CN115850314B - Peroxynitrite and lipid drop dual-response probe and preparation method and application thereof - Google Patents
Peroxynitrite and lipid drop dual-response probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a peroxynitrite and lipid drop dual-response probe, a preparation method and application thereof, wherein the structural formula of the probe is as follows: The preparation method comprises the following steps: reacting 4-fluoro-2-hydroxybenzaldehyde and 4- (bromomethyl) phenylboronic acid pinacol ester in a first solvent in the presence of alkali metal carbonate to obtain an intermediate product CN-1; reacting an intermediate product CN-1 with benzothiazole-2-acetonitrile in a second solvent in the presence of triethylamine to obtain an intermediate product CN-2; intermediate CN-2 and 3, 5-dimethyl piperidine react in a third solvent to obtain the peroxynitrite and lipid drop dual-response probe. The fluorescent probe has strong selectivity to ONOO ‑ and LDS MIMICS, and can emit strong light in a system in which ONOO ‑ and LDS MIMICS coexist, so that the generation of foam cells can be accurately judged.
Description
Technical Field
The invention relates to the technical field of fluorescent probes, in particular to a peroxynitrite and lipid drop dual-response probe, and a preparation method and application thereof.
Background
Foam cells are one of the main cells that make up the core of atherosclerotic plaques, and are converted by macrophages to phagocytose oxidized low density lipoprotein (ox-LDL). Excessive accumulation of ox-LDL triggers the formation of Lipid Droplets (LDs) in foam cells. Lipid droplets are an organelle in cells responsible for lipid metabolism, consisting mainly of neutral lipids, which are important markers for foam cells. Thus, currently available fluorescent probes for identifying foam cells typically rely on lipid droplet markers such as oil red O, AIEgens and other types of lipid droplet probes and the like. In addition, since macrophages in plaque are stimulated by ox-LDL inflammatory signals, and excessive Reactive Oxygen Species (ROS) are generated, the foaming of macrophages can also be judged to some extent by ROS probes.
However, lipid drop probes are not selective for other lipid-bearing organelles and tissue cells, such as being unable to distinguish between foam cells and fat cells, and thus adipose tissue near the arterial wall can severely interfere with detection of foam cells in plaque, causing false positive results. In addition, in addition to ox-LDL, various inflammatory signals (such as LPS, PMA or cytokines, etc.) can stimulate macrophages to produce ROS, and the detection of false positive results from foam cells using ROS probes is more widespread. Thus, there is still a lack of a highly selective fluorescent probe for foam cells.
Disclosure of Invention
The invention aims to provide a peroxynitrite and lipid drop dual response probe which has strong selectivity to peroxynitrite (ONOO -) and LDs, and is an ONOO - and LDs dual response probe which has more accurate foam cell recognition capability.
The invention also aims to provide a preparation method of the peroxynitrite and lipid drop dual-response probe, which is simple and has controllable parameters, and is suitable for industrial mass production.
The third object of the invention is to provide the application of the peroxynitrite and lipid drop dual response probe in foam cell specificity detection, and the probe has more accurate recognition capability when being used for detecting foam cells.
The invention solves the technical problems by adopting the following technical scheme.
The invention provides a peroxynitrite and lipid drop dual-response probe, which has the structural formula as follows:
The invention provides a preparation method of a peroxynitrite and lipid drop dual-response probe, which comprises the following steps:
S1, in the presence of alkali carbonate, 4-fluoro-2-hydroxybenzaldehyde and 4- (bromomethyl) phenylboronic acid pinacol ester react in a first solvent for 22-26 h to obtain an intermediate product CN-1;
s2, reacting the intermediate product CN-1 with benzothiazole-2-acetonitrile in a second solvent for 1.5-2.5 h in the presence of triethylamine to obtain an intermediate product CN-2;
S3, reacting the intermediate product CN-2 and 3, 5-dimethylpiperidine in a third solvent to obtain the peroxynitrite and lipid drop dual-response probe.
The invention also provides application of the peroxynitrite and lipid drop dual-response probe in foam cell specificity detection.
