth noting that the fluorescent probe JL-JC can correctly feedback the changes in mitochondrial viscosity of living cells caused by drug treatment or environmental changes; at the same time, it can also be used to prepare mitochondrial staining products in vivo and products for monitoring changes in mitochondrial viscosity in vivo. It can be seen that the fluorescent probe JL-JC of the present invention can be used as a unique potential tool for detecting changes in mitochondrial viscosity in organisms, and also provides new ideas for the subsequent research of rotor-type mitochondrial viscosity response probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1:(a) Absorption spectra of fluorescent probe JL-JC (10μM) in solutions of different polarities; (b) Fluorescence emission spectra of fluorescent probe JL-JC (10μM) in solutions of different polarities; (c) Fluorescence spectra of fluorescent probe JL-JC (10μM) in glycerol-water solutions of different concentrations; (d) Linear fitting curve of fluorescence intensity of fluorescent probe JL-JC (10μM) at 560nm and viscosity of glycerol solution in H2O; (e) Fluorescence emission of fluorescent probe JL-JC (10μM) at 560nm in buffer solutions of different pH values; (f) Fluorescence intensity of fluorescent probe JL-JC in the presence of multiple analytes.

[0042] Figure 2 : Survival rate of H9c2 cells under different incubation concentrations of fluorescent probe JL-JC.

[0043] Figure 3 : Fluorescence images obtained under a confocal microscope after cells were stained with different concentrations of fluorescent probe JL-JC.

[0044] Figure 4 :(a) Fluorescence imaging at different imaging time points Figure 4 (b) Statistics of average fluorescence intensity; (b) After H9c2 cells were stained with 1 μM fluorescent probe JL-JC, they were immediately imaged under a microscope and confocal images obtained at different observation time points.

[0045] Figure 5 :(a) After H9c2 cells were stained with 1 μM fluorescent probe JL-JC for 10 min, the H9c2 cells were counterstained with mitochondrial probe (MTDR), lysosomal probe (LTDR), and nuclear probe (Hoechst 33342) to obtain confocal imaging images; (b) Figure 5 (a) Correlation diagram between the fluorescence intensity and distance distribution of the fluorescent probe JL-JC and the mitochondrial probe, enzymosome probe, and nucleus probe, respectively, in the area indicated by the white arrow in the Merged image.

[0046] Figure 6 :(a) Confocal images obtained under a microscope after H9c2 cells were stained with mitochondrial probes JL-JC, MTDR, and MTG; (b) Figure 6 SNR statistics calculated for the yellow marked area in (a).

[0047] Figure 7 : Microscope images of H9c2 cells stained with 1 μM JL-JC for 10 min, after the excess staining solution was washed away with PBS. (a) shows the mitochondrial fragmentation process in the enlarged area, and (b) shows the dynamic fusion process of mitochondria in the enlarged area.

[0048] Figure 8 :(a) Under the same conditions, confocal microscopy images of the cell pathology model established by different concentrations (10μM, 20μM) of nystatin and cells cultured under normal conditions (0μM) were stained with the fluorescent probe JL-JC; (b) Figure 8 (a) Statistics of the average fluorescence intensity of fluorescent probe JL-JC in cells.

[0049] Fig. 9 :(a) Imaging of cells cultured normally (37℃) and cells pre-cooled (0℃) under a confocal microscope after being stained with the fluorescent probe JL-JC (1μM) for 10 minutes; (b) Statistical graph of the average fluorescence intensity of the fluorescent probe JL-JC in cells.

[0050] Fig.10 : Confocal microscopy images of 3-day-old zebrafish treated with different concentrations of nystatin (0 μM, 10 μM, 20 μM) and then stained with the fluorescent probe JL-JC (5 μM, 1 h). DETAILED DESCRIPTION

[0051] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0052] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0053] The reagents used in the embodiments and test examples are all existing commonly used commercial reagents, and the testing instruments are all existing commonly used commercial instruments, and the specific sources are not repeated here.

