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

Abstract

The application discloses a kind of high signal-to-noise ratio detection biological in-vivo, in-vitro mitochondrial viscosity change fluorescence probe development and its application, it is related to fluorescence probe technical field.The chemical name of the fluorescence probe is (E)-5,6-dichloro-1,3-diethyl-2-(8-hydroxyju Longoni 9-vinyl) benzimidazole-3-iodonium.The fluorescence probe has the characteristics of low dyeing concentration, fast dyeing time, high biocompatibility, specific target, high signal-to-noise ratio, can be used for dyeing mitochondria in extrachromosomal living cell, mitochondria viscosity monitoring in extrachromosomal living cell, preparation for product and monitoring product of biological in-vivo mitochondrial viscosity change, the fluorescence probe JL-JC is a powerful tool for real-time, in-situ tracking mitochondrial dynamic change and monitoring mitochondrial viscosity change, helps mitochondrial dynamics and its potential application in drug development, provides important reference for in-depth understanding of mitochondrial related diseases.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, specifically to the development and application of a high signal-to-noise ratio fluorescent probe for detecting changes in mitochondrial viscosity in vivo and in vitro. Background Technology

[0002] Mitochondrial metabolic homeostasis is essential for the normal functioning of eukaryotic cells. As cells expand and eventually divide, they require more "fuel," and therefore more mitochondria. As indispensable energy generators for maintaining tissue homeostasis and pathways for programmed apoptosis, the core functions of mitochondria necessitate strict control over their quality and quantity. Among the many indicators used to assess mitochondria, viscosity stands out as a key parameter, representing the diffusion rate of various species within the mitochondria. Furthermore, the biodynamics of many bioactive macromolecules, such as protein folding, signal transduction, and enzyme catalysis, typically exhibit stable viscosity variations under normal conditions. Therefore, changes in mitochondrial viscosity can provide clues to the state and function of these organelles. Conversely, abnormal changes in mitochondrial viscosity can disrupt cellular function, leading to various diseases. Therefore, real-time tracking and monitoring of mitochondrial viscosity changes are necessary for the research and diagnosis of diseases related to mitochondrial viscosity. However, the lack of mature technologies and methods in this process remains an ongoing challenge.

[0003] Fluorescence microscopy with fluorescent probes is considered one of the most attractive methods in biosensing, enabling real-time visualization of information 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 offers advantages such as ease of operation, high sensitivity, high spatiotemporal resolution, and strong real-time in-situ imaging capabilities. When combined with fluorescence lifetime microscopy, super-resolution fluorescence microscopy, and two-photon imaging microscopy, it allows for more convenient and non-destructive detection of relevant molecules in organisms, showing promising applications in the biomedical field.

[0004] Changes in mitochondrial viscosity are frequently associated with tumors, atherosclerosis, and other diseases. In recent years, many fluorescent probes have been developed to detect disease-related intracellular mitochondrial viscosity. Although probes have made some progress in reporting changes in intracellular mitochondrial viscosity, viscosity changes under complex in vivo environments have not been thoroughly studied.

[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 viscosity changes of mitochondria in cells and organisms under different conditions, and to observe the dynamic processes of mitochondria in situ. Summary of the Invention

[0006] To address the aforementioned limitations of existing technologies, the present invention aims to develop and apply a high signal-to-noise ratio fluorescent probe for detecting changes in mitochondrial viscosity in vivo and in vitro. The high signal-to-noise ratio mitochondrial viscosity-responsive fluorescent probe obtained by this invention is chemically named (E)-5,6-dichloro-1,3-diethyl-2-(2-(8-hydroxyjulonidin-9-vinyl)-benzimidazole-3-iodide, abbreviated as JL-JC. It can detect mitochondria in extrachromosomal live cells and, with excellent photostability, can monitor mitochondrial breakage and fusion in situ. It can sensitively reflect changes in mitochondrial viscosity in live cells caused by drug induction and environmental changes. Furthermore, the fluorescent probe can be used to prepare products for mitochondrial staining in vivo and for monitoring changes in mitochondrial viscosity in vivo. Due to its molecular structure, the fluorescent probe is sensitive to environmental viscosity in vivo and in vitro, exhibiting a signal-to-noise ratio superior to commercially available mitochondrial probes.

