CN112500382B - Ratiometric fluorescent probe for reversible detection of sulfur dioxide/formaldehyde, preparation method and application - Google Patents
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Abstract
The invention relates to a ratiometric fluorescent probe for reversibly detecting sulfur dioxide/formaldehyde, a preparation method and application, in particular to a ratiometric fluorescent probe for measuring, detecting or screening sulfur dioxide/formaldehyde and living cell fluorescence imaging, particularly to reversible colorimetric ratiometric detection of sulfur dioxide/formaldehyde, wherein the probe can realize at least one of the following technical effects: the kit can be used for reversibly detecting sulfur dioxide/formaldehyde, detecting colorimetric ratio, identifying sulfur dioxide/formaldehyde with high selectivity, rapidly responding to sulfur dioxide/formaldehyde, sensitively analyzing sulfur dioxide/formaldehyde, detecting sulfur dioxide/formaldehyde under physiological level conditions, and has the advantages of simple synthesis and stable property.
Description
Technical Field
The invention belongs to the field of fluorescent probes, and particularly relates to a coumarin compound-based fluorescent probe for reversibly detecting sulfur dioxide/formaldehyde and application thereof in a method for measuring, detecting or screening sulfur dioxide/formaldehyde and live cell fluorescence imaging; the invention also provides a method for preparing the fluorescent probe.
Background
In a living system, formaldehyde plays an important role in the process of maintaining carbon cycle metabolism as an active carbonyl substance, and the formaldehyde is generated endogenously by demethylase and oxidase, such as lysine specific demethylase and amino-sensitive amine oxidase. The concentration levels of formaldehyde in organisms are associated with spatial memory and cognitive abilities, however, abnormal accumulation of formaldehyde may cause protein and DNA damage, which in turn causes a variety of diseases including alzheimer's disease, embryonic malformations, respiratory diseases, heart diseases, and cancer. Formaldehyde is also reported to occur naturally in human cells and in various organisms, and under normal physiological conditions, up to 0.5mM in certain organelles can occur at relatively high concentrations in cells, and normal levels of formaldehyde in human blood range from about 0.06 mM to about 0.08mM, and because of the hazard and importance of formaldehyde in biological systems, there is a need to search for sensitive methods for detecting formaldehyde in the hope of achieving in situ detection of formaldehyde in a living organism.
In the living body, SO 2 And formaldehyde, to maintain homeostasis. Nevertheless, the potential interaction is due to lack of on-site monitoring of SO 2 And formaldehyde, SO in complex biological systems 2 The interaction with formaldehyde is still unknown. Therefore, powerful chemical tools were developed to study SO 2 And formaldehyde are very urgent and important. In a living system. These molecular tools are for understanding SO 2 And the pathophysiology of the role and relationship of formaldehyde in the organism are of great importance.
Due to high sensitivity and significant spatiotemporal resolution, fluorescent probes are used as a non-invasive tool for analyte determination in biological imaging. So far, the fluorescent probe with reversible ratio for detecting sulfur dioxide/formaldehyde in organisms is relatively lack, and the exploration of a novel high-efficiency fluorescent probe for rapidly detecting formaldehyde and sulfur dioxide in environment and living cells is still a hot problem. Therefore, the development of a rapid, highly sensitive, and highly selective fluorescent probe, especially a reversible colorimetric ratiometric fluorescent probe, is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the present invention aims to provide a sulfur dioxide/formaldehyde fluorescent probe with a reversible colorimetric ratio, which is simple to prepare, rapid, highly sensitive, and highly selective, and a preparation method and use thereof, which have the characteristics of simple synthesis, good selectivity, high sensitivity, and rapid response, and can effectively measure, detect, or screen sulfur dioxide/formaldehyde under physiological level conditions, especially can qualitatively and quantitatively analyze sulfur dioxide/formaldehyde with a colorimetric ratio.
Specifically, the invention provides a compound having a structure represented by formula (I):
in the formula (I), R 1 ，R 2 ，R 3 ，R 4 ，R 5 ，R 6 ，R 7 ，R 8 And R 9 Is independently selected from the group consisting of a hydrogen atom, a linear or branched alkyl group, a linear or branched alkoxy group, a sulfonic acid group, an ester group and a carboxyl group; and wherein R is 1 ，R 2 ，R 3 ，R 4 ，R 5 ，R 6 ，R 7 ，R 8 And R 9 May be the same or different.
In some embodiments of the invention, the compound of the invention is R 1 ，R 2 ，R 3 ，R 4 ，R 5 ， R 6 ，R 7 ，R 8 And R 9 A compound of formula (I) each being a hydrogen atom, having the formula:
the invention also provides a process for the preparation of a compound of formula (i) comprising the steps of: reacting a compound of formula (III) with hydrazine hydrate to produce a compound of formula (I) having the formula:
in the formulae (I), (IV) and (III): r 1 ，R 2 ，R 3 ，R 4 And R 5 Is independently selected from the group consisting of a hydrogen atom, a linear or branched alkyl group, a linear or branched alkoxy group, a sulfonic acid group, an ester group and a carboxyl group; and wherein R is 1 ，R 2 ，R 3 ，R 4 And R 5 May be the same or different.