The peroxynitrite and lipid drop dual-response probe and the preparation method and application thereof have the beneficial effects that:
The invention designs AND synthesizes an AND logic fluorescent probe with double responses of peroxynitrite AND lipid droplets. The fluorescent probe has strong selectivity to ONOO - and LDS MIMICS, which can emit strong light in a system where ONOO - and LDS MIMICS are present simultaneously. The peroxynitrite and lipid drop dual-response probe provided by the invention can judge the generation of foam cells more accurately through the cross identification of the lipid drop and the peroxynitrite.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a 1 H-NMR spectrum of CN-1 of example 1 of the present invention;
FIG. 2 is a 13 C-NMR spectrum of CN-1 of example 1 of the present invention;
FIG. 3 is a high resolution mass spectrum of CN-1 of example 1 of the present invention;
FIG. 4 is a 1 H-NMR spectrum of CN-2 of example 1 of the present invention;
FIG. 5 is a 13 C-NMR spectrum of CN-2 of example 1 of the present invention;
FIG. 6 is a high resolution mass spectrum of CN-2 of example 1 of the present invention;
FIG. 7 is a 1 H-NMR spectrum of CNP2-B of example 1 of the present invention;
FIG. 8 is a 13 C-NMR spectrum of CNP2-B of example 1 of the present invention;
FIG. 9 is a high resolution mass spectrum of CNP2-B of example 1 of the present invention;
FIG. 10A is a graph showing the comparison of fluorescence intensity of CNP2-B of example 1 of the present invention in different response systems;
FIG. 10B is a graph showing the trend of the effect of reaction time on the fluorescence intensity of CNP2-B of example 1;
FIG. 11A is a plot of the trend of LDS MIMICS concentration changes on the fluorescence intensity of CNP2-B of example 1;
FIG. 11B is a graph showing the effect of ONOO - concentration change on the fluorescence intensity of CNP2-B of example 1;
FIG. 12 is a graph showing the selectivity of CNP2-B to organic bioactive molecules according to example 1 of the present invention;
FIG. 13 is a graph showing the selectivity of CNP2-B to inorganic ions according to example 1 of the present invention;
FIG. 14 is a fluorescence image of stained mouse macrophages with the probe CNP2-B of example 1 and the probe HPF of comparative example 1, respectively, under different stimuli.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The peroxynitrite and lipid drop dual-response probe and the preparation method and application thereof are specifically described below.
The structural formula of the peroxynitrite and lipid drop dual-response probe provided by the embodiment of the invention is as follows:
The peroxynitrite and lipid drop dual response probe of the present invention has strong selectivity for ONOO - and LDS MIMICS. Compared with the existing single-response lipid drop probe or ONOO-probe, the peroxynitrite and lipid drop dual-response probe can emit strong light in a system with ONOO - and LDS MIMICS simultaneously, so that foam cells can be identified more accurately. Compared with the existing peroxynitrite KC-ONOO probe, the peroxynitrite and lipid drop dual-response probe has stronger fluorescence intensity in a system in which ONOO - and LDS MI MICS coexist, so that foam cells can be better identified.
The invention also provides a preparation method of the peroxynitrite and lipid drop dual-response probe, which comprises the following steps:
s1, reacting 4-fluoro-2-hydroxybenzaldehyde and 4- (bromomethyl) phenylboronic acid pinacol ester in a first solvent for 22-26 h in the presence of alkali metal carbonate to obtain an intermediate product CN-1. Wherein the reaction temperature is room temperature. The room temperature is generally understood in the art to be 10 to 30 ℃.
Further, in a preferred embodiment of the present invention, the alkali metal carbonate is selected from potassium carbonate, the first solvent is selected from acetonitrile, and the mass-to-volume ratio of the 4-fluoro-2-hydroxybenzaldehyde, the potassium carbonate, the acetonitrile and the 4- (bromomethyl) phenylboronic acid pinacol ester is 4.8 to 5.2: 12-13: 1: 10 to 11 (mg/mg/mL/mg).
Further, in a preferred embodiment of the present invention, the reaction further comprises the following steps: the first solvent is removed from the reacted product under reduced pressure, and then the product is purified by a silica gel column to obtain an intermediate product CN-1, wherein the purified eluent is a mixed solution of dichloromethane/methanol (DCM/MeOH), and the volume ratio of dichloromethane to methanol in the mixed solution of dichloromethane/methanol is 100:1.