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

[0008] In a first aspect, the present invention provides a high signal-to-noise ratio mitochondrial viscosity-responsive fluorescent probe, the chemical structure of which 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-hydroxyjulonidin-9-vinyl)-benzimidazole-3-iodide, abbreviated as JL-JC.

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

[0012] The fluorescent probe exhibits a sensitive response to environmental viscosity, with a fluorescence emission intensity increasing tens of times in solution testing. The fluorescent probe molecule has a cationic salt structure, enabling rapid targeting of mitochondria in living cells via electrostatic interactions. Compared to commercial mitochondrial probes, the fluorescent probe molecule has a higher signal-to-noise ratio, allowing for clearer imaging of mitochondria in living cells. Furthermore, the fluorescent probe accurately reflects changes in mitochondrial viscosity in both in vitro living cells and in vivo organisms, and with its excellent photostability, it allows for real-time observation of dynamic changes in mitochondria within living cells.

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

[0014] (1) Mix 5,6-dichloro-2-methylbenzimidazole and iodoethane and dissolve them in acetonitrile. React the mixture and wash the reaction product to obtain compound C1.

[0015] (2) After mixing compound C1 and 8-hydroxyjulonidin-9-carboxaldehyde, dissolve them in ethanol to obtain a mixture. Then add piperidine and react. After the reaction is complete, remove the ethanol and purify to obtain the fluorescent probe.

[0016] Preferably, in step (1), the ratio of the amount of 5,6-dichloro-2-methylbenzimidazole, iodoethane and acetonitrile added 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℃ and the reaction time is 10-14h.

[0018] Preferably, in step (2), the ratio of compound C1, 8-hydroxyjulonidin-9-carboxaldehyde and piperidine added 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℃ and the reaction time is 10-14h.

[0020] The chemical reaction formulas involved in the above preparation method are shown below:

[0021]

[0022] A third aspect of the present invention provides the application of the above-mentioned high signal-to-noise ratio mitochondrial viscosity-responsive fluorescent probe on mitochondria in stained live cells.

[0023] Preferably, the live 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, causing the mitochondria to display a green fluorescent pseudocolor.

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

[0026] Further preferred, 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 within the cell can be clearly observed. Compared with commercial mitochondrial probes MTDR and MTG, the fluorescent probe JL-JC of this invention has a higher imaging signal-to-noise ratio.

[0028] Specifically, commercially available mitochondrial probes MTDR and MTG exhibit high background signals during confocal imaging, with signal-to-noise ratios (SNRs) of 4.3 and 23.3 times, respectively, in the labeled region. In contrast, the SNR of the fluorescent probe JL-JC of this invention is 37 times. Therefore, the imaging SNR capability of probe JL-JC is significantly higher than that of probes MTDR and MTG. The superior SNR imaging capability of probe JL-JC enables long-range tracking of dynamic changes in mitochondria under a fixed field of view. Furthermore, probe JL-JC exhibits good biocompatibility.

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

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

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

[0032] In a fifth aspect, the present invention provides the application of the above-described high signal-to-noise ratio mitochondrial viscosity-responsive fluorescent probe in viscosity response.

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

[0034] Specifically, in vitro spectroscopy revealed that the fluorescence emission intensity of the fluorescent probe JL-JC of this invention significantly increased with increasing glycerol content in solvents with different water-to-glycerol ratios. This indicates its sensitive response to environmental viscosity. When cells were treated with different concentrations of nystatin to induce an increase in mitochondrial viscosity, the fluorescence intensity of the probe was significantly enhanced in cells treated with nystatin compared to the control group (without nystatin), and this enhancement was positively correlated with the nystatin concentration. Furthermore, low-temperature treatment of the same batch of cells also resulted in increased fluorescence intensity due to increased mitochondrial viscosity.

[0035] In a sixth aspect, the present invention provides the application of the above-described high signal-to-noise ratio mitochondrial viscosity-responsive fluorescent probe in the preparation of in vivo mitochondrial staining products and products for monitoring changes in mitochondrial viscosity in vivo.

