CN109503435B - Novel dual-emission fluorescent dye probe and preparation and application thereof - Google Patents
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Abstract
The invention relates to a dual-emission fluorescent dye probe and a preparation method and application thereof, wherein the structural formula of the probe is shown as the following formula I:
the probe takes two-side isophorone malononitrile as a framework and 2, 4-dinitrobenzenesulfonyl as a biological thiol recognition site. Compared with the prior art, the probe is unique in that the probe realizes ratio type detection of biological thiol instead of traditional off-on type, and successfully performs fluorescence confocal imaging of biological thiol in living cells.
Description
Technical Field
The invention belongs to the field of small molecule fluorescent probes, relates to a ratio type biological sulfhydryl fluorescent probe and a preparation method and application thereof, and particularly relates to a ratio type fluorescent probe based on an isophorone malononitrile structure and a preparation method and application thereof.
Background
Biological thiols are widely found in cells or organisms and include cysteine (Cys), Glutathione (GSH) and homocysteine (Hcy). They are key factors in regulating cellular function by maintaining microenvironment redox homeostasis. Abnormal levels of biological thiols will therefore lead to a number of diseases. For example, GSH plays an important role as the most abundant intracellular thiol (1-10mM) in endogenous antioxidant activity, and decreased GSH levels may be associated with aids, cancer, and many neurological diseases, Cys deficiency may lead to diseases such as slow growth in children, skin lesions, and weakness, and Hcy is a risk factor for heart disease and alzheimer's disease. Therefore, it is increasingly important to detect and quantify thiols in biological systems.
The small molecular fluorescent probe is a biosensor, has the advantages of high sensitivity, convenience, low cost, no invasion and the like, can detect in real time in a life system, and is simple to operate. Therefore, fluorescence detection has become one of the most convenient methods in various detection techniques. Ratiometric fluorescence is an analytical method for determining a target by measuring the ratio of the intensities of fluorescence at two different wavelengths. The construction of ratiometric fluorescent probes has attracted considerable interest to researchers, as the measured fluorescence ratio signals can greatly reduce the effects of light source intensity fluctuations and instrument stability variations, giving the method greater sensitivity and accuracy, and a large number of fluorescent probes for the detection of biological thiols, mostly of the Turn-ON type, have been developed, such as coumarins with blue emission, fluoresceins with green emission, naphthalimides with green or yellow emission, etc., mostly with only a single wavelength signal. The development of fluorescent dyes with dual emission is therefore of great interest anyway.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a dual-emission fluorescent dye probe and preparation and application thereof. The probe is a ratio type fluorescent probe which takes DNBS (2, 4-dinitrobenzenesulfonyl) as a recognition group of biological sulfydryl and bilateral isophorone malononitrile as a fluorophore framework, and the fluorescent dye can be applied to living cell staining.
The purpose of the invention can be realized by the following technical scheme: a dual-emission fluorescent dye probe is characterized in that the structural formula of the probe is shown as the following formula I:
the preparation method of the dual-emission fluorescent dye probe is characterized by comprising the following steps of:
(1) adding isophorone, malononitrile and a catalyst into an organic solvent, carrying out reflux reaction for 10-20 h under the protection of nitrogen, cooling, pouring the reaction liquid into ice water, separating out a solid, and filtering to obtain white solid isophorone malononitrile (formula IV);
(2) adding the isophorone malononitrile, 4-hydroxy isophthalaldehyde and piperidine obtained in the step (1) into absolute ethyl alcohol, performing reflux reaction for 2-6 hours under the protection of nitrogen, cooling, removing the solvent under reduced pressure, and performing column chromatography by using dichloromethane to obtain an orange-red solid (formula VI);
(3) adding 2, 4-dinitrobenzenesulfonyl chloride, the compound of formula VI obtained in the step (2) and triethylamine into anhydrous acetonitrile, and reacting the reaction mixture for 0.2-1 hour at 30-50 ℃ under the protection of nitrogen. Tracking the reaction by TLC, separating out light yellow solid, and filtering to obtain a target product;
the catalyst in the step (1) comprises piperidine, and the organic solvent comprises ethanol.
The molar ratio of the isophorone, the malononitrile, the catalyst and the organic solvent in the step (1) is 1: (1-2): (0.2-0.4): (25-35).