S2, reacting the intermediate product CN-1 with benzothiazole-2-acetonitrile in a second solvent for 1.5-2.5 h in the presence of triethylamine to obtain an intermediate product CN-2. Wherein the reaction temperature is room temperature.
Further, in a preferred embodiment of the present invention, the second solvent is selected from a mixed solution of ethanol and dichloromethane (EtOH/DCM), wherein the volume ratio of ethanol to dichloromethane is 1:1, wherein the mass volume ratio of the intermediate product CN-1 to the triethylamine to the ethanol/dichloromethane mixed solution to the benzothiazole-2-acetonitrile is 195-205: 1:38 to 42: 97-99 (mg/mL/mL/mg).
Further, in a preferred embodiment of the present invention, the reaction further comprises the following steps: the product after the reaction was subjected to removal of the second solvent under reduced pressure, suspended with ethyl acetate, and filtered to obtain intermediate CN-2.
S3, reacting the intermediate product CN-2 and 3, 5-dimethylpiperidine in a third solvent to obtain the peroxynitrite and lipid drop dual-response probe.
Further, in a preferred embodiment of the present invention, the mass-to-volume ratio of the intermediate product CN-2, the 3, 5-dimethylpiperidine and the third solvent is 195 to 205:1:18 to 22 (mg/mL/mL), wherein the third solvent is selected from N, N-Dimethylformamide (DMF).
Further, in the preferred embodiment of the present invention, the reaction temperature is 45 to 55℃and the reaction time is 22 to 26 hours.
Further, in a preferred embodiment of the present invention, the reaction further comprises the following steps: extracting the reacted product with ethyl acetate and water, removing the ethyl acetate under reduced pressure, and purifying by a silica gel column to obtain the peroxynitrite and lipid drop dual response probe, wherein the purified eluent is petroleum ether/ethyl acetate (PE/EA) mixed solution, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether/ethyl acetate mixed solution is 20:1.
The specific synthetic route of the peroxynitrite and lipid drop dual response probe is as follows:
The invention also provides application of the peroxynitrite and lipid drop dual-response probe in foam cell specificity detection.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The peroxynitrite and lipid drop dual-response probe provided by the embodiment is prepared according to the following method:
(1) Compound 4-fluoro-2-hydroxybenzaldehyde (100 mg, 0.719 mmol) and K 2CO3 (246.59 mg,1.78 mmol) were added to 20mL acetonitrile, stirred for 5 minutes, followed by the addition of compound 4- (bromomethyl) phenylboronic acid pinacol ester (211.97 mg,0.73 mmol) and stirred for 24 hours overnight. The solvent was then removed under reduced pressure. The product was purified using a silica gel column (DCM/meoh=100:1) to give intermediate CN-1 (167.2 mg, yield: 65.77%) as a pale yellow solid.
(2) Compound CN-1 (100.0 mg,0.280 mmol) and 0.5mL of Et 3 N were added to a mixed solution of EtOH/DCM (10 mL:10 mL) and stirred at room temperature for 5 minutes, then benzothiazole-2-acetonitrile (48.91 mg,0.280 mmol) was added to the reaction solution and reacted at room temperature for 2h. After removal of the solvent under reduced pressure, the product was suspended with 20mL of EA and then filtered to give an orange solid, intermediate CN-2 (128.91 mg, yield: 89.61%).
(3) Compound CN-2 (100.0 mg,0.195 mmol) and 0.5mL of 3, 5-dimethylpiperidine were added to 10mL of DMF and reacted at 50℃for 24h. The solvent was then extracted with EA and water, and EA was removed under reduced pressure. Finally, the product was purified by a silica gel column (PE/ea=20:1) to give an orange solid, namely peroxynitrite and lipid drop dual response probe (CNP 2-B) (47.28 mg, yield: 40%).