[0054] Example 1: Preparation of mitochondrial viscosity-responsive fluorescent probe JL-JC with high signal-to-noise ratio

[0055] The preparation reaction equation of the fluorescent probe JL-JC is as follows:

[0056]

[0057] The specific preparation method is as follows:

[0058] (1) 2 mmol of 5,6-dichloro-2-methylbenzimidazole and 2.2 mmol of iodoethane were mixed and dissolved in 10 mL of acetonitrile, and the mixture was heated under reflux at 80° C. for 12 h to obtain a reaction product, which was then rinsed with petroleum ether three times to obtain compound C1;

[0059] (2) 1 mmol of compound C1 and 1.5 mmol of 8-hydroxyljulidine-9-carboxaldehyde were mixed and dissolved in ethanol to obtain a mixture, and 100 μL of piperidine was added. The mixture was stirred and reacted at 85° C. for 12 h. After the reaction was completed, the mixture was cooled to room temperature and the remaining ethanol was removed by distillation under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain 137 mg of a purple-red solid, which was the fluorescent probe JL-JC, with a yield of 23.5%.

[0060] The prepared fluorescent probe JL-JC was analyzed by hydrogen spectrum, carbon spectrum and mass spectrum, and the simplified spectrum data are as follows:

[0061] 1 H NMR (400MHz, DMSO-d6): δ (ppm) 9.03 (s, 1H), 8.42 (s, 2H), 7.93 (d, J = 16.2Hz, 1H), 7.44 (s, 1H), 6.92 (d, J = 16.2Hz, 1H), 4.53 (q,J=7.1Hz,4H),3.30-3.23(m,4H),2.69(t,J=6.3Hz,2H),2.61(t,J=6.4Hz,2H),1.91-1.83(m,4H),1.45(t,J=7.1Hz,6H). 13 CNMR (101MHz, DMSO-d6): δ (ppm) 154.62, 151.04, 147.91, 143.08, 131.50, 128.85, 126.03, 115.26, 114.4 6,110.99,107.33,96.14,50.01,49.28,41.63,27.35,21.79,21.50,20.94,14.54.HRMS(m / z):[JL-JC-I - ] + calculated for C 25 H 28 N3Cl2O + ,456.1604;found,456.1610.

[0062] Experimental Example 1: Optical property test of fluorescent probe JL-JC in different solvents

[0063] Six solvents with different polarities, namely toluene, tetrahydrofuran, acetonitrile, methanol, dimethyl sulfoxide and water, were used to prepare 10 μM JL-JC solutions. The UV absorption spectra of JL-JC in solvents with different polarities were obtained using a HITACHI U-2910 UV spectrophotometer. Figure 1(a) as shown; the fluorescence emission spectrum of JL-JC was obtained under 470nm excitation using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp, as shown in FIG. Figure 1 (b)

[0064] A 10 μM JL-JC solution was prepared using a water-glycerol mixed solvent containing different volumes (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) of glycerol, and the fluorescence emission spectra of JL-JC were obtained under excitation at 470 nm using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450 W Xe lamp, as shown in FIG. Figure 1 (c) As shown; the mathematical model based on the Hoffmann equation is used to establish the correlation between the glycerol viscosity coefficient (η) and the fluorescence intensity at 560 nm, expressed as log F = 1.01 + 0.57logη, as shown in Figure 1 (d)

[0065] Buffer solutions with different pH values ​​(3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) were prepared, and the fluorescence emission spectra of JL-JC were obtained under 470 nm excitation using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450 W Xe lamp, and a dot-line graph was made based on the fluorescence intensity at 560 nm, as shown in the figure. Figure 1 (e)

[0066] Different ion solutions (KCl, NaCl, Zn(NO3)2, CuSO4, Cu(NO3)2, KNO3, CaCl2, MgCl2, Ni(NO3)2, K2CO3, Na2HPO4, Cys) with a concentration of 0.1 mM were prepared, and the above solutions were used to finally prepare a JL-JC solution with a concentration of 10 μM. The fluorescence emission spectrum of JL-JC was obtained under excitation at 470 nm using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450 W Xe lamp, and a bar graph was made of the fluorescence intensity at 560 nm, as shown in FIG. Figure 1 (f) as shown.

