A fluorescent probe and its preparation method and application

Abstract

The present invention discloses a fluorescent probe, a preparation method thereof, and an application thereof, and belongs to the technical field of fluorescent probes. The preparation method of the fluorescent probe provided by the present invention comprises the following steps: the preparation method comprises stirring a compound I as shown in I and 6-methoxyquinoline in DMF, and separating and purifying the fluorescent probe compound. The fluorescence of the fluorescent probe synthesized by the present invention can be selectively quenched by chloride ions, and near physiological conditions, the Stern-Volmer constant for chloride ions is not affected by pH. The compound has good Golgi apparatus targeting ability, and its fluorescence intensity changes with the change of chloride ion concentration in the Golgi apparatus, proving that the probe can be used to detect changes in chloride ion concentration in the Golgi apparatus.

Description

Technical Field

[0001] The present invention relates to the technical field of novel small molecule fluorescent probes, and in particular to a fluorescent probe and a preparation method and application thereof. Background Art

[0002] Chloride is the most abundant anion in the human body, distributed throughout all cell types. It plays a crucial role in regulating cell volume, membrane potential, and maintaining cellular pH. Typically, the extracellular chloride concentration (120 mM) is much higher than the cytoplasmic chloride concentration (5-40 mM).

[0003] The Golgi apparatus, also known as the Golgi apparatus or Golgi complex, is a weakly acidic subcellular organelle ubiquitous in eukaryotic cells, with a pH range of 6.0 to 6.5 under physiological conditions. The Golgi apparatus's primary function is to process, sort, and package various proteins synthesized by the endoplasmic reticulum, then transport them to specific locations within the cell or secrete them outside the cell. As the final processing and packaging site for cellular secretions (such as proteins), the Golgi apparatus participates in numerous biological processes, including cell polarization, stress response, directional migration, mitosis, metabolism, autophagy, apoptosis, and DNA repair. When cells are stimulated or damaged by external stimuli, causing changes in the content of substances within the organelle, the organelle's primary function is affected, thereby impacting the overall physiological function of the cell. Imbalances in chloride ion concentration within the Golgi apparatus may impair its ability to precisely process and sort proteins to specific regions of neurons, thereby inducing apoptosis. At the same time, the imbalance of chloride ion concentration causes changes in the pH of the Golgi apparatus, which can directly reduce glycosylation and change the structure and function of the Golgi apparatus, thereby triggering a series of Golgi apparatus-related diseases, such as Alzheimer's disease, cystic fibrosis, Parkinson's disease, and liver disease.

[0004] Therefore, it is of great significance to design and synthesize a fluorescent probe that can detect changes in chloride ion concentration in the Golgi apparatus in real time. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a fluorescent probe that detects chloride ions by quenching fluorescence upon collision with chloride ions. The probe can also specifically target the Golgi apparatus and detect changes in chloride ion concentration within the Golgi apparatus.

[0006] The present invention also provides a method for preparing the fluorescent material.

[0007] The present invention also provides applications of the fluorescent material.

[0008] The first aspect of the present invention provides a fluorescent probe, the structural formula of which is shown in II:

[0009]

[0010] The fluorescent probe according to the first embodiment of the present invention has at least the following beneficial effects:

[0011] The fluorescent probe provided by the present invention uses 6-methoxyquinoline as a chloride ion recognition site and p-aminobenzenesulfonamide as a Golgi apparatus targeting group to synthesize a fluorescent probe. Specifically, the interaction between the quinoline group and the chloride ion causes fluorescence quenching of the quinoline structure, that is, the fluorescence of the fluorescent probe of the present invention can be selectively quenched by chloride ions. The fluorescent probe has a moderate affinity for chloride ions, and the Stern-Volmer constant for chloride ions is not affected by pH. The Stern-Volmer constant is approximately 60M at physiological pH. -1 .

[0012] The fluorescent probe provided by the present invention responds very quickly to chloride ions and has good specificity and anti-interference properties. In addition, the fluorescent probe has good biocompatibility, low toxicity, and little damage to cells.

[0013] The fluorescent probe provided by the present invention has good stability and good Golgi apparatus targeting ability, and the fluorescence intensity of the fluorescent probe changes with the change of chloride ion concentration in the Golgi apparatus.

