CN111574506A - Preparation method of ratio-type acidic pH fluorescent probe - Google Patents

Preparation method of ratio-type acidic pH fluorescent probe Download PDF

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CN111574506A
CN111574506A CN202010335218.8A CN202010335218A CN111574506A CN 111574506 A CN111574506 A CN 111574506A CN 202010335218 A CN202010335218 A CN 202010335218A CN 111574506 A CN111574506 A CN 111574506A
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姜舒
韩志湘
许海
董良欢
代晓婷
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Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a preparation method and application of a ratio-type acidic pH fluorescent probe. The invention provides a preparation method for synthesizing a fluorescent probe by 4-methylquinoline and 7-diethylaminocoumarin-3-aldehyde under the action of trimethylchlorosilane; the ratio-type acidic pH fluorescent probe prepared by the invention has good selectivity on pH, high sensitivity and quick response; the probe is respectively used for detecting the pH in living cells, in escherichia coli or in zebra fish, and a satisfactory result is obtained.

Description

Preparation method of ratio-type acidic pH fluorescent probe
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to preparation of a ratio-type acidic pH fluorescent probe and application of the ratio-type acidic pH fluorescent probe in detection of pH in living cells, escherichia coli or zebra fish for non-diagnosis and treatment purposes.
Background
pH plays an important regulatory role in various cell behaviors, such as enzyme activity, proliferation and apoptosis of cells, information transmission and drug resistance, etc., which are important parameters affecting cell behaviors. Normally H in the extracellular fluid and blood of the human body+The concentration of the active ingredient was 40nM of the active ingredient,the pH was 7.4 and fluctuated no more than 5 nM. However, pH abnormality in a living body may cause abnormal cell and tissue functions, which may lead to various diseases, such as inflammation, tumor, cardiopulmonary or nervous system diseases (e.g., Alzheimer's disease), cystic fibrosis, and the like. Therefore, accurate, in situ and real-time monitoring and tracking of the dynamic changes in pH in tissues and cells can be used for diagnosis of certain diseases, which helps to better understand the important role of pH in various processes such as physiology and pathology.
Compared with other methods for detecting intracellular pH, the fluorescence probe method has the advantages of simple operation, high sensitivity, good selectivity, quick response, no damage to samples, in-situ detection and visual imaging, and thus becomes a powerful tool for monitoring intracellular pH dynamic change. Currently, pH fluorescent probes reported in the literature can be divided into two categories: one for responding to neutral pH (pH 6-8) and the other for responding to weakly acidic organelles, such as lysosomes, functioning in the pH range of 4.5-6.0. But relatively few fluorescent probes are used in response to pH less than 4.5. In eukaryotic cells, an acidic environment is particularly important for many organelles, especially along secretory and endocytic pathways. For example, gastric acid in gastric juice has a pH of about 1.0, which plays an important role in the digestion of food. Some microorganisms, such as acidophilus bacteria and helicobacter pylori, can survive under extremely acidic conditions. Enteric bacteria, such as E.coli and Salmonella, can survive through the highly acidic mammalian stomach. Therefore, it is very important to obtain intracellular pH under extremely acidic conditions. However, the determination of intracellular extreme acidic pH remains a great challenge due to the lack of suitable probes.
The ratio type fluorescent probe detects by recording the ratio change of two different emission intensities, can avoid the influence of factors such as environmental change, the concentration of the probe, the performance of an instrument and the like, and greatly improves the detection accuracy. Coumarin fluorescent dyes have good optical properties such as high fluorescence quantum yield, good light stability, absorption and emission spectra in the visible light region, large stokes shift, and the like, and are widely used for developing fluorescent probes. Meanwhile, the structure is easy to modify, the optical performance can be adjusted by changing the electron gain and loss capacity of 3-position or 7-position, and the fluorescent probe is often used as a fluorophore of a ratiometric fluorescent probe. In view of this, it is feasible and highly desirable to develop a coumarin derivative-based ratiometric fluorescent probe for the determination of extremely acidic pH values.