[0036] Preferably, the organism is a zebrafish.

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

[0038] The beneficial effects of this invention are:

[0039] This invention discloses a high signal-to-noise ratio, viscosity-responsive mitochondrial long-range tracking fluorescent probe JL-JC. Utilizing the -180mV potential of the mitochondrial inner membrane, JL-JC targets mitochondria via electrostatic interactions, exhibiting a superior signal-to-noise ratio compared to commercial probes MTDR and MTG. Furthermore, the JL-JC probe requires low cell staining concentrations, has a rapid staining time, excellent biocompatibility, and demonstrates a sensitive response to mitochondrial viscosity only in complex environments.

[0040] The fluorescent probe JL-JC of this invention can detect mitochondria in extrachromosomal live cells and, under the premise of excellent photostability, can monitor the dynamic changes of mitochondrial breakage and fusion in live cells in situ. Notably, the fluorescent probe JL-JC can accurately reflect changes in mitochondrial viscosity in live cells caused by drug treatment or environmental changes; furthermore, it can be used to prepare in vivo mitochondrial staining products and products for monitoring changes in mitochondrial viscosity in vivo. Therefore, the fluorescent probe JL-JC of this invention can serve as a unique potential tool for detecting changes in mitochondrial viscosity in organisms and provides new ideas for subsequent research on rotor-type mitochondrial viscosity-responsive probes. Attached Figure Description

[0041] Figure 1(a) Absorption spectrum of fluorescent probe JL-JC (10 μM) in solutions of different polarities; (b) Fluorescence emission spectrum of fluorescent probe JL-JC (10 μM) in solutions of different polarities; (c) Fluorescence spectrum of fluorescent probe JL-JC (10 μM) in glycerol-water solutions of different concentrations; (d) Linear fitting curve of fluorescence intensity of fluorescent probe JL-JC (10 μM) at 560 nm versus viscosity of glycerol solution in H2O; (e) Fluorescence emission of fluorescent probe JL-JC (10 μM) at 560 nm 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 Fluorescent images obtained under a confocal microscope after staining cells with different concentrations of the fluorescent probe JL-JC.

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

[0045] Figure 5 (a) Confocal images obtained by restaining H9c2 cells with 1 μM fluorescent probe JL-JC for 10 min, followed by probe counterstaining with mitochondrial probe (MTDR), lysosomal probe (LTDR), and nuclear probe (Hoechst 33342), respectively; (b) Figure 5 (a) Correlation between fluorescence intensity and distance distribution of fluorescent probe JL-JC and mitochondrial probe, enzyme probe, and nuclear probe in the region indicated by the white arrow in the Merged image.

[0046] Figure 6 (a) Confocal images of H9c2 cells stained with mitochondrial probes JL-JC, MTDR, and MTG, obtained under a microscope; (b) Figure 6 (a) The signal-to-noise ratio statistics calculated from the yellow highlighted area.

[0047] Figure 7 Microscopic images of H9c2 cells after staining with 1 μM JL-JC for 10 min and washing off excess staining solution with PBS. (a) shows the mitochondrial fragmentation process in the magnified area, and (b) shows the dynamic fusion process of mitochondria in the magnified area.

[0048] Figure 8 (a) Confocal microscopy images of cell pathological models established with different concentrations (10 μM, 20 μM) of nystatin using the fluorescent probe JL-JC, and cells cultured under normal conditions (0 μM), after staining under the same conditions; (b) Figure 8 (a) Statistical analysis of the average fluorescence intensity of fluorescent probe JL-JC in cells.

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

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

[0051] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0052] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0053] All reagents used in the examples and test cases are commercially available and commonly used reagents, and all testing instruments are commercially available and commonly used instruments. Their specific sources will not be repeated here.

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

[0055] The reaction equation for the preparation of the fluorescent probe JL-JC is shown below:

[0056]

[0057] The specific preparation method is as follows:

[0058] (1) Mix 2 mmol of 5,6-dichloro-2-methylbenzimidazole and 2.2 mmol of iodoethane and dissolve them in 10 mL of acetonitrile. Heat the mixture under reflux at 80 °C for 12 h to obtain the reaction product. Wash the reaction product three times with petroleum ether to obtain compound C1.