[0014] Therefore, the probe provided by the present invention detects chloride ions by colliding with chloride ions to cause fluorescence quenching, and can specifically target the Golgi apparatus and detect changes in the chloride ion concentration in the Golgi apparatus.

[0015] A second aspect of the present invention provides a method for preparing a fluorescent material, the method comprising the following steps:

[0016] The preparation method comprises the steps of stirring the compound I shown in FIG1 and 6-methoxyquinoline in DMF to react and separate and purify the fluorescent probe compound;

[0017]

[0018] The method for preparing the fluorescent material according to the second embodiment of the present invention has at least the following beneficial effects:

[0019] The preparation method of the fluorescent probe of the present invention has simple and mild conditions, can be completed in only two steps, and the subsequent processing is also relatively simple.

[0020] According to some embodiments of the present invention, the synthesis method of compound I as shown in I comprises stirring p-aminobenzenesulfonamide and bromoacetyl bromide in acetone for reaction.

[0021] According to some embodiments of the present invention, the compound I is 2-bromo-N-(4-aminosulfonylphenyl)acetamide, with a CAS number of 5332-70-7.

[0022] According to some embodiments of the present invention, the yield of compound I is about 30-36%.

[0023] Preferably, the yield of compound I is about 35%.

[0024] According to some embodiments of the present invention, the molar ratio of p-aminobenzenesulfonamide to bromoacetyl bromide is 1:(0.9-1).

[0025] Preferably, the molar ratio of p-aminobenzenesulfonamide to bromoacetyl bromide is about 1:0.95.

[0026] According to some embodiments of the present invention, the mass volume ratio of p-aminobenzenesulfonamide and acetone is (0.08-0.2) g:1 ml.

[0027] Preferably, the mass volume ratio of p-aminobenzenesulfonamide to acetone is about 0.17 g:1 mL.

[0028] According to some embodiments of the present invention, the stirring reaction temperature in the synthesis method of Compound I is 50-55°C.

[0029] Preferably, the stirring reaction temperature in the synthesis method of compound I is 52°C.

[0030] According to some embodiments of the present invention, the stirring reaction time in the synthesis method of Compound I is 10 to 30 minutes.

[0031] Preferably, the stirring reaction time in the synthesis method of compound I is 20 minutes.

[0032] According to some embodiments of the present invention, the synthesis method of Compound I further comprises the step of adding 10 to 20 mL of water after the stirring reaction, and continuing the stirring reaction for 8 to 12 hours.

[0033] Preferably, the synthesis method of compound I further comprises the step of adding 15 mL of water after the stirring reaction and continuing the stirring reaction for 10 hours.

[0034] According to some embodiments of the present invention, the method for synthesizing compound I further includes a purification and separation operation after the stirring reaction.

[0035] According to some embodiments of the present invention, the purification and separation method is: washing the solid in the system with water at 2-10° C. and then recrystallizing it with ethanol.

[0036] According to some embodiments of the present invention, the solid after recrystallization by ethanol further includes the steps of filtering and vacuum drying.

[0037] According to some embodiments of the present invention, the water comprises at least one of distilled water and deionized water.

[0038] According to some embodiments of the present invention, the temperature of the water is 2 to 10° C. under standard atmospheric pressure.

[0039] Preferably, the temperature of the water is 4°C under standard atmospheric pressure.

[0040] According to some embodiments of the present invention, the molar ratio of compound I to 6-methoxyquinoline is 1:(2-4).

[0041] Preferably, the molar ratio of compound I to 6-methoxyquinoline is about 1:2.

[0042] According to some embodiments of the present invention, the mass volume ratio of the 6-methoxyquinoline to the DMF solution is (0.02-0.09) g:1 ml.

[0043] Preferably, the mass volume ratio of the 6-methoxyquinoline to the DMF solution is about 0.04 g:1 ml.

[0044] According to some embodiments of the present invention, the temperature of the stirring reaction in the method for preparing the fluorescent probe compound is 90-110°C.

[0045] Preferably, the stirring reaction temperature in the fluorescent probe compound preparation method is 100°C.

[0046] According to some embodiments of the present invention, the stirring reaction time in the method for preparing the fluorescent probe compound is 8 to 12 hours.

[0047] Preferably, the stirring reaction time in the fluorescent probe compound preparation method is 10 hours.