Disclosure of Invention
Based on the proposed requirements, the present inventors have made extensive literature research and intensive studies and have provided a ratiometric acidic pH fluorescent probe.
The technical scheme of the invention is that a ratio-type acidic pH fluorescent probe has the following molecular structure:
Figure BDA0002466325760000021
a preparation method of a ratio-type acidic pH fluorescent probe comprises the following steps:
(1) completely dissolving 7-diethylaminocoumarin-3-aldehyde and 4-methylquinoline in anhydrous N, N-dimethylformamide according to a certain proportion, then adding trimethylchlorosilane, and wrapping a bottle body with aluminum foil to prevent light; in N2Under the protection condition, an oil bath kettle is used for heating, stirring and refluxing;
(2) cooling the reaction solution, adding water to quench the reaction, and using Na2CO3Adjusting the pH value of the solution; then adding dichloromethane for extraction, collecting organic phase and using anhydrous Na2SO4Drying is carried out; removing the solvent by rotation to obtain a crude product, and finally performing silica gel column chromatography purification by using petroleum ether and ethyl acetate as eluent to obtain a reddish brown solid which is a probe molecule compound JS-1; namely the ratio type acid pH fluorescent probe to be prepared by the invention.
In the step (1), the dosage relationship of the 7-diethylaminocoumarin-3-aldehyde, the 4-methylquinoline, the anhydrous N, N-dimethylformamide and the trimethylchlorosilane is 1 mmol: 1.2 mmol: 10mL of: 10 mmol.
In the step (1), the heating, stirring and refluxing time is 24 hours, and the heating temperature is 100 ℃.
In the step (2), the Na2CO3The concentration of the solution was 1.0M, Na was used2CO3The solution adjusted the solution pH to 8. In the step (2), the volume ratio of the petroleum ether to the ethyl acetate is 2: 1.
the ratio-type acidic pH fluorescent probe prepared by the invention is used for detecting the pH in living cells, escherichia coli or zebra fish for non-diagnosis and treatment purposes.
The invention has the beneficial effects that:
(1) the N atom on the quinoline group in the ratio-type acidic pH fluorescent probe JS-1 prepared by the invention is the reaction recognition site of pH. When in the environment H+The concentration of N atom and H on the quinoline group is increased continuously+After being combined, the probe is protonated, so that the electronic arrangement of the probe molecules is changed, the Intramolecular Charge Transfer (ICT) effect is enhanced, and therefore, the probe is in a ratio emission mode, and the detection accuracy is improved.
(2) The ratio-type acidic pH fluorescent probe JS-1 prepared by the invention shows good spectral response performance to pH. The ultraviolet absorption spectrum of the probe was first studied. At pH 8.4, the probe absorbs strongly at 432 nm. When the pH was lowered from 8.4 to 2.1, the red-shift was to 520 nm. Next, the fluorescence spectrum properties of the probe were investigated. When the pH was equal to 8.4, the fluorescence of the probe at 568nm increased significantly. When the pH was lowered from 8.4 to 2.1, the red-shift was to 689 nm. Then, the selectivity of the probe was investigated. Examine the probe for various metal ions (K)+、Ca2+、Mg2+、Cu2+、Zn2+、Cd2+、Mn2+、Co2+And Fe3+) And the fluorescence response of biologically relevant substances (glucose, vitamin C, glutathione and cysteine). The results show that there is no significant change in the emission intensity of the probe when various interferents are added. Next, the response time of the probe to different pH values was investigated within 2 minutes. Finally, the reversible response of the probe to changes in pH was investigated. The probe was able to detect pH changes back and forth in solution 7 times.
(3) The ratio-type acidic pH fluorescent probe JS-1 prepared by the invention has been successfully used for the fluorescence imaging visual detection of pH in HepG2 cells.