[0059] (2) 1 mmol of compound C1 and 1.5 mmol of 8-hydroxyjulonidin-9-carboxaldehyde were mixed and dissolved in ethanol to obtain a mixture. Then 100 μL of piperidine was added and the mixture was stirred 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 vacuum distillation to obtain the crude product. The crude product was purified by column chromatography to obtain 137 mg of purple-red solid, which is the fluorescent probe JL-JC, with a yield of 23.5%.

[0060] The prepared fluorescent probe JL-JC was analyzed by proton, carbon, and mass spectrometry. The simplified spectral 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 Properties Testing of Fluorescent Probe JL-JC in Different Solvents

[0063] Six 10 μM JL-JC solutions were prepared using six solvents of different polarities: toluene, tetrahydrofuran, acetonitrile, methanol, dimethyl sulfoxide, and water. The UV absorption spectra of JL-JC in these different solvents were obtained using a HITACHI U-2910 UV spectrophotometer. Figure 1As shown in (a), the fluorescence emission spectrum of JL-JC was obtained using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp under excitation at 470 nm, as follows. Figure 1 As shown in (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. The fluorescence emission spectra of JL-JC were obtained using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp at 470 nm excitation. Figure 1 As shown in (c); a mathematical model based on the Hoffmann equation was 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 follows. Figure 1 As shown in (d);

[0065] Buffer solutions with different pH values ​​(3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) were prepared. Fluorescence emission spectra of JL-JC were obtained using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp at 470 nm excitation. A dotted-line plot of the fluorescence intensity at 560 nm was then created. Figure 1 As shown in (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 a JL-JC solution with a concentration of 10 μM was finally prepared using the above solutions. The fluorescence emission spectrum of JL-JC was obtained using a HITACHI F-2700 fluorescence spectrophotometer equipped with a 450W Xe lamp under excitation at 470 nm, and a bar graph was plotted based on the fluorescence intensity at 560 nm. Figure 1 As shown in (f).

[0067] Depend on Figure 1The results showed that, despite differences in solution polarity, the maximum absorption wavelength of the probe was between 470-500 nm, and the fluorescence emission peak was between 560-580 nm. Fluorescence emission measurements in solutions with different glycerol percentages revealed that the fluorescence emission intensity of the probe was extremely low in aqueous solution. However, the fluorescence emission intensity increased by 51.4 times with increasing glycerol percentage, indicating that in glycerol-water solutions with different volume percentages, the fluorescence emission intensity of the fluorescent probe JL-JC significantly increased with increasing glycerol volume percentage. 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. Furthermore, in complex environments containing multiple ions, the fluorescence emission intensity of JL-JC remained almost unchanged. This demonstrates that the response of the fluorescent probe JL-JC to viscosity is unaffected by pH and other complex analytes in the solution.

[0068] Therefore, it can be seen that, due to the ICT effect, the probe JL-JC exhibits a sensitive response to environmental viscosity, and can respond to minute changes in environmental viscosity through variations in fluorescence emission intensity. Notably, this viscosity response is unaffected by environmental pH and complex ions.

[0069] Experimental Example 2: Preparation of Live Cell Samples for Testing

[0070] H9c2 cells were cultured in high-glucose medium (H-DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. They were passaged every 2-3 days in a 37°C, 5% CO2 saturated humidity incubator. Once the cells reached the logarithmic growth phase, they were grafted onto coverslips for culture: ① Coverslips were immersed in anhydrous ethanol for 30 min, dried under an alcohol lamp, and placed in disposable 35mm culture dishes for later use; ② The confluent cells in 100mL cell culture flasks were washed three times with PBS, digested with 1mL of 0.25% trypsin for 3-5 minutes, the trypsin was carefully poured off, fresh culture medium was added, and the cells were repositioned and counted. The cell density was controlled by the amount of culture medium added, aiming for a final cell concentration of 1×10⁶ cells / mL. 5 Then, the cells were seeded into the culture dishes containing coverslips and placed in a 5% CO2 incubator at 37°C to allow the cells to adhere 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 effect of JL-JC on cell viability was investigated using the standard MTT assay. Specifically,