[0048] According to some embodiments of the present invention, the method for separating and purifying the fluorescent probe compound is: extracting with dichloromethane and water, collecting the water layer, and freeze-drying to obtain the fluorescent probe compound.

[0049] According to some embodiments of the present invention, the yield of the fluorescent probe compound is about 80-85%.

[0050] Preferably, the yield of the fluorescent probe compound is about 83%.

[0051] A third aspect of the present invention provides an application of a fluorescent probe in detecting chloride ions in organelles.

[0052] According to some embodiments of the invention, the use comprises detecting changes in chloride ion concentration within a cell organelle.

[0053] According to some embodiments of the invention, the organelle comprises at least one of a Golgi apparatus, a mitochondria, a lysosome and an endoplasmic reticulum.

[0054] Preferably, the organelle is the Golgi apparatus.

[0055] The application of the fluorescent probe according to the third aspect of the present invention has at least the following beneficial effects:

[0056] The fluorescent probe of the present invention can detect the change of chloride ion concentration in the Golgi body in real time, which is of great significance for clarifying the physiological function of the Golgi body and studying the occurrence and development mechanism of diseases related to the Golgi body.

[0057] Unless otherwise specified, the term “about” in the present invention means that the error is allowed to be within the range of ±2%. For example, about 100 is actually 100±2%×100.

[0058] Additional features and advantages of the present invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0060] Figure 1 This is the synthetic route of the preparation method described in Example 1 of the present invention;

[0061] Figure 2 The fluorescent probe prepared by the preparation method of Example 1 of the present invention is 1 H NMR spectrum;

[0062] Figure 3 The fluorescent probe prepared by the preparation method of Example 1 of the present invention is 13 C NMR spectrum;

[0063] Figure 4 is the HR-ESI-MS spectrum of the fluorescent probe prepared by the preparation method of Example 1 of the present invention;

[0064] Figure 5 is the compound Ⅰ prepared by the preparation method of Example 1 of the present invention 1 H NMR spectrum;

[0065] Figure 6 is the compound Ⅰ prepared by the preparation method of Example 1 of the present invention 13 C NMR spectrum;

[0066] Figure 7 is the HR-ESI-MS spectrum of compound Ⅰ prepared by the preparation method of Example 1 of the present invention;

[0067] Figure 8 The UV-visible absorption spectrum and fluorescence emission spectrum of the fluorescent probe of the present invention in the presence or absence of chloride ions;

[0068] (a) is the UV-visible absorption spectrum, (b) is the fluorescence emission spectrum; λ ex =350nm;

[0069] Figure 9 is a graph showing the photostability and response of the fluorescent probe to chloride ions in the presence or absence of chloride ions, λ ex / λ em =350 / 453nm;

[0070] Figure 10 This is a graph showing the fluorescence spectrum and fluorescence intensity changes of the fluorescent probe of the present invention at different pH values in the absence of chloride ions;

[0071] (a) is the fluorescence spectrum, (b) is the change of fluorescence intensity; λ ex =453nm;

[0072] Figure 11 The fluorescent probe of the present invention is characterized by different pH and different Cl - Fluorescence spectra in 100 mM phosphate buffer at 240 nm ion concentrations;

[0073] Figure 12 The fluorescence of the fluorescent probe of the present invention is Cl - Stern-Volmer plot of quenching;

[0074] Figure 13 This is a graph showing the selectivity and specificity of the fluorescent probe of the present invention for common anions and cations in organisms;

[0075] Figure 14 The fluorescent probe of the present invention is Cl at different concentrations - Fluorescence spectrum under

[0076] Figure 15 The fluorescence intensity of the fluorescent probe at 453 nm varies with Cl - Concentration change curve;

[0077] Figure 16 The fluorescence intensity of the fluorescent probe of the present invention at 453 nm is related to Cl - Linear relationship graph between concentrations;

[0078] Figure 17 This is a graph showing the cell viability of HeLa cells after the fluorescent probe of the present invention was applied for 12 hours;

[0079] Figure 18 This is a diagram showing the results of a co-localization imaging experiment between the fluorescent probe of the present invention and HeLa cells;

[0080] Figure 19 These are photos of HeLa cells stained with the fluorescent probe of the present invention at different concentrations of chloride ions;

[0081] (a) Chloride ion concentration is 0 mM; (b) Chloride ion concentration is 9 mM; (c) Chloride ion concentration is 18 mM; (d) Chloride ion concentration is 36 mM; (e) Chloride ion concentration is 72 mM; (f) Chloride ion concentration is 144 mM.