[0067] Depend on Figure 1The results show that despite the different polarities of the solutions, the maximum absorption wavelength of the probe is between 470-500nm, and the fluorescence emission peak is between 560-580nm. The fluorescence emission in solutions with different glycerol ratios was measured and it was found that the fluorescence emission intensity of the probe in aqueous solution was extremely low, and the fluorescence emission intensity increased by 51.4 times with the increase of glycerol ratio, that is, in glycerol-water solutions with different volume ratios, when the volume ratio of glycerol increased, the fluorescence emission intensity of the fluorescent probe JL-JC was significantly enhanced. When the solution pH increased from 3.0 to 9.0, the fluorescence emission intensity of JL-JC fluctuated slightly within a relatively stable range, and in a complex environment with multiple ions, the fluorescence emission intensity of JL-JC hardly changed. It can be seen that the response of the fluorescent probe JL-JC to viscosity is not interfered by pH and other complex analytes in the body.

[0068] It can be seen that due to the ICT effect, the probe JL-JC has a sensitive response to the environmental viscosity and can respond to slight changes in the environmental viscosity through changes in fluorescence emission intensity. It is worth mentioning that this viscosity response is not affected by environmental pH and complex ions.

[0069] Experimental Example 2: Preparation of living cell samples for testing

[0070] H9c2 cells were cultured in a high-glucose medium (H-DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The cells were subcultured every 2-3 days in an incubator at 37°C and 5% CO2 with saturated humidity. When the cells grew to the logarithmic phase, they were cultured by grafting: ① Soak the coverslip in anhydrous ethanol for 30 minutes, dry it with an alcohol lamp, and place it in a disposable 35mm culture dish for later use; ② Wash the cells in the 100mL cell bottle three times with PBS, digest them with 1mL 0.25% trypsin for 3-5 minutes, carefully pour out the trypsin, add fresh culture medium, blow evenly, and count the cells. The cell density is controlled by the amount of culture medium added, so that the final cell concentration is 1×10 per ml. 5 Then, the cells were inoculated into the culture dish containing the coverslip and placed in a 5% CO2 incubator at 37°C to allow the cells to adhere to the wall and grow. H9c2 cells incubated for 48 hours can be used for subsequent imaging experiments.

[0071] Experimental Example 3: Cytotoxicity test of probe JL-JC

[0072] The standard MTT assay was used to study the effect of JL-JC on cell viability.

[0073] After 48 hours of growth, H9c2 cells were seeded into 96-well plates and cultured for 24 hours. JL-JC solution concentrations of 100nM, 200nM, 500nM, 1μM, 2μM, 5μM, 10μM, and 20μM were added to the 96-well plates and used as experimental groups (100μL), and the negative control group was added with DMEM without dye. The cells were incubated at 37°C and 5% CO2 for 24 hours. MTT (5mg / mL in DMEM) was then added to each well. After incubation at 37°C for 4 hours, 100μL DMSO was added to dissolve the purple crystals. After incubation for 20 minutes, the optical density reading at a wavelength of 490nm was read using an enzyme reader. The experiment was repeated 3 times. The experimental results are as follows Figure 2 shown.

[0074] Depend on Figure 2 It can be seen that at a working concentration of 1 μM, the survival rate of H9c2 cells is above 90%, and even after incubation with a JL-JC solution with a concentration of 20 μM for 24 hours, the survival rate of H9c2 cells is still over 70%. This shows that the JL-JC probe has good biocompatibility.

[0075] Experimental Example 4: Testing the staining ability of probes JL-JC at different staining concentrations on live H9c2 cells

[0076] H9c2 cells cultured in the same batch were stained with different concentrations (0 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM) of JL-JC and incubated in a cell culture incubator for 30 min. The cells were imaged using a fluorescence confocal microscope. Figure 3 shown.

[0077] Depend on Figure 3 It can be seen that when the concentration of probe JL-JC staining is 0.2 μM, a weak green fluorescence signal can be observed in the cells. When the concentration increases to 1 μM, JL-JC can clearly stain the filamentous structure in living cells. Therefore, 1 μM was selected as the working concentration of probe JL-JC for staining mitochondria in living H9c2 cells.

[0078] Experimental Example 5: Testing the staining ability of probe JL-JC at different staining times on live H9c2 cells

[0079] The H9c2 cells cultured in the same batch were stained with 1 μM JL-JC and immediately observed under a microscope to obtain confocal imaging images at different time points, and the average fluorescence intensity was statistically analyzed. Figure 4 shown.