The molar ratio of the isofluranone, the malononitrile, the catalyst and the organic solvent is preferably 1: 1.5: 0.35: 32.
the molar ratio of the isophorone malononitrile to 4-hydroxyisophthalaldehyde in the step (2) is 1: (1-3);
the molar volume ratio of the isophorone malononitrile to the piperidine is as follows: 1, (0.5-2) mmol/d;
the molar volume ratio of the isophorone malononitrile to the absolute ethyl alcohol is 1: 8-12 mmol/ml.
The molar ratio of the VI compound in the step (3), the 2, 4-dinitrobenzene sulfonyl chloride and the triethylamine is 1: (1-2): (1.5-2.5);
the molar volume ratio of the VI compound to the anhydrous acetonitrile is 1: 35-40 mmol/ml.
The preferable molar ratio of the VI compound in the step (3), the 2, 4-dinitrobenzenesulfonyl chloride and the triethylamine is 1: 1.4: 2.
the application of the dual-emission fluorescent dye probe is characterized in that the probe is used for detecting in-vitro biological sulfhydryl and biological sulfhydryl in a biological living cell.
Compared with the prior art, the probe is a fluorescent probe which is constructed by modifying two identical isophorone malononitrile structures on a benzene ring and using DNBS (2, 4-dinitrobenzenesulfonyl) as a recognition group and has ratio detection on biological sulfydryl. Due to the quenching effect of DNBS (2, 4-dinitrobenzenesulfonyl), the fluorescence of p-isophorone malononitrile is quenched, while the quenching effect of p-isophorone malononitrile is relatively small. Thus, the emission wavelength of probe CHT before addition of the bio-thiol was 497 nm. When the biological thiol is added, the quenching group DNBS is separated, the fluorescence of the para-isophorone malononitrile is recovered, the electron cloud is redistributed, the emission at 497nm disappears, and the conjugated system is lengthened due to the exposure of hydroxyl. The emission wavelength is therefore red shifted to 568nm, enabling ratiometric detection of the biological thiol groups (497nm to 568 nm).
The isophorone malononitrile dye product has the advantages of low toxicity, easily available raw materials, simple structure, concise synthetic route, simple post-treatment, easy industrialization, high selectivity to biological sulfydryl and quick response time.
Drawings
FIG. 1 shows the UV absorption spectrum of the prepared probe CHT in a mixed solution of tetrahydrofuran and PBS (pH 7.4,0.01M), and the UV absorption spectrum of the prepared probe CHT after Cys, GSH and Hcy are added respectively;
FIG. 2 is a fluorescence emission spectrum of the prepared probe CHT in a mixed solution of tetrahydrofuran and PBS (pH 7.4,0.01M), and a fluorescence emission spectrum of the probe CHT after Cys, GSH and Hcy are added respectively;
FIG. 3 is a graph showing the selectivity of the probe CHT;
FIG. 4 is a scattergram of the change in fluorescence intensity of the time response of probe CHT to Cys, GSH, Hcy (based on the peak height at the maximum, 568nm, 497 nm);
FIG. 5 is a graph showing the cytotoxicity test of the prepared probe CHT;
FIG. 6 is an image of a live cell from which the probe CHT was prepared.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
The chemicals and solvents used in examples 1-6 were purchased from commercial sources and used without further purification. Thin Layer Chromatography (TLC) was performed on silica gel plates, and column chromatography was performed using 300-400 mesh silica gel (HaiLang, Qingdao). Bruker AV-400 spectrometer (in deuterated dimethyl sulfoxide, 1H and 13C NMR were recorded using Me4Si as an internal standard) in ppm to indicate chemical shifts.
Example 1:
synthesis of fluorescent Probe CHT:
(1) preparation of 2- (3,5, 5-trimethylcyclohex-2-en-1-ylidene) malononitrile (a):
in a 50mL round-bottom flask, isophorone (1.9g, 13.7mmol), malononitrile (1.36g, 20.6 mmol), piperidine (450. mu.l), ethanol (25mL) were added, respectively, and the mixture was refluxed for 12 hours under nitrogen atmosphere. After cooling, the reaction solution was poured into 50ml of ice water to precipitate a solid, which was then filtered to obtain a white solid (2- (3,5, 5-trimethylcyclohex-2-en-1-ylidene) malononitrile (A)) with a yield of 85%.