[0082] Figure 20 It is a bar graph of relative fluorescence intensity after HeLa cells are stained with the fluorescent probe of the present invention. DETAILED DESCRIPTION

[0083] The following will clearly and completely describe the concept of the present invention and the technical effects produced in conjunction with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels.

[0084] The raw materials and instruments used in the examples of the present invention are:

[0085] Instrument model and company:

[0086] Fluorescence spectrometer: Perkin Elmer FL8500 fluorescence spectrophotometer, Perkin Elmer, USA;

[0087] UV-visible photometer: U-3900, HITACHI;

[0088] Nuclear magnetic resonance spectrometer: Bruker Avance AV 500 MHz NMR spectrometer, Bruker GmbH, Switzerland;

[0089] Mass spectrometer: Thermo Scientific TM Orbitrap Fusion TMMass spectrometer, Thermo Fisher Scientific, USA;

[0090] Laser confocal microscope: LEICA / TCS SP8, Leica, Germany;

[0091] Reagent source and manufacturer:

[0092] Golgi apparatus dye Golgi-Tracker Red, mitochondrial dye Mito-Tracker Red, lysosome dye Lyso-Tracker Red, and endoplasmic reticulum dye ER-Tracker Red: Beyotime, Shanghai Beyotime Biotechnology Co., Ltd.;

[0093] p-Aminobenzenesulfonamide: CAS: 63-74-1; Guangzhou Shuopu Biotechnology Co., Ltd.;

[0094] Bromoacetyl bromide: CAS: 598-21-0; Beijing Bailingwei Technology Co., Ltd.;

[0095] Acetone: CAS: 67-64-1; Guangdong Guangshi Reagent Technology Co., Ltd.;

[0096] Ethanol: CAS: 64-17-5; Anhui Zesheng Technology Co., Ltd.;

[0097] 6-Methoxyquinoline: CAS: 5263-87-6; Beijing Bailingwei Technology Co., Ltd.;

[0098] DMF: CAS: 68-12-2; Taifan Technology (Guangzhou) Co., Ltd.

[0099] Dichloromethane: CAS: 75-09-2; Taifan Technology (Guangzhou) Co., Ltd.

[0100] Example 1

[0101] The present invention prepares a fluorescent probe by the following steps:

[0102] Sulfonamide (1.72 g, 10 mmol) was weighed and suspended in acetone (10 mL). Bromoacetyl bromide (0.82 mL, 9.5 mmol) was added dropwise at 0°C. The reaction mixture was stirred at 55°C for 10 minutes. 20 mL of ice water was added and the reaction was stirred for 8 hours. The collected solid was washed with ice water and recrystallized from ethanol. The solid was collected by filtration and dried under vacuum to obtain Compound I. The yield of Compound I was 980 mg, a 35% yield.

[0103] The test results of the specific obtained compounds are: 1H NMR (500MHz, DMSO-d6) δ10.72(s,1H),7.79(d,J=8.9Hz,2H),7.74(d,J=8.9Hz,2H),7.29(s,2H),4.07(s,2H); 13 C NMR (125MHz, DMSO-d6) δ172.0,141.9,138.9,127.0,119.6,62.3; HR-ESI-MS for C8H9N2O3S([M+H] + )Calcd: 292.9592, Found: 292.9590. This indicates that the compound I was indeed obtained. The specific results are as follows Figures 2-4 shown.

[0104] Compound I (235 mg, 0.8 mmol) was weighed and added to a DMF solution (6 mL) of 6-methoxyquinoline (255 mg, 1.6 mmol) and stirred at 100°C for 8 h. The mixture was extracted with dichloromethane and water, and the aqueous layer was collected and freeze-dried to obtain 300 mg of solid product with a yield of 83%. The synthetic route of the fluorescent probe in this example is as follows: Figure 1 shown.