Example 2
The peroxynitrite and lipid drop dual-response probe provided by the embodiment is prepared according to the following method:
(1) Compound 4-fluoro-2-hydroxybenzaldehyde (100 mg, 0.719 mmol) and K 2CO3 (246.59 mg,1.78 mmol) were added to 20mL acetonitrile, stirred for 5 minutes, followed by the addition of compound 4- (bromomethyl) phenylboronic acid pinacol ester (211.97 mg,0.73 mmol) and stirred for 24 hours overnight. The solvent was then removed under reduced pressure. The product was purified using a silica gel column (DCM/meoh=100:1) to give a pale yellow solid, intermediate CN-1.
(2) Compound CN-1 (100.0 mg,0.280 mmol) and 0.5mL of Et 3 N were added to a mixed solution of EtOH/DCM (10 mL:10 mL) and stirred at room temperature for 5 minutes, then benzothiazole-2-acetonitrile (48.91 mg,0.280 mmol) was added to the reaction solution and reacted at room temperature for 2h. After removal of the solvent under reduced pressure, the product was suspended with 20mL EA and then filtered to give an orange solid, intermediate CN-2.
(3) Compound CN-2 (100.0 mg,0.195 mmol) and 0.5mL of 3, 5-dimethylpiperidine were added to 10mL of DMF and reacted at 45℃for 26h. The solvent was then extracted with EA and water, and EA was removed under reduced pressure. Finally, the product was purified by a silica gel column (PE/ea=20:1) to give an orange solid, namely peroxynitrite and lipid drop dual response probe (CNP 2-B).
Comparative example 1
This comparative example provides an ONOO - single-response probe HPF, which is commercially available, for example, from beijing coupling technologies, inc.
Test example 1
This experimental example uses hydrogen, carbon, and high resolution mass spectra to characterize the structures of intermediate CN-1, intermediate CN-2, and CNP2-B of example 1, respectively.
FIG. 1 shows the 1 H-NMR spectrum of CN-1 of example 1; FIG. 2 shows the 13 C-NMR spectrum of CN-1 of example 1; FIG. 3 shows a high resolution mass spectrum of CN-1 of example 1. As can be seen from fig. 1 to 3, the intermediate CN-1 was successfully synthesized in this example 1, and the structural formula thereof is:
FIG. 4 shows the 1 H-NMR spectrum of CN-2 of example 1; FIG. 5 shows the 13 C-NMR spectrum of CN-2 of example 1; FIG. 6 shows a high resolution mass spectrum of CN-2 of example 1. As can be seen from fig. 4 to 6, the intermediate CN-2 was successfully synthesized in this example 1, and the structural formula thereof is as follows:
FIG. 7 shows 1 H-NMR spectrum of CNP2-B of example 1; FIG. 8 shows 13 C-NMR spectrum of CNP2-B of example 1; fig. 9 shows a high resolution mass spectrum of CNP2-B of example 1. As can be seen from fig. 7 to 9, in this example 1, CNP2-B was successfully synthesized, and the structural formula thereof was as follows:
test example 2
This test example analyzes the dual response characteristic and time-dependent response of CNP2-B of example 1 in vitro, and comprises the following specific steps:
ONOO - preparation: 10mL of 0.7M H 2O2 was added to 10mL of 0.6M NaNO 2, stirred at 0deg.C for 5 minutes, 10mL of 0.6M HCl was added quickly, then 20mL of 1.5M NaOH was added quickly, stirring was continued for 5 minutes and mixing was complete, 500mg of manganese dioxide was added and stirred for 10 minutes to remove excess H 2O2, and the mixed solution was filtered to remove manganese dioxide. Finally, ONOO - is obtained, and the product is stored at-20 ℃ for standby after split charging. The concentration of ONOO - was determined by measuring the absorption of the solution at 302nm before use using the extinction coefficient epsilon=1670 cm -1M-1.
1MM of the CNP2-B probe stock of example 1 was prepared for use. CNP2-B final concentration was made to 10. Mu.M in a 3mL system, 10. Mu.M of ONOO - and/or 200. Mu.g/ML LDS MIMICS were added, respectively, and the reaction was carried out for 120 minutes, and the emission spectrum and fluorescence intensity of the probe were measured under excitation light at 430 nm.