(4) The ratio-type acidic pH fluorescent probe JS-1 prepared by the invention can be used for visual detection of pH in Escherichia coli.
(5) The ratio-type acidic pH fluorescent probe JS-1 prepared by the invention has been successfully used for the visual detection of pH in zebra fish.
Drawings
FIG. 1 shows a synthetic route of a fluorescent probe JS-1.
FIG. 2 shows UV-VIS absorption spectra of fluorescent probe JS-1(10 μ M) in response to different pH values (2.1-8.4). Illustration is shown: the probe molecule JS-1 responds to the colour of the solutions (both containing 40% acetonitrile as co-solvent) at pH 8.4 (left) and pH 2.1 (right).
FIG. 3 shows fluorescence spectra of fluorescent probe JS-1(10 μ M) in response to different pHs (2.1-8.4). The inset is a fitted curve of the ratio response of fluorescent probe JS-1(10 μ M) to pH change. Pictures are the color of the solution (both containing 40% acetonitrile as co-solvent) when probe molecule JS-1 responds to pH 8.4 (left) and pH 2.1 (right) under 365nm hand-held uv lamp.
FIG. 4 is a graph showing the fluorescence intensity ratio response (λ) of fluorescent probe JS-1(10 μ M) after the reaction with each reactant at pH 3.7 and 4.3, respectivelyex＝495nm，λem1＝568nm；λem2689nm), 1-14 represents probes JS-1, K respectively+(5mM)、Ca2+(5mM)、Mg2+(5mM)、Cu2+(50μM)、Zn2+(50μM)、Cd2+(50μM)、Mn2+(50μM)、Co2+(50μM)、Fe3+(50. mu.M), glucose (100. mu.M), vitamin C (100. mu.M), glutathione (100. mu.M) and cysteine (100. mu.M).
FIG. 5 is a graph showing the change of fluorescence intensity ratio with time (λ. lambda. according to the pH of the fluorescent probe JS-1(10 μ M) at pH 3.1, 3.5, 5.5 and 7.5, respectivelyex＝495nm，λem1＝568nm；λem2＝689nm)。
FIG. 6 is a line graph showing the change in the fluorescence intensity ratio during 7 back-and-forth shifts of the fluorescent probe JS-1 (10. mu.M) between pH 5.0 and 3.0. (lambdaex＝495nm，λem1＝568nm；λem2＝689nm)。
FIG. 7 is a graph showing the evaluation of cytotoxicity of probes by MTT method.
FIG. 8 is a fluorescent image of pH detection in HepG2 cell by the fluorescent probe JS-1; (a-c) images of HepG2 cells incubated with JS-1 (10. mu.M) for 30min and further incubated at pH 2.1 for 30 min; (d-f) images of HepG2 cells incubated with JS-1 (10. mu.M) for 30min and further incubated at pH 5.0 for 30 min; (g-i) images of HepG2 cells incubated with JS-1 (10. mu.M) for 30min and further incubated at pH 5.0 for 30 min; wherein (a, d, g) is a bright field, (b, e, h) is a red fluorescent channel, and (c, f, i) is a green fluorescent channel.
FIG. 9 is a fluorescent image of pH detection in E.coli by the fluorescent probe JS-1; (a-c) images of E.coli incubated with JS-1 (30. mu.M) for 2h at pH 2.1; (d-f) images of E.coli incubated with JS-1 (30. mu.M) for 2h at pH 8.3; wherein (a, d) is bright field, (b, e) is red fluorescence channel, and (c, f) is green fluorescence channel.
FIG. 10 is a fluorescent image of the fluorescent probe JS-1 detecting pH in zebra fish;
(a-c) images of zebrafish incubated with JS-1(20 μ M) for 2h at pH 3.0; (d-f) images of zebrafish incubated with JS-1(20 μ M) for 2h at pH 8.3; wherein (a, d) is bright field, (b, e) is red fluorescence channel, and (c, f) is green fluorescence channel.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples.