[0105] The test results of the product obtained in this embodiment are: 1 H NMR (500MHz, DMSO-d6) δ11.20(s,1H),9.39(d,J=4.3Hz),9.23(d,J=8.1Hz,1H),8.41(d,J=9.6Hz,1H),8.22(dd,J=8.1,4.3Hz,1H),7. 95(d,J=2.9Hz,1H),7.90(dd,J=9.6,2.9Hz,1H),7.79(d,J=8.9Hz,2H),7.75(d,J=8.9Hz,2H),7.31(s,2H),6.08(s,2H),4.01(s,3H); 13 C NMR (500MHz, DMSO-d6) δ164.0,159.7,148.8,147.0,141.4,139.7,134.8,131.9,128.6,127.4,122.8,121.0,119.5,108.6,59.9,56.9; HR-ESI-MS calcd for C 18 H 18 N3O4S + ([M-Br] + )372.1012, found372.1010. It can be seen from this that the fluorescent probe compound represented by Formula II was indeed prepared in this example. The specific results are as follows Figures 5-7shown.

[0106] Test Case

[0107] Ultraviolet absorption spectrum and fluorescence emission spectrum test of fluorescent probe

[0108] Solution preparation:

[0109] ①HEPES (4-hydroxyethylpiperazineethanesulfonic acid) buffer solution: Weigh a certain amount of HEPES and dissolve it in ultrapure water to a final concentration of 50 mM, and adjust the pH to 7.4 with sodium hydroxide;

[0110] ② Fluorescent probe stock solution: Dissolve the fluorescent probe compound in HEPES buffer solution (50 mM, pH 7.4) to make the final concentration of the fluorescent probe stock solution 10 mM;

[0111] ③ Sodium chloride solution: Weigh a certain amount of sodium chloride and dissolve it in HEPES buffer solution (50 mM, pH 7.4) to make the final concentration of the sodium chloride solution 5 M.

[0112] Take an appropriate amount of fluorescent probe stock solution and add it to HEPES buffer solution (50mM, pH7.4) with or without chloride ions, so that the concentration of fluorescent probe in the test system is 100μM, and the chloride ion concentration is 0 or 50mM respectively. Test the ultraviolet absorption spectrum and fluorescence emission spectrum of the fluorescent probe. The fluorescent probe has strong absorption at 320nm and 350nm. The absorption spectrum does not show obvious changes after the reaction with chloride ions. Figure 8 (a) Compared with the UV absorption spectrum, the fluorescence intensity of the fluorescent probe at 453 nm is significantly weakened after adding chloride ions under 350 nm excitation, as shown in Figure 8 (b) This result shows that chloride ions can quench the fluorescence of the fluorescent probe.

[0113] Response time and photostability testing

[0114] Take an appropriate amount of fluorescent probe mother solution, use HEPES buffer solution (50mM, pH7.4) to prepare a test solution containing the fluorescent probe (100μM), and use a fluorescence spectrophotometer to track the fluorescence intensity of the fluorescent probe (100μM) in HEPES buffer solution (50mM, pH7.4). At 30min, add sodium chloride solution to make the chloride ion concentration 50mM, and continue to observe the changes in fluorescence intensity. Under 350nm excitation, after the addition of chloride ions, the fluorescence intensity of the fluorescent probe drops rapidly and stabilizes instantly, indicating that the response of the fluorescent probe to chloride ions is very fast and can be used to monitor the immediate changes of chloride ions, such as Figure 9On the other hand, the fluorescence intensity of the fluorescent probe at 453 nm did not change significantly within the monitoring time of 30 min in the presence or absence of chloride ions, which indicates that the fluorescent probe has good photostability.

[0115] Effect of pH

[0116] Solution preparation:

[0117] Phosphate buffer:

[0118] Solution A: 23.996 g NaH2PO4 dissolved in 1 L ultrapure water to a final concentration of 0.2 M;

[0119] Solution B: 28.392 g Na2HPO4 dissolved in 1 L ultrapure water to a final concentration of 0.2 M;

[0120] Take a certain volume of solution A and solution B, mix them, add ultrapure water to dilute them so that the phosphate concentration in the test system is 0.1M, and then adjust the pH to 3, 4, 5, 6, 7, and 8 with 1M H3PO4 and 2M NaOH respectively.

[0121] In order to confirm that the fluorescent probe can work under the pH conditions in the Golgi apparatus, the fluorescence spectra of the fluorescent probe in phosphate buffer solutions (100 μM) with different pH values were tested, as shown in Figure 5. Figure 10 (a) As shown. The fluorescence intensity of the fluorescent probe decreases with increasing pH, as shown in Figure 10 (b) shown.