FIG. 10A is a graph showing the comparison of fluorescence intensity of CNP2-B of example 1 in different response systems. FIG. 10B is a graph showing the trend of the effect of reaction time on the fluorescence intensity of CNP2-B of example 1. It can be seen from FIGS. 10A AND 10B that in the presence of ONO - or lipid drop mimics (LDS MIMICS,200 μg/mL) alone, CNP2-B does not fluoresce, but only in the system where ONO - AND LDS MIMICS are present, CNP2-B emits intense light, demonstrating that CNP2-B is a typical "AND" logic probe for both ONO - AND LDS MIMICS.
Test example 3
This test example analyzes the concentration-dependent response of CNP2-B of example 1 in vitro, and comprises the following specific steps:
1mM of the CNP2-B probe stock of example 1 was prepared for use. CNP2-B final concentration reaches 10 mu M in 3mL system, and ONO - and/or ONO ML LDS MIMICS are/is added respectively to react for 120 min, and the emission spectrum and fluorescence intensity of the probe are detected under 430nm excitation light.
FIG. 11A is a graph showing the trend of LDS MIMICS concentration changes on the fluorescence intensity of CNP2-B of example 1. FIG. 11B is a graph showing the effect of ONOO - concentration change on the fluorescence intensity of CNP2-B of example 1. As can be seen from fig. 11A and 11B, in the case of controlling the input of one of the responders, the fluorescence intensity of CNP2-B was enhanced with the increase of the concentration of the other one, exhibiting good linear correlation.
Test example 4
This test example analyzes the response selectivity of CNP2-B of example 1 in vitro, specifically comprising the following steps:
1mM of the CNP2-B probe stock of example 1 was prepared for use. In a 3mL system, CNP2-B is brought to a final concentration of 10 mu M, and 200 mu g/mL of LDS MIMICS, BSA, HSA, DNA, RNA, cys, GSH, ADP, ATP and Glucose are added in the presence of 10 mu M of ONOO -; 100μM ClO-,H2O2,ClO-,IO4 -,NO2 -,Ca2+,Cu2+,Fe3+,K+,Zn2+, was added thereto in the presence of 200. Mu.g/ML LDS MIMICS for reaction for 120 minutes, and the emission spectrum and fluorescence intensity of the probe were measured under excitation light of 430 nm.
FIG. 12 is a graph showing the selectivity of CNP2-B for organic bioactive molecules of example 1. As can be seen from fig. 12, CNP2-B only responds to ONOO - in the presence of LDS MIMICS.
FIG. 13 is a graph showing the selectivity of CNP2-B to inorganic ions of example 1. As can be seen from fig. 13, CNP2-B only has a strong response to LDS MIMICS in the presence of ONOO -, demonstrating that CNP2-B has a strong selectivity to ONOOs - and LDS MIMICS.
Test example 5
This experimental example separately analyzes the foam cell specific recognition imaging effect of the CNP2-B of example 1 and the ONOO - single-response probe HPF of comparative example 1, and specifically includes the steps of:
RAW264.7 mouse macrophages were cultured at the cellular level, transformed into foam cells by ox-LDL-stimulating cells, and inflammatory responses were produced by stimulating cells with LPS or PMA to give ox-LDL-stimulated groups, which were then stained with the probe CNP2-B of example 1 and the probe HPF of comparative example 1, respectively. The ox-LDL+NAC stimulated group was additionally taken and intracellular ROS were cleared using NAC, and then stained and fluorescence imaged using probes CNP2-B of example 1 and probe HPF of comparative example 1, respectively.
RAW264.7 mouse macrophages were cultured at the cellular level, and cells were stimulated with LPS, PMA and PBS, respectively, to produce an LPS-stimulated group, a PMA-stimulated group and a PBS-stimulated group, which were then stained and fluorescence imaged using probes CNP2-B of example 1 and probe HPF of comparative example 1, respectively.