(2) Preparation of 2,2'- (((1E, 1' E) - (4-hydroxy-1, 3-phenylene) bis (ethylene-2, 1-diyl)) bis (5, 5-dimethyl-2-en-3-yl-1-ylidene)) dipropionitrile (B):
in a 25mL round-bottom flask, 4-hydroxy isophthalaldehyde (0.300g, 2mmol), 2- (3,5, 5-trimethylcyclohex-2-en-1-ylidene) malononitrile (A) (0.744g, 4mml), piperidine (2d), and absolute ethanol (20mL) were added, and the mixture was refluxed for 4 hours under nitrogen protection. Cooled and the solvent removed under reduced pressure. Column chromatography with dichloromethane gave an orange-red solid with a yield of 45%. 1H NMR (400MHz, DMSO-d6), δ 10.72(s,1H),8.13(d, J ═ 1.2Hz,1H),7.53(d, J ═ 6.8Hz,1H),7.44(d, J ═ 3.2Hz,2H),7.26(d, J ═ 16.4Hz,2H),6.93(d, J ═ 8.4Hz,1H),6.84(d, J ═ 10.8Hz,2H),2.60(s,4H),2.53(s,4H),1.02(d, J ═ 3.6Hz, 12H);
(3) preparation of 2, 4-bis ((E) -2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl 2, 4-dinitrobenzenesulfonate (CHT):
2, 4-dinitrobenzenesulfonyl chloride (0.770g, 0.29mmol), the compound B2, 2'- (((1E, 1' E) - (4-hydroxy-1, 3-phenylene) bis (ethylene-2, 1-diyl)) bis (5, 5-dimethyl-2-en-3-yl-1-ylidene)) dipropionitrile (0.100g, 0.21mmol) obtained in step (2), triethylamine (0.042g, 0.40mmol) were mixed in anhydrous acetonitrile (8ml), and the reaction mixture was reacted at 40 ℃ for 0.5 hour under nitrogen. The reaction was followed by TLC and a pale yellow solid precipitated which was filtered to give the desired product in 40% yield.
The basic data for the probe are as follows:
pale yellow solid powder
1H NMR(400MHz,DMSO-d6),δ9.179(d,J＝2.4,1H),8.612(d,J＝2Hz,1H),8.248(d,J＝1.6Hz,2H),7.757(d,J＝2Hz,1H),7.534(d,J＝16Hz,1H),7.383(d,J＝8.8Hz,1H),7.326(d,J＝3.6Hz,1H),7.286(d,J＝4Hz,1H),7.020(d,J＝16.4Hz,1H),6.940(s,1H),6.816(s,1H),2.503(d,J＝3.2Hz,8H),1.011(d,J＝14Hz,12H)；
13CNMR(400MHz，DMSO-d6)，δ170.70，170.47，155.52，154.40，152.10，148.59，146.90，136.88，134.09，133.60,128.21，128.00，124.84，124.13，121.25，114.09，113.92，113.40，113.13，78.69，77.88，42.75，42.57，38.70，38.46，32.16，27.89，27.75HRMS(ES+):C39H33N6O7S[M]Calculated values: 716.2053, actual value: 716.1953.
example 2:
uv absorption spectrum of probe CHT in a mixed solution of tetrahydrofuran and PBS (pH 7.4,0.01M), and change in uv absorption of probe CHT after addition of Cys, GSH, Hcy, respectively.
Probe CHT was dissolved in tetrahydrofuran and the mixture was prepared at 1X 10﹣3M probe stock solution, 30 μ l of the probe stock solution was added to a mixed solution of 3mL tetrahydrofuran and PBS (pH 7.4,0.01M) at a volume ratio of 1:1 so that the final concentration of the probe was 10 μ M, and the ultraviolet absorption spectrum of the probe was measured to have a maximum absorption at 375 nm. When Cys, GSH, Hcy (500. mu.M) was added, the UV absorption of the probe gradually decreased at 375nm, gradually increased at 475nm, and isosbestic points appeared at 425nm, as shown in FIG. 1.
Example 3:
fluorescence emission spectrum of probe CHT in a mixed solution of tetrahydrofuran and PBS (pH 7.4,0.01M), and change in fluorescence emission of probe CHT after addition of Cys, GSH, Hcy, respectively.