[0073] H9c2 cells were seeded into 96-well plates after 48 hours of growth and cultured for 24 hours. JL-JC solutions at concentrations of 100 nM, 200 nM, 500 nM, 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM were added to each well as an experimental group (100 μL), while the negative control group received dye-free DMEM. Cells were incubated at 37°C and 5% CO2 for 24 hours. Then, MTT (5 mg / mL in DMEM) was added to each well. After incubation at 37°C for 4 hours, 100 μL of DMSO was added to dissolve the purple crystals. After 20 minutes of incubation, the optical density was read at 490 nm using a microplate reader. The experiment was repeated three times. The results are shown below. Figure 2 As 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 over 90%, and even after incubation with a 20 μM JL-JC solution for 24 hours, the survival rate of H9c2 cells still exceeds 70%. This demonstrates that the JL-JC probe has good biocompatibility.

[0075] Experiment Example 4: Staining ability of different staining concentrations of probe JL-JC on live H9c2 cells

[0076] H9c2 cells from 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 then imaged using a fluorescence confocal microscope. The results are as follows: Figure 3 As shown.

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

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

[0079] H9c2 cells cultured in the same batch were stained with 1 μM JL-JC and immediately observed under a microscope. Confocal images were obtained at different time points, and the average fluorescence intensity was statistically analyzed. Results are as follows: Figure 4 As shown.

[0080] Depend on Figure 4As can be seen, a weak fluorescence signal can be observed in the cells at 4 min, and the filamentous structure in the cells is clearly visible at 10 min. Extending the staining time to 15 min and observing the intracellular fluorescence signal, and statistically analyzing the average fluorescence intensity, revealed that the fluorescence signal in the cells hardly changed after 10 min. Therefore, the optimal staining time for probe JL-JC is 10 min.

[0081] Experiment 6: Co-localization experiment of probe JL-JC with commercial mitochondrial probe MTDR, lysosomal probe LTDR, and nuclear probe Hoechst33342

[0082] First, a DMSO solution of 1 mM probe JL-JC was prepared as the stock solution. After the cell culture dish was confluent, the cells were stained with commercially available probes (MTDR, LTDR: 200 nM, 20 min; Hoechst 33342: 5 μM, 20 min). After staining, excess probe was washed away. Then, the cells were incubated again with 1 μM JL-JC for 10 min, and excess probe was washed away after staining. The staining was observed under a laser confocal fluorescence microscope, and the changes in fluorescence intensity of the two channels in the white arrow area were statistically analyzed (for imaging 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 as follows: Figure 5 As shown.

[0083] Depend on Figure 5 As can be seen, in live H9c2 cells, the fluorescence signal of probe JL-JC in the 550-650nm channel overlaps well with the fluorescence signal of MTDR in the 655-755nm channel, with a co-localization coefficient as high as 0.90; however, it shows almost no overlap with the fluorescence signals of MTDR channels Hoechst33342, with Pearson coefficients of 0.34 and 0.04, respectively. Furthermore, the fluorescence intensity changes in the arrow regions also show that the fluorescence signal positions of JL-JC and MTDR almost completely overlap. This indicates that probe JL-JC can specifically stain mitochondria in live cells, rather than other organelles.

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

[0085] Live H9c2 cells cultured in the same batch were stained and incubated with different probes: 1 μM JL-JC for 10 min; 200 nM MTDR for 20 min; and 200 nM MTG for 20 min. After incubation, the cells were washed twice with PBS and then imaged under a microscope. The results are as follows: Figure 6 As shown.

[0086] The excitation wavelengths of JL-JC, MTDR, and MTG are 488 nm, 635 nm, and 488 nm, respectively, and their light-gathering ranges are 550-650 nm, 655-755 nm, and 490-590 nm, respectively. Figure 6 As can be seen, JL-JC has a 2-fold to 9-fold improvement in imaging capability compared to commercially available mitochondrial probes. This demonstrates that JL-JC has a superior signal-to-noise ratio imaging capability compared to commonly used commercial mitochondrial probes.