Example 1:
synthesis of fluorescent probes
The synthetic route is shown in figure 1. In a 250mL round bottom flask, 7-diethylaminocoumarin-3-aldehyde (200mg, 0.82mmol) and 4-methylquinoline (141mg, 0.98mmol) were dissolved in 10mL DMF, p-trimethylchlorosilane (1.1mL,8.2mmol) was added, and the body was wrapped with aluminum foil and protected from light. In N2Heating and stirring for 24h under the protection and 100 ℃. The reaction was stopped, cooled to room temperature, and 100ml of water was added thereto using 1.0M Na2CO3The solution was adjusted to a solution pH of 8. Extracted three more times with 100ml dichloromethane, the organic layers were collected and 3g anhydrous Na was added2SO4And (5) drying. Removing the solvent by rotation, and mixing the crude product by using a volume ratio of 2: the petroleum ether and the ethyl acetate in the step 1 are used as eluent to carry out silica gel column chromatography purification, and reddish brown solid (100mg, 32.9%) is obtained through purification and separation, namely the probe molecule compound JS-1.1H NMR(400MHz,CDCl3)(ppm):8.88(d,J＝8.4Hz,1H),8.36(d,J＝16.0Hz,1H),8.30(d,J＝8.4Hz,1H),8.13(d,J＝8.4Hz,1H),7.72-7.76(m,2H),7.59-7.62(m,2H),7.34(d,J＝4.8Hz,1H),7.24(s,1H),6.61(d,J＝8.8Hz,1H),6.52(s,1H),3.44(q,J＝7.0Hz,4H),1.24(t,J＝6.8Hz,6H)；13C NMR(100MHz,CDCl3)(ppm):12.50,44.94,97.05,108.87,109.36,116.35,116.59,112.08,123.37,123.88,124.09,126.41,126.53,126.64,129.32,129.46,129.59,130.15,130.26,141.23,149.70,151.11,156.02,160.99；MS(ESI):m/z:calcd for C24H22N2O2:371.18[M+H]+.Found:371.44.
Example 2:
preparing a fluorescent probe and solutions with different pH values;
1. and dissolving a proper amount of probe molecule JS-1 in chromatographic pure acetonitrile to prepare a 1.0mM stock solution of the fluorescent probe molecule.
2. Weighing appropriate amount of KH2PO4、Na2HPO4Fixing the volume of NaCl and KCl with distilled water to obtain a phosphate buffer solution with the concentration of 0.01M and the pH value of 7.4;
3. adjusting the pH of the phosphate buffer solution by using 1.0M hydrochloric acid or 1.0M sodium hydroxide solution to obtain phosphate buffer solutions with various required pH values; and according to the acetonitrile: PBS 4: 6 to prepare acetonitrile/PBS detection solutions with different pH values;
4. and (3) adding the fluorescent probe molecule stock solution obtained in the step (1) into the detection solution obtained in the step (3) to obtain a mixed solution to be detected, wherein the concentration of the fluorescent probe molecules in the mixed solution to be detected is 10 mu M, and the pH value is 2.1-8.4.
Example 3:
ultraviolet-visible absorption spectrum determination of the action of the fluorescent probe and different pH values;
FIG. 2 shows UV-visible absorption spectra of fluorescent probe JS-1(10 μ M) in response to different pH values (2.1-8.4). The instrument used for UV-Vis absorption Spectroscopy was the Shimadzu UV-2600 UV-Vis spectrophotometer. As shown in FIG. 2, when the pH was 8.4, the probe showed strong absorption at 432 nm; as the pH is lowered from 8.4 to 2.1, the absorption maximum wavelength of the probe red shifts to 520 nm. This is due to the protonation of the N atom in the quinoline group. The solution changed color from light yellow (pH 8.4) to pink (pH 2.1), and the color changed significantly and was visible to the naked eye.