FIG. 14 shows fluorescence images of stained mouse macrophages with the probe CNP2-B of example 1 and the probe HPF of comparative example 1, respectively, under different stimuli. FIG. 14A is a fluorescent image of stained mouse macrophages with probes CNP2-B and DAPI of example 1 under different stimuli; fig. 14B shows fluorescence images of mouse macrophages stained with the probe HPF of comparative example 1 and DAPI under different stimuli. As can be seen from FIG. 14A, only the ox-LDL-stimulated group was strongly colored with the double response probe CNP2-B, and fluorescence was not observed in the ox-LDL-stimulated group after NAC treatment. As can be seen from fig. 14B, the ONOO - single-response probe HPF failed to distinguish between LPS or PMA-induced simple inflammatory response and ox-LDL-induced foam cell formation, demonstrating that the dual-response probe CNP2-B had more precise foam cell recognition capability than the single-response lipid drop probe or ONOO-probe.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Claims (9)
1. The peroxynitrite and lipid drop dual response probe is characterized by comprising the following structural formula:
。
2. a method of preparing a peroxynitrite and lipid drop dual response probe according to claim 1 comprising the steps of:
S1, in the presence of alkali metal carbonate, reacting 4-fluoro-2-hydroxybenzaldehyde and 4- (bromomethyl) phenylboronic acid pinacol ester in a first solvent for 22-26 hours to obtain an intermediate product CN-1; wherein, the structural formula of the intermediate product CN-1 is as follows: ；
S2, reacting the intermediate product CN-1 with benzothiazole-2-acetonitrile in a second solvent for 1.5-2.5 hours in the presence of triethylamine to obtain an intermediate product CN-2; wherein, the structural formula of the intermediate product CN-2 is as follows: ；
S3, reacting the intermediate product CN-2 and 3, 5-dimethylpiperidine in a third solvent to obtain the peroxynitrite and lipid drop dual-response probe.
3. The preparation method according to claim 2, wherein in the step S1, the alkali metal carbonate is selected from potassium carbonate, the first solvent is selected from acetonitrile, and the mass-volume ratio of the 4-fluoro-2-hydroxybenzaldehyde, the potassium carbonate, the acetonitrile and the 4- (bromomethyl) phenylboronic acid pinacol ester is 4.8-5.2: 12-13: 1: 10-11 mg/mg/mL/mg.
4. The method according to claim 2, wherein in step S1, the reaction further comprises the steps of: removing the first solvent from the reacted product under reduced pressure, and purifying the product by a silica gel column to obtain an intermediate product CN-1, wherein the purified eluent is a dichloromethane/methanol mixed solution, and the volume ratio of dichloromethane to methanol in the dichloromethane/methanol mixed solution is 100:1.
5. The preparation method according to claim 2, wherein in step S2, the second solvent is selected from an ethanol/dichloromethane mixed solution, wherein in the ethanol/dichloromethane mixed solution, the volume ratio of ethanol to dichloromethane is 1:1, the mass volume ratio of the intermediate product CN-1, the triethylamine, the ethanol/dichloromethane mixed solution and the benzothiazole-2-acetonitrile is 195-205: 1: 38-42: 97-99 mg/mL/mL/mg.
6. The method according to claim 2, wherein in step S2, the reaction further comprises the steps of: the second solvent was removed from the reacted product under reduced pressure, and then suspended with ethyl acetate and filtered to obtain intermediate CN-2.
7. The preparation method according to claim 2, wherein in step S3, the mass-to-volume ratio of the intermediate CN-2, the 3, 5-dimethylpiperidine and the third solvent is 195-205: 1: 18-22 mg/mL, wherein the third solvent is selected from N, N-dimethylformamide.
8. The preparation method according to claim 2, wherein in the step S3, the reaction temperature is 45-55 ℃ and the reaction time is 22-26 h.
9. The method according to claim 2, wherein in step S3, the reaction further comprises the steps of: after extracting the reacted product with ethyl acetate and water, removing the ethyl acetate under reduced pressure, and purifying by a silica gel column to obtain the peroxynitrite and lipid drop dual-response probe, wherein the purified eluent is petroleum ether/ethyl acetate mixed solution, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether/ethyl acetate mixed solution is 20:1.
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| CN110964022A (en) * | 2019-12-24 | 2020-04-07 | 济南大学 | Fluorescent probe for detecting peroxynitrite ions and preparation method and application thereof |
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