Probe CHT was dissolved in tetrahydrofuran and the mixture was prepared at 1X 10﹣3M probe mother liquor, 30 mul of the probe mother liquor is added into 3mL of mixed solution of tetrahydrofuran and PBS (pH 7.4,0.01M) during testing, wherein the volume ratio of the tetrahydrofuran to the PBS is 1:1, so that the final concentration of the probe is 10 mul, the absorption wavelength of an isoabsorption point is 425nm as an excitation wavelength, the probe is found to have a maximum emission peak at 497nm, the emission peak at 497nm of the probe is gradually reduced after Cys, GSH and Hcy (500 muM) are added, and the emission peak at 568nm is gradually increased, as shown in figure 2, the probe body shows good ratiometric response to biological sulfydryl.
Example 4:
selective testing of probe CHT.
Next, to test whether other substances interfere with the detection, we further investigated the fluorescent response of probe CHT to other analytes. There was no significant change in fluorescence for probe CHT in the presence of glycine (Gly), L-proline (Pro), aspartic acid (Asp), L-tyrosine (Tyr), L. Arginine (arginine), L-glutamic acid (Glu), L-alanine (Ala), L-threonine (Thr), phenylalanine (Phe), DL-serine (Ser), L-methionine (Met), glutamine (Gln). In contrast, upon addition of the thiol-containing amino acid (Cys, GSH, Hcy), a red shift of the emission wavelength from the original 497nm to 568nm was seen. The same result can be expressed in the ratio of fluorescence intensities and is more intuitive, as shown in fig. 3. The above experiments demonstrate that probe CHT has high selectivity for three thiol-containing amino acids.
Example 5:
and preparing the time response of the probe CHT to Cys, GSH and Hcy.
We investigated the time-dependent fluorescence change of probe CHT for Cys, GSH, Hcy, as shown in FIG. 4. After 500 μ M of bio-thiol was added to PBS and THF buffer solution containing 10 μ M CHT, we can rapidly observe the change of CHT fluorescence intensity ratio within 6min, respond to Cys faster than GSH and Hcy, and reach steady state within 90 s. The probe CHT is proved to have a quick response mechanism to biological sulfhydryl.
Example 6:
cytotoxicity test of probe CHT.
The cytotoxicity of probe CHT was tested by MTT method, and log phase CHO-k1 cells were collected, plated, and 100. mu.l complete medium (10% calf serum and 90% F-12 medium), 5% CO, was added to each well of 96-well plate2Incubation at 37 ℃ until cell monolayers adhere to the walls, adding a probe CHT with a concentration gradient, incubating for 12h and 24h respectively, adding 100. mu.l of MTT (1g/L) solution into each well, incubating for 4h in an oven at 37 ℃, absorbing the MTT solution, adding 150. mu.l of DMSO solution into each well, shaking for 10min in a shaking table, measuring absorbance by a microplate reader, and processing data by origin to obtain a graph 5.
As can be seen from FIG. 5, when the concentration of the probe is 70 μ M, the cell survival rate is still above 80% after incubation for 24h, which indicates that the biocompatibility of the probe is high, and the application of the probe in the subsequent biological sample test is facilitated.
Example 7:
confocal imaging of living cells to produce probe CHT.
CHO-K1 cells were first incubated with 50 μ M probe CHT for 30 minutes, allowed to enter the cells, washed three times with PBS, and observed by confocal laser microscopy. The green fluorescence emission channel (channel 1: 450-. After incubating the cells with probe CHT for 10min, and then adding Cys (200. mu.M) for 20 min, the green channel was darkened and the red channel was brightened by confocal laser microscopy. When CHO-K1 cells were pretreated with NEM (200. mu.M) for 30 minutes and then incubated with addition of probe CHT for 30 minutes, the red channel darkened and the green channel brightened as observed by confocal laser microscopy. When Cys was added to CHO-K1 cells previously treated with NEM, the red channel reopened and the green channel darkened as observed by confocal laser microscopy. The other two thiol-containing amino acids (GSH, Hcy) showed the same behavior, and the cell diagram is shown in fig. 6.
The verification result shows that the probe CHT can realize ratio detection of the sulfhydryl-containing amino acid in the organism.
Wherein NEM isN-ethyl maleimide can eliminate the substance containing sulfhydryl in cell.
Example 8
A preparation method of a dual-emission fluorescent dye probe comprises the following steps:
(1) adding isophorone, malononitrile and a catalyst into an organic solvent, carrying out reflux reaction for 10-20 h under the protection of nitrogen, cooling, pouring the reaction liquid into ice water, separating out a solid, and filtering to obtain white solid isophorone malononitrile (formula IV); the catalyst comprises piperidine, and the organic solvent comprises ethanol; the molar ratio of the isofluranone to the malononitrile to the catalyst to the organic solvent is 1: (1-2): (0.2-0.4): (25-35).