Example 4:
measuring the action of the fluorescent probe and different pH values by fluorescence spectrum;
FIG. 3 shows fluorescence spectra of fluorescent probe JS-1(10 μ M) in response to different pHs (2.1-8.4). The excitation spectrum used in the experiment is 495nm, and the emission wavelength range is 515-800 nm. The excitation and emission slit widths were both 10nm, and the fluorescence measurement instrument used was a Thermo Fisher Lumina spectrofluorometer. As shown in FIG. 3, the fluorescence at 568nm is strongest when the pH is equal to 8.4. When the pH is reduced from 8.4 to 2.1, the fluorescence intensity of the probe at 568nm is significantly reduced, while the fluorescence at 689nm is significantly increased. The apparent red shift in emission spectra is due to H+With the increased concentration, the N atom in the quinoline group is protonated, so that the Intramolecular Charge Transfer (ICT) effect is enhanced, and the fluorescence emission spectrum is red-shifted. The inset is a fitted graph plotting probe JS-1 as it changes in intensity of ratio signals at 568nm and 689nm, in response to changes in pH. Fluorescence emission ratio (I) when pH was lowered from 8.4 to 2.1568：I689) A reduction from 15.8 to 0.025 produced a 631-fold significant change. According to the Henderson-Hasselbalch type mass action equation: log [ (I)max-I)/(I-Imin)]＝pH-pKaAnd taking the intensity change of the ratio signal as a function of pH to obtain the pK of the probeaIs 4.11 and is very sensitive to acidic pH. In addition, the change of the ratio signal has a good linear relation with the pH in the range of 3.49 to 4.58, and the regression equation is I568/I689＝-31.2306+9.6277×pH，R20.9917. Under 365nm hand-held ultraviolet lamp irradiation, the solution color changed from blue-green (pH 8.4) to red (pH 2.1), and the color changed remarkably.
Example 5:
selectivity of the fluorescent probe for pH determination;
FIG. 4 is a graph of selectivity of fluorescent probes for pH determination. Examination of fluorescent probe JS-1 (10. mu.M) against the measured substances (KCl, LiCl, MgSO) at concentrations of 0.05-5 μ M M under pH of 3.7 and 4.3, respectively4、ZnCl2、Cu(NO3)2、FeCl3、CdSO4、MnSO4、CoCl2Ascorbic acid, GSH, Cys, Hcy and Glucose). As shown in FIG. 4, when a certain amount of the above-mentioned substance was added to the probe solution, the ratio of fluorescence intensities at 568nm and 689nm was not significantly changed in the solutions of pH 3.7 and 4.3. The fluorescent probe JS-1 is shown to have high selectivity in response to pH, and is not affected by competitive species.
Example 6:
measuring the response time of the fluorescent probe acting with the pH;
FIG. 5 is a graph showing the time response of fluorescent probe JS-1 (10. mu.M) at pH 3.1, 3.5, 5.5 and 7.5, respectively. As shown in FIG. 5, under the above pH condition, the fluorescence intensity ratio of the solution at 568nm and 689nm both changes rapidly within 2min to the maximum and does not change significantly within 30min, which indicates that the response of the probe to the pH is very rapid and can be used for monitoring the fluctuation of the pH in the actual sample.
Example 7:
measuring a pH reversible response pair by using a fluorescent probe;
FIG. 6 is a graph showing the reversible response of fluorescent probes to pH after adjusting the pH of the solution back and forth between 5.0 and 3.0 using 1.0M concentrated HCl and 1.0M sodium hydroxide solution 7 times. Reversibility of the probe to pH is obtained by measuring the ratio of fluorescence emission intensity of the fluorescent probe JS-1 at 568nm and 689nm under excitation of 495 nm. As shown in FIG. 6, when the pH of the solution was varied back and forth between 5.0 and 3.0, the ratio of fluorescence intensities was also varied back and forth, and the process was completely reversible. The response and recovery in the solutions with different pH values are completed quickly, which shows that the probe has the capability of monitoring the dynamic change of the pH value in real time and has high reversible response to the pH value.