[0080] Depend on Figure 4It can be seen that a weak fluorescence signal can be observed in the cells at 4 minutes, and the filamentous structure in the cells is clearly visible at 10 minutes. The fluorescence signal in the cells is observed by increasing the staining time to 15 minutes and statistically analyzing the average fluorescence intensity. It is found that the fluorescence signal in the cells has almost no change after 10 minutes. Therefore, the optimal staining time for probe JL-JC is 10 minutes.

[0081] Experimental Example 6: Co-localization experiment of probe JL-JC with commercial mitochondrial probe MTDR, lysosomal probe LTDR and cell nucleus probe Hoechst33342

[0082] First, prepare a 1mM DMSO solution of the probe JL-JC as the mother solution. After the cell culture dish is full of cells, stain the cells with commercial probes (MTDR, LTDR: 200nM, 20min; Hoechst33342: 5μM, 20min), and wash away the excess probes after staining. Then incubate the cells again with 1μM JL-JC for 10min, and wash away the excess probes after staining. The cell culture dish was observed for staining under a laser confocal fluorescence microscope and the changes in fluorescence intensity of the two channels in the white arrow area were statistically analyzed (in order to image the channels, JL-JC was given red pseudo-color light, MTDR and LTDR were given green pseudo-color light, and Hoechst 33342 was given blue pseudo-color light). The results are shown in the figure. Figure 5 shown.

[0083] Depend on Figure 5 It can be seen that in living H9c2 cells, the light of probe JL-JC in the 550-650nm channel can overlap well with the fluorescence signal of MTDR in the 655-755nm channel, and the colocalization coefficient is as high as 0.90; there is almost no overlap with the fluorescence signal of the LTDR channel Hoechst33342 channel, and the Pearson coefficients are 0.34 and 0.04 respectively. And from the change of fluorescence intensity in the arrow area, it can be observed that the positions of the fluorescence signals of JL-JC and MTDR are almost completely overlapped. This shows that probe JL-JC can specifically stain mitochondria in living cells instead of other organelles.

[0084] Experimental Example 7: Signal-to-noise ratio imaging capability test of probe JL-JC and commercial mitochondrial probes (MTDR, MTG)

[0085] The same batch of living H9c2 cells were stained and incubated with different probes: 1μM JL-JC for 10min; 200nM MTDR for 20min; 200nM MTG for 20min. After incubation, they were rinsed twice with PBS and then imaged under a microscope. The results are shown in Figure 2. Figure 6 shown.

[0086] The excitation wavelengths of JL-JC, MTDR, and MTG are 488nm, 635nm, and 488nm, respectively, and the light receiving ranges are 550-650nm, 655-755nm, and 490-590nm, respectively. Figure 6 It can be seen that compared with commercial mitochondrial probes, JL-JC has an imaging capability that is 2 times or even 9 times higher. This shows that compared with the commonly used commercial mitochondrial probes on the market, JL-JC has a better signal-to-noise ratio imaging capability.

[0087] Experimental Example 8: After staining cells with probe JL-JC, the dynamic changes of cells were observed in real time under a microscope

[0088] After incubation in a cell culture dish for 48 h, H9c2 cells were stained with 1 μM probe JL-JC for 10 min and then washed three times with PBS buffer solution. The dynamic changes of mitochondria in cells were observed under a fixed field of view under a microscope and confocal images were obtained at different time points. Figure 7 shown.

[0089] Depend on Figure 7 It can be seen that during the imaging process, the filamentous structure of mitochondria is clearly visible and dynamic processes such as breakage and fusion can be observed in the area indicated by the arrow.

[0090] Experimental Example 9: Response of probe JL-JC to pathological cell model treated with nystatin

[0091] The same batch of H9c2 cells were treated with different concentrations of nystatin (0μM, 10μM, 20μM) for 30 minutes in advance to induce changes in mitochondrial viscosity. The pretreated cells were stained with probe JL-JC (1μM, 30min) in turn, and rinsed twice with PBS after staining. The luminescence of JL-JC was observed under the same imaging conditions of the microscope. The results are shown in Figure 2. Figure 8 shown.

[0092] Depend on Figure 8 It can be seen that with the gradual increase of nystatin treatment concentration, the average fluorescence intensity of probe JL-JC in cells under the same conditions continued to increase and showed concentration dependence, indicating that JL-JC can respond to changes in mitochondrial viscosity in cells through changes in fluorescence intensity.