The molar ratio of the isofluranone, the malononitrile, the catalyst and the organic solvent is preferably 1: 1.5: 0.35: 32.
(2) adding the isophorone malononitrile, 4-hydroxy isophthalaldehyde and piperidine obtained in the step (1) into absolute ethyl alcohol, performing reflux reaction for 2-6 hours under the protection of nitrogen, cooling, removing the solvent under reduced pressure, and performing column chromatography by using dichloromethane to obtain an orange-red solid (formula VI);
the molar ratio of the isophorone malononitrile to the 4-hydroxy isophthalaldehyde is 1: (1-3);
the molar volume ratio of the isophorone malononitrile to the piperidine is as follows: 1, (0.5-2) mmol/d;
the molar volume ratio of the isophorone malononitrile to the absolute ethyl alcohol is 1: 8-12 mmol/ml.
(3) Adding 2, 4-dinitrobenzenesulfonyl chloride, the compound of formula VI obtained in the step (2) and triethylamine into anhydrous acetonitrile, and reacting the reaction mixture for 0.2-1 hour at 30-50 ℃ under the protection of nitrogen. Tracking the reaction by TLC, separating out light yellow solid, and filtering to obtain a target product;
the molar ratio of the VI compound to the 2, 4-dinitrobenzene sulfonyl chloride to the triethylamine is 1: (1-2): (1.5-2.5);
the molar volume ratio of the VI compound to the anhydrous acetonitrile is 1: 35-40 mmol/ml.
The preferable molar ratio of the VI compound in the step (3), the 2, 4-dinitrobenzenesulfonyl chloride and the triethylamine is 1: 1.4: 2.
the obtained probe is used for detecting in vitro biological sulfhydryl and biological sulfhydryl in biological living cells.
Example 8
A preparation method of a dual-emission fluorescent dye probe comprises the following steps:
(1) adding isophorone, malononitrile and a catalyst into an organic solvent, carrying out reflux reaction for 10 hours under the protection of nitrogen, cooling, pouring the reaction liquid into ice water, separating out a solid, and filtering to obtain white solid isophorone malononitrile (formula IV); the catalyst comprises piperidine, and the organic solvent comprises ethanol; the molar ratio of the isofluranone to the malononitrile to the catalyst to the organic solvent is 1: 1: 0.2: 25.
(2) adding the isophorone malononitrile, 4-hydroxy isophthalaldehyde and piperidine obtained in the step (1) into absolute ethyl alcohol, carrying out reflux reaction for 2 hours under the protection of nitrogen, cooling, removing the solvent under reduced pressure, and carrying out column chromatography by using dichloromethane to obtain an orange-red solid (formula VI);
the molar ratio of the isophorone malononitrile to the 4-hydroxy isophthalaldehyde is 1: 1;
the molar volume ratio of the isophorone malononitrile to the piperidine is as follows: 1:0.5 mmol/d;
the molar volume ratio of the isophorone malononitrile to the absolute ethyl alcohol is 1:8 mmol/ml.
(3) Adding 2, 4-dinitrobenzenesulfonyl chloride, the compound of formula VI obtained in the step (2) and triethylamine into anhydrous acetonitrile, and reacting the reaction mixture at 30 ℃ for 1 hour under the protection of nitrogen. Tracking the reaction by TLC, separating out light yellow solid, and filtering to obtain a target product;
the molar ratio of the VI compound to the 2, 4-dinitrobenzene sulfonyl chloride to the triethylamine is 1: 1: 1.5;
the molar volume ratio of the VI compound to the anhydrous acetonitrile is 1: 35 mmol/ml.
The obtained probe is used for detecting in vitro biological sulfhydryl and biological sulfhydryl in biological living cells.
Example 9
A preparation method of a dual-emission fluorescent dye probe comprises the following steps:
(1) adding isophorone, malononitrile and a catalyst into an organic solvent, carrying out reflux reaction for 20 hours under the protection of nitrogen, cooling, pouring the reaction liquid into ice water, separating out a solid, and filtering to obtain white solid isophorone malononitrile (formula IV); the catalyst comprises piperidine, and the organic solvent comprises ethanol; the molar ratio of the isofluranone to the malononitrile to the catalyst to the organic solvent is 1: 2: 0.4: 35.