[0087] Experimental Example 8: Real-time observation of dynamic changes in cells under a microscope after staining with probe JL-JC.

[0088] H9c2 cells were incubated in cell culture dishes for 48 hours, stained with 1 μM probe JL-JC for 10 minutes, washed three times with PBS buffer, and the dynamic changes of mitochondria within the cells were observed under a fixed microscope. Confocal images were obtained at different time points. The results are as follows: Figure 7 As shown.

[0089] Depend on Figure 7 As can be seen, the filamentous structure of mitochondria is clearly visible during imaging, 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 a pathological cell model treated with nystatin

[0091] H9c2 cells from the same batch were pretreated with different concentrations of nystatin (0 μM, 10 μM, 20 μM) for 30 min to induce changes in mitochondrial viscosity. The pretreated cells were then stained sequentially with the probe JL-JC (1 μM, 30 min), and rinsed twice with PBS after staining. The luminescence of JL-JC was observed under identical microscopic imaging conditions. Results are as follows: Figure 8 As 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 continuously increases and exhibits concentration dependence. This indicates that JL-JC can respond to changes in mitochondrial viscosity in cells through changes in fluorescence intensity.

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

[0094] H9c2 cells from the same batch were cultured in two ways: half were pre-chilled at 0°C for 1 hour and then stained with the JL-JC probe; the other half were pre-treated at 37°C for 1 hour and then stained with JL-JC in the same manner. Microscopic imaging parameters were identical, and the fluorescence of JL-JC in the two pre-treated cells was observed. Results are as follows: Figure 9 As shown.

[0095] Depend on Figure 9 It can be seen that the mitochondrial fluorescence intensity of pre-cooled cells was significantly enhanced after JL-JC staining compared to normal cells. This indicates that the JL-JC probe can respond sensitively to changes in cellular viscosity caused by environmental factors.

[0096] Experimental Example 11: Zebrafish Experimental Test

[0097] Three-day-old zebrafish juveniles from the same batch were stained with the probe JL-JC (5 μM, 1 h). After staining, the culture medium was replaced with a probe-free medium, and fish anesthetic was added to anesthetize the live fish. The staining phenomenon was then observed under a microscope. In addition, zebrafish were pretreated with 10 μM and 20 μM nystatin for 1 h to induce changes in mitochondrial viscosity in the juveniles. They were then stained with JL-JC (5 μM, 1 h) and incubated under the same conditions, followed by confocal imaging. The results are as follows: Figure 10 As shown.

[0098] Depend on Figure 10 As can be seen, relatively weak fluorescence was observed in normally cultured zebrafish. After treatment with nystatin, the mitochondrial viscosity in zebrafish increased, and the fluorescence of JL-JC also showed a significant concentration-dependent increase. These experimental results indicate that the probe JL-JC has low biotoxicity, can stain mitochondria in living organisms, and can reflect changes in viscosity in living organisms through changes in fluorescence.

[0099] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

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

2. A method for preparing the high signal-to-noise ratio mitochondrial viscosity-responsive fluorescent probe according to claim 1, characterized in that, Includes the following steps: (1) 5,6-Dichloro-1-ethyl-2-methylbenzimidazole and iodoethane were mixed and dissolved in acetonitrile. The reaction was carried out, and the reaction product was washed to obtain compound C1. The structural formula of compound C1 is: ; (2) After mixing compound C1 and 8-hydroxyjulonidin-9-carboxaldehyde, dissolve them in ethanol to obtain a mixture. Then add piperidine and react. After the reaction is complete, remove the ethanol and purify to obtain the fluorescent probe.

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

4. The preparation method according to claim 2, characterized in that, In step (2), the ratio of compound C1, 8-hydroxyjulonidin-9-carboxaldehyde and piperidine added is (0.8-1.2) mmol:(1.0-2.0) mmol:(90-110) μL; the reaction temperature is 80-90℃ and the reaction time is 10-14h.

5. The use of the fluorescent probe of claim 1 in the preparation of products for staining mitochondria in vivo and for monitoring changes in mitochondrial viscosity in vivo.

Images