[0093] Experimental Example 10: Cell Imaging Test Probe JL-JC's Sensitive Response to Environmental Viscosity

[0094] Half of the H9c2 cells cultured in the same batch were pre-cooled in a 0℃ environment for 1 hour, and then stained with probe JL-JC; the other half of the cells were pre-treated in a 37℃ incubator for 1 hour and then stained with JL-JC. The microscope imaging parameters were set the same, and the fluorescence of JL-JC in the two different pre-treated cells was observed. The results are shown in Figure 2. Fig. 9 shown.

[0095] Depend on Fig. 9 It can be seen that the mitochondrial fluorescence intensity of the pre-cooled cells after JL-JC staining is significantly enhanced compared with that of normal cells, indicating that the probe JL-JC can respond sensitively to changes in cell viscosity caused by the environment.

[0096] Experimental Example 11: Zebrafish Experimental Test

[0097] Three-day-old zebrafish larvae cultured in the same batch were selected for staining with the probe JL-JC (5μM, 1h). After staining, the culture medium without the probe was replaced with fish anesthetic to anesthetize the live fish, and then the staining phenomenon of the probe was observed under a microscope. In addition, the zebrafish were pretreated with 10μM and 20μM nystatin for 1h, respectively, to induce changes in mitochondrial viscosity in the larvae. Similarly, confocal imaging observation was performed under the same conditions after staining and incubation with JL-JC (5μM, 1h). The results are shown in Figure 2. Fig.10 shown.

[0098] Depend on Fig.10 It can be seen that relatively weak fluorescence can be observed in zebrafish cultured normally. After being treated with nystatin, the viscosity of mitochondria in zebrafish increased, and the fluorescence of JL-JC also showed a significant concentration-dependent increase. The above experimental results show that the probe JL-JC has low biological toxicity, can stain mitochondria in living organisms, and can reflect changes in viscosity in organisms through changes in fluorescence.

[0099] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A mitochondrial viscosity-responsive fluorescent probe with high signal-to-noise ratio, characterized in that: The chemical structure of the fluorescent probe is shown in formula (I):

2. A method for preparing a mitochondrial viscosity-responsive fluorescent probe with a high signal-to-noise ratio according to claim 1, characterized in that: The following steps are involved: (1) mixing 5,6-dichloro-2-methylbenzimidazole and iodoethane, dissolving the mixture in acetonitrile, reacting the mixture, and washing the reaction product to obtain compound C1; (2) Compound C1 and 8-hydroxyljulidine-9-carboxaldehyde are mixed and dissolved in ethanol to obtain a mixture, and piperidine is added to react. After the reaction is completed, the ethanol is removed and the mixture is purified to obtain a fluorescent probe.

3. The preparation method according to claim 2, characterized in that: In step (1), the added amount ratio of 5,6-dichloro-2-methylbenzimidazole, iodoethane and acetonitrile is (1.8-2.2) mmol: (2.0-2.4) mmol: (8-12) mL; the reaction temperature is 75-85° C., and the reaction time is 10-14 h.

4. The preparation method according to claim 2, characterized in that: In step (2), the addition ratio of the compound 1, 8-hydroxyljulidine-9-carboxaldehyde and piperidine is (0.8-1.2) mmol: (1.0-2.0) mmol: (90-110) μL; the reaction temperature is 80-90° C., and the reaction time is 10-14 h.

5. Use of the mitochondrial viscosity-responsive fluorescent probe with high signal-to-noise ratio according to claim 1 in staining mitochondria in living cells.

6. The use according to claim 5, characterized in that The living cells are rat embryonic cardiomyocytes.

7. Use of the mitochondrial viscosity-responsive fluorescent probe with a high signal-to-noise ratio as claimed in claim 1 in in situ monitoring of dynamic changes of mitochondria in living cells.

8. Use of the mitochondrial viscosity-responsive fluorescent probe with high signal-to-noise ratio according to claim 1 in viscosity response.

9. The use according to claim 8, characterized in that The fluorescent probe sensitively responds to viscosity changes caused by living cell pathological models and / or environmental changes.

10. Use of the fluorescent probe according to claim 1 in preparing a product for staining mitochondria in an organism and a product for monitoring changes in mitochondrial viscosity in an organism.

Images