(2) adding the isophorone malononitrile, 4-hydroxy isophthalaldehyde and piperidine obtained in the step (1) into absolute ethyl alcohol, carrying out reflux reaction for 6 hours under the protection of nitrogen, cooling, removing the solvent under reduced pressure, and carrying out column chromatography by using dichloromethane to obtain an orange-red solid (formula VI);
the molar ratio of the isophorone malononitrile to the 4-hydroxy isophthalaldehyde is 1: 3;
the molar volume ratio of the isophorone malononitrile to the piperidine is as follows: 1:2 mmol/d;
the molar volume ratio of the isophorone malononitrile to the absolute ethyl alcohol is 1:12 mmol/ml.
(3) Adding 2, 4-dinitrobenzenesulfonyl chloride, the compound of formula VI obtained in the step (2) and triethylamine into anhydrous acetonitrile, and reacting the reaction mixture at 50 ℃ for 0.2 hour under the protection of nitrogen. Tracking the reaction by TLC, separating out light yellow solid, and filtering to obtain a target product;
the molar ratio of the VI compound to the 2, 4-dinitrobenzene sulfonyl chloride to the triethylamine is 1: 2: 2.5;
the molar volume ratio of the VI compound to the anhydrous acetonitrile is 1: 40 mmol/ml.
The obtained probe is used for detecting in vitro biological sulfhydryl and biological sulfhydryl in biological living cells.
Claims (9)
1. A dual-emission fluorescent dye probe is characterized in that the structural formula of the probe is shown as the following formula I:
2. a method for preparing the dual emission fluorescent dye probe of claim 1, comprising the steps of:
(1) adding isophorone, malononitrile and a catalyst into an organic solvent, carrying out reflux reaction for 10-20 h under the protection of nitrogen, cooling, pouring the reaction liquid into ice water, separating out a solid, and filtering to obtain white solid isophorone malononitrile (formula IV);
(2) adding the isophorone malononitrile, 4-hydroxy isophthalaldehyde and piperidine obtained in the step (1) into absolute ethyl alcohol, performing reflux reaction for 2-6 hours under the protection of nitrogen, cooling, removing the solvent under reduced pressure, and performing column chromatography by using dichloromethane to obtain an orange-red solid (formula VI);
(3) adding 2, 4-dinitrobenzenesulfonyl chloride, the compound of formula VI obtained in the step (2) and triethylamine into anhydrous acetonitrile, and reacting the reaction mixture for 0.2-1 hour at 30-50 ℃ under the protection of nitrogen; tracking the reaction by TLC, separating out light yellow solid, and filtering to obtain a target product;
3. the method for preparing the dual emission fluorescent dye probe as claimed in claim 2, wherein the catalyst in step (1) is piperidine, and the organic solvent is ethanol.
4. The method for preparing a dual emission fluorescent dye probe as set forth in claim 2, wherein the molar ratio of the isophorone, the malononitrile, the catalyst and the organic solvent in step (1) is 1: (1-2): (0.2-0.4): (25-35).
5. The method for preparing a dual emission fluorescent dye probe as set forth in claim 4, wherein the molar ratio of the isophorone, the malononitrile, the catalyst and the organic solvent is 1: 1.5: 0.35: 32.
6. the method of preparing a dual emission fluorescent dye probe as set forth in claim 2, wherein the molar ratio of isophorone malononitrile to 4-hydroxyisophthalaldehyde in step (2) is 1: (1-3);
the molar volume ratio of the isophorone malononitrile to the piperidine is as follows: 1, (0.5-2) mmol/drop;
the molar volume ratio of the isophorone malononitrile to the absolute ethyl alcohol is 1: 8-12 mmol/ml.
7. The method for preparing the dual-emission fluorescent dye probe as claimed in claim 2, wherein the molar ratio of the compound VI in the step (3), the 2, 4-dinitrobenzenesulfonyl chloride and the triethylamine is 1: (1-2): (1.5-2.5);
the molar volume ratio of the VI compound to the anhydrous acetonitrile is 1: 35-40 mmol/ml.
8. The method for preparing the dual-emission fluorescent dye probe as claimed in claim 7, wherein the molar ratio of the compound VI in the step (3), the 2, 4-dinitrobenzenesulfonyl chloride and the triethylamine is 1: 1.4: 2.
9. use of the dual emission fluorescent dye probe of claim 1 for the non-disease diagnosis and treatment of biological thiols in vitro and biological thiols in living cells.
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