[0122] In order to quantitatively characterize the response of the fluorescent probe to chloride ions at different pH values, the concentration of the compound was fixed at 100 μM and the concentration of chloride ions was gradually changed in the range of 0 to 250 mM. It can be observed that the fluorescence intensity of the fluorescent probe showed a regular decrease, such as Figure 11 As shown, the Stern-Volmer constant is obtained from the Stern-Volmer equation.

[0123] F0 / F=1+K sv [Cl - ];

[0124] Where F0 represents the fluorescence intensity of the fluorescent probe when no chloride ions are added, and F represents the fluorescence intensity after chloride ions are added. - ]Draw a graph and perform linear fitting to obtain a straight line, such as Figure 12 The slope of the straight line equation is the Stern-Volmer constant under the test conditions, as shown in Table 1. In summary, the response ability of the fluorescent probe to chloride ions is basically the same under various pH conditions.

[0125] Table 1 Fluorescent probes for Cl -Stern-Volmer constant

[0126]

[0127] Selectivity and interference resistance experiments

[0128] Common anions and cations in organisms were selected for specificity testing of fluorescent probes. Anions and cations include Cl - Br - , I - 、F - 、SO4 2- 、NO3 - 、Na + , Ca 2+ Mg 2+ and NH4 + 4 mL of the fluorescent probe test solution (50 mM HEPES, pH 7.4) was prepared, with a final concentration of 100 μM for the fluorescent probe and 100 mM for each anion and cation. Each experiment was repeated three times in parallel.

[0129] The orange bars represent the F0 / F ratios of the fluorescent probe in the presence of various ions (mean ± sd, n = 3). The green bars represent the F0 / F ratios of the fluorescent probe in the coexistence of chloride ions and competing ions (mean ± sd, n = 3). Figure 13 As shown, the fluorescent probe can be quenched by chloride, iodide, and bromide ions, while other anions and cations have little quenching effect on the fluorescence of the fluorescent probe. When chloride ions coexist with other anions and cations, the fluorescent probe's response to chloride ions is unaffected. Typically, the concentration of chloride ions in the cytoplasm can reach 5–40 mM, while the concentrations of iodide and bromide ions in cells are in the μM range, far lower than the concentration of chloride ions. Therefore, this probe has the potential to be used for chloride ion detection in cells. In summary, the fluorescent probe exhibits excellent specificity and interference resistance for chloride ions.

[0130] Detection limit

[0131] In order to test the response of the fluorescent probe to chloride ions, the fluorescent probe was mixed with chloride ions of different concentrations and fluorescence spectra were measured. Figure 14 It can be seen that with the increase of chloride ion concentration, the fluorescence intensity of the fluorescent probe gradually decreases; the fluorescence intensity increases with the increase of Cl - The concentration increases and decreases. - When the concentration exceeds 150 mM, the fluorescence is almost completely quenched. Figure 15 It is known.

[0132] The fluorescence intensity at 453 nm was used to analyze the added Cl -The concentration was plotted and linear fitting was performed. When pH was 7.4, the fluorescence intensity was related to Cl - There is a good linear relationship between the concentrations of 0.1 and 10 mM, and the linear equation is y = -1307[Cl - ] + 51201, the detection limit was calculated using the formula LOD = 3σ / k, where σ represents the standard deviation of the blank probe tested 15 times in parallel, and k represents the slope of the fitting curve, as shown in Figure 16 As shown in Figure 2, the detection limit of the fluorescent probe for chloride ions is LOD = 34 μM.

[0133] Cytotoxicity assay

[0134] After the HeLa cells were cultured to the logarithmic growth phase, they were digested with trypsin and prepared into a cell suspension. 96-well plates were plated (8000 cells / well). After 24 hours, the culture medium was aspirated and replaced with 100 μL of culture medium containing different concentrations of fluorescent probes (the DMSO content in each well was kept at 1%, and 4 replicates of the fluorescent probe were set up). After further culture for 12 hours, 10 μL of MTT solution (5 mg / mL) was added to each well. After 4 hours, the culture medium in the well was aspirated and 100 μL of DMSO was added to each well. After shaking, the absorbance value at 570 nm was tested using a microplate reader.

[0135] Even when the concentration of fluorescent probe is higher than 3mM, the cell survival rate is still as high as 90%. Figure 17 As shown, the fluorescent probe showed little cytotoxicity.