Example 8:
evaluating cytotoxicity of the fluorescent probe;
HepG2 cells were cultured at 4 × 104The density of individual cells/well was seeded in each well of a 96-well plate and cultured for 24h until the cells were adherent. The stock solutions of fluorescent probe molecules were diluted with DMEM medium containing 10% fetal bovine serum to give final concentrations of 5, 10, 15, 20, 30 and 50. mu.M probe in solution, respectively. 100 μ L of the above solution was added to each well of a 96-well plate, incubated for 24h, and washed 3 times with PBS solution to remove extracellular probe molecules. mu.L of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide solution (5mg ∙ mL)-1HEPES) was added to each well and placed in an incubator and incubated at 37 ℃ for 4h to form blue-violet formazan crystals. Absorbing the redundant solution, adding 100 mu L of dimethyl sulfoxide into each well to dissolve crystals, and finally measuring the absorbance at 490nm in a Synergy H5 microplate detector to obtain the cytotoxicity of the probe. It can be seen from FIG. 7 that the activity of HepG2 cell was always above 90% when the concentration of fluorescent probe JS-1 was increased from 0 to 30. mu.M. When the concentration was increased to 50. mu.M, the activity of HepG2 cells was slightly decreased but still remained above 80%. This result indicates that probe JS-1 has low cytotoxicity, and is able to further detect pH in live cells and zebrafish.
Example 9:
the fluorescent probe is applied to the fluorescent imaging detection of the pH in HepG2 cells;
HepG2 cells 2 × 104Cell density per well was seeded on 24-well plates and DMEM complete medium containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin was added. Culturing in a carbon dioxide incubator at 37 deg.C for 24 hr to make the cells adhere to the wall. Dead cells were removed by washing them three times with 1.0mL of PBS buffer. 1mL of serum-free DMEM medium containing 10. mu.M of fluorescent probe molecule JS-1 was added to each well and incubated at 37 ℃ for 40min, and washed three times with PBS buffer solution to wash out extracellular probe molecules. PBS buffer solutions containing 10. mu.g/ml nigericin at pH 2.1, 5.0 and 8.3, respectively, were added. After incubation at 37 ℃ for 30min, 1.0mL of serum-free DMEM medium was added, and finally imaging was performed with a 20-fold objective lens on a Zeiss GmbH37081 inverted fluorescence microscope. As shown in FIG. 8, when the cell pH was 2.1, fluorescence was clearly observed in the red channel (655 to 755nm), and negligible fluorescence was observed in the green channel (460 to 510 nm). When the cell pH was 5.0, the fluorescence intensity in the red channel decreased, while the fluorescence intensity in the green channel increased. At a cell pH of 8.27, the red channel showed negligible fluorescence, while a significant fluorescence enhancement occurred in the green channel. These results indicate that the probe has good cell membrane permeability while being able to image pH fluctuation changes in living cells.
Example 10:
the fluorescent probe is applied to the fluorescence imaging detection of pH in escherichia coli;
escherichia coli was cultured at 37 ℃ by using Luria-Bertani (LB) liquid medium. 100. mu.L of the activated bacterial suspension was inoculated into 100mL of fresh LB liquid medium and cultured for 12 hours at 180rpm using a KYC 100B shaker. The mixture was then centrifuged for 5min at 5000rpm using a large scale AvaniJ-25 centrifuge. Discarding the supernatant, and collecting the lower layer of Escherichia coli. The LB medium was removed by washing twice with 50ml of physiological saline. Resuspending Escherichia coli in PBS buffer solution with pH of 2.08 and 8.26 for 5min, and controlling Escherichia coli concentration to OD6000.1. Fluorescent probe molecules JS-1 solution were added thereto, respectively, to bring the probe concentration to 30. mu.M and incubated for 2 hours, then coated on a glass slide and imaged by a Zeiss GmbH37081 type inverted fluorescence microscope with a 40-fold objective lens. As shown in fig. 9, the red channel showed strong fluorescence and the green channel showed very weak fluorescence at pH 2.1. When the pH was increased to 8.3, the fluorescence intensity of the red channel dropped sharply, while the fluorescence intensity of the green channel increased. The results indicate that the probe has the ability to image pH changes in e.
Example 11:
the fluorescent probe is applied to the fluorescent imaging detection of the pH in the zebra fish;
dividing 3 days old zebra fish into 2 groups, culturing in zebra fish embryo culture solution (0.1% NaCl, 0.003% KCl, 0.004% CaCl) containing 20 μ M fluorescent probe molecule JS-1 and having pH of 3.0 and 8.0, respectively2·H2O and 0.008% MgSO4) And (4) incubating for 40 minutes. And then washed with zebrafish embryo culture solution which does not contain the probe molecules and has the above pH to remove the residual probe molecules. Fluorescence imaging was observed on a SZX2-ILLT type stereofluorescence microscope. As shown in fig. 10, it can be observed that the fluorescence intensity of the red channel decreases as the pH increases from 3.0 to 8.0, while the fluorescence intensity of the green channel increases, consistent with the trend in HepG2 cells and e. It is shown that probe JS-1 has the ability to image pH dynamics in zebra fish.
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.

Claims (7)

1. A ratio-type acidic pH fluorescent probe is characterized in that the structure is as follows:
Figure FDA0002466325750000011
2. the method for preparing a ratiometric acidic pH fluorescent probe of claim 1, wherein the reaction steps are as follows:
(1) completely dissolving 7-diethylaminocoumarin-3-aldehyde and 4-methylquinoline in anhydrous N, N-dimethylformamide according to a certain proportion, then adding trimethylchlorosilane, and wrapping a bottle body with aluminum foil to prevent light; in N2Under the protection condition, an oil bath kettle is used for heating, stirring and refluxing;
(2) cooling the reaction solution, adding water to quench the reaction, and using Na2CO3Adjusting the pH value of the solution; then adding dichloromethane for extraction, collecting organic phase and using anhydrous Na2SO4Drying is carried out; removing solvent by rotary to obtain crude product, and finally using stonePerforming silica gel column chromatography purification by using oil ether and ethyl acetate as eluent to obtain a reddish brown solid which is a probe molecule compound JS-1; namely the ratio type acid pH fluorescent probe.
3. The method for preparing a ratio-type acidic pH fluorescent probe according to claim 2, wherein in the step (1), the relationship between the amounts of the 7-diethylaminocoumarin-3-aldehyde, the 4-methylquinoline, the anhydrous N, N-dimethylformamide and the trimethylchlorosilane is 1 mmol: 1.2 mmol: 10mL of: 10 mmol.
4. The method for preparing a ratiometric acidic pH fluorescent probe according to claim 2, wherein in the step (1), the heating, stirring and refluxing time is 24h, and the heating temperature is 100 ℃.
5. The method for preparing a ratiometric acidic pH fluorescent probe according to claim 2, wherein in step (2), the Na is2CO3The concentration of the solution was 1.0M, Na was used2CO3The solution adjusted the solution pH to 8.
6. The method for preparing a ratiometric acidic pH fluorescent probe according to claim 2, wherein in the step (2), the volume ratio of petroleum ether to ethyl acetate is 2: 1.
7. use of a ratiometric acidic pH fluorescent probe according to claim 1, for the detection of pH in living cells, in escherichia coli or in zebrafish for non-diagnostic and therapeutic purposes.
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CN112480052A (en) * 2020-12-08 2021-03-12 山西大学 Ratio type near-infrared fluorescent probe for detecting pH, preparation method and application
CN112480052B (en) * 2020-12-08 2022-05-31 山西大学 Ratio type near-infrared fluorescent probe for detecting pH, preparation method and application

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