[0136] Cell colocalization experiments

[0137] To verify the specific targeting of the fluorescent probe to the Golgi region, four subcellular organelle-specific dyes, including Golgi-Tracker Red, Mito-Tracker Red, Lyso-Tracker Red, and ER-Tracker Red, were co-stained with the fluorescent probe in HeLa cells. Colocalization imaging experiments were performed to investigate the fluorescent probe's ability to target specific subcellular organelles. The concentrations of the fluorescent probes were 2 mM, Golgi-Tracker Red was 150 μg / mL, and the concentrations of the other three dyes were 1 μM.

[0138] The fluorescent probe showed a good colocalization coefficient with the Golgi apparatus dye Golgi-Tracker Red, with a colocalization coefficient of 0.814. It showed relatively poor colocalization effects with the other three dyes, with colocalization coefficients of 0.401 for mitochondria, 0.627 for lysosomes, and 0.314 for endoplasmic reticulum. Figure 18 These results demonstrate that the fluorescent probe has good Golgi targeting ability.

[0139] Detection of Chloride Ion Concentration Changes in the Golgi Body

[0140] HeLa cells were cultured at 5×10 4 The cells were seeded into a confocal culture dish at a density of 100 cells / well and incubated for 24 hours. After the incubation, the supernatant was removed and washed with HBSS solution without chloride ions (1mL×3). Then 1mL of HBSS solution without chloride ions containing 10% FBS was added and cultured in a 37°C incubator for 6 hours. After 6 hours, the supernatant was removed and washed with HBSS solution without chloride ions (1mL×3). HBSS solutions with different chloride ion concentrations containing fluorescent probes (2mM) were then added to the culture dish and cultured in a 37°C incubator for 2.5 hours. After 2.5 hours, the culture dish was taken out, the supernatant was removed, and the cells were washed with HBSS solutions with corresponding chloride ion concentrations (1mL×3), and then 1mL of HBSS solutions with corresponding chloride ion concentrations were added. Finally, the staining of the cells was observed under a laser confocal microscope and appropriate areas were selected for photographing, such as Figure 19 shown.

[0141] ImageJ software was used to quantitatively calculate the blue fluorescence intensity of the cell images. The higher the chloride ion concentration, the lower the intracellular fluorescence intensity, and it decreased in a concentration-dependent manner. Figure 20 This result shows that the fluorescent probe can be used to detect changes in chloride ion concentration in the Golgi apparatus.

[0142] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A fluorescent probe, characterized in that The structural formula of the fluorescent probe is shown in II:

2. A method for preparing a fluorescent probe according to claim 1, characterized in that: The preparation method comprises the steps of stirring the compound I shown in FIG1 and 6-methoxyquinoline in DMF to react and separate and purify the fluorescent probe compound; 3. The method for preparing a fluorescent probe according to claim 2, wherein: The synthesis method of compound I shown in FIG1 comprises stirring p-aminobenzenesulfonamide and bromoacetyl bromide in acetone for reaction.

4. The method for preparing a fluorescent probe according to claim 3, wherein: The molar ratio of p-aminobenzenesulfonamide to bromoacetyl bromide is 1:(0.9-1).

5. The method for preparing a fluorescent probe according to claim 3, wherein: The mass volume ratio of the p-aminobenzenesulfonamide and acetone is (0.08-0.2) g:1 ml.

6. The method for preparing a fluorescent probe according to claim 2, wherein: The molar ratio of the compound I to 6-methoxyquinoline is 1:(2-4).

7. The method for preparing a fluorescent probe according to claim 2, wherein: The mass volume ratio of the 6-methoxyquinoline to the DMF solution is (0.02-0.04) g:1 ml.

8. The method for preparing a fluorescent probe according to claim 2, wherein: The stirring reaction conditions are: reaction temperature of 90-110° C.; reaction time of 8-12 h.

9. The method for preparing a fluorescent probe according to claim 2, wherein: The separation and purification method comprises the following steps: extracting with dichloromethane and water, collecting the water layer, and freeze-drying the layer to obtain the fluorescent probe compound.

10. Use of the fluorescent probe according to claim 1 in preparing a reagent for detecting chloride ions in organelles.

11. The use according to claim 10, characterized in that The cell organelles include at least one of a Golgi apparatus, a mitochondria, a lysosome, and an endoplasmic reticulum.

Images