Specifically, dissolving a compound of a formula (III) and a compound of a formula (IV) in methanesulfonic acid, performing reflux reaction, cooling to room temperature after the reaction is finished, pouring into ice water, adding perchloric acid, and performing suction filtration to obtain a crude product; the crude product is further separated by chromatography using dichloromethane as eluent to obtain the pure compound of formula (I).
In some embodiments of the invention, the molar ratio of the compound of formula (III) to hydrazine hydrate is from 1:1 to 1: 5.
In some embodiments of the invention, the reaction time is from 0.5 to 24 hours.
The invention also provides a fluorescent probe composition for measuring, detecting or screening sulfur dioxide/formaldehyde, which comprises the compound shown in the formula (I) in the invention.
In some embodiments of the invention, the compound of formula (I) has the following structure:
in some embodiments of the invention, the fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.
The present invention also provides a method for detecting the presence of or determining the amount of sulphur dioxide/formaldehyde in a sample comprising:
a) contacting the compound of formula (I) or formula (ii) with a sample to form a compound that changes fluorescence;
b) determining the fluorescent properties of the fluorescence-altered compound.
In some embodiments of the invention, the sample is a chemical sample or a biological sample.
In some embodiments of the invention, the sample is a biological sample comprising water, blood, microorganisms, or animal cells or tissues.
The invention also provides a kit for detecting the presence of or determining the amount of sulphur dioxide/formaldehyde in a sample, comprising a compound of formula (I) or formula (II).
The invention also provides application of the compound shown in the formula (I) or the formula (II) in cell fluorescence imaging.
Compared with the prior art, the invention has the following remarkable advantages and effects:
(1) reversible ratio detection of sulfur dioxide/formaldehyde
The sulfur dioxide/formaldehyde fluorescent probe can effectively detect the content of sulfur dioxide/formaldehyde at the same time, can detect the sulfur dioxide and the formaldehyde in a ratio manner, and effectively reduces background interference.
(2) Colorimetric detection of sulfur dioxide/formaldehyde
The sulfur dioxide/formaldehyde fluorescent probe disclosed by the invention is convenient for qualitative analysis.
(3) High selectivity and high anti-interference ability
The sulfur dioxide/formaldehyde fluorescent probe can selectively and specifically react with sulfur dioxide/formaldehyde to generate a product with fluorescence change, compared with other common metal ions and other substances in a living body, including but not limited to blank, sodium chloride, potassium chloride, calcium chloride, formaldehyde, magnesium sulfate, zinc nitrate, ferric sulfate, nickel sulfate, copper sulfate, chromium sulfate, sodium bromide, potassium fluoride, sodium nitrate, sodium nitrite, cysteine, glutathione, hydrogen peroxide, tert-butyl peroxide free radical, potassium superoxide, singlet oxygen, hydroxyl free radical, peroxynitrite, sodium hydrosulfide, acetaldehyde and the like, the sulfur dioxide/formaldehyde fluorescent probe shows higher selectivity and strong anti-interference capability.
(4) High sensitivity and quick response
The sulfur dioxide/formaldehyde fluorescent probe disclosed by the invention is very sensitive to the reaction with sulfur dioxide/formaldehyde and is quick in response, so that the detection of sulfur dioxide/formaldehyde is facilitated.
(5) Can be applied under physiological level condition
The sulfur dioxide/formaldehyde fluorescent probe can be applied under the condition of physiological level, and metal ions and other substances which are common in organisms have small interference on the probe, so that the probe can be applied to living cell fluorescence imaging.
(6) Good stability
The sulfur dioxide/formaldehyde fluorescent probe has good stability and can be stored and used for a long time.
(7) Simple synthesis
The sulfur dioxide/formaldehyde fluorescent probe is simple to synthesize and beneficial to commercial popularization and application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1a is the response time at 485nm after addition of probe (5. mu.M) to sodium bisulfite (10. mu.M).
FIG. 1b is the response time at 685nm after addition of probe (5. mu.M) to sodium bisulfite (10. mu.M).
FIG. 2a is the response time at 485nm after formaldehyde (200. mu.M) was added to the solution after the probe (5. mu.M) was reacted with sodium bisulfite (10. mu.M).
FIG. 2b is the response time at 685nm after adding formaldehyde (200. mu.M) to the solution after adding sodium bisulfite (10. mu.M) to the probe (5. mu.M).
FIG. 3 is a graph showing the change in fluorescence spectrum of a fluorescent probe (5. mu.M) added to sodium bisulfite (0 to 5. mu.M).
FIG. 4 is a graph of the linear relationship of different concentrations of sodium bisulfite (0-5. mu.M) versus probe (5. mu.M).
FIG. 5 is a graph showing the change in fluorescence spectrum of a solution obtained by reacting a fluorescent probe (5. mu.M) with sodium bisulfite (10. mu.M) after addition of formaldehyde (0 to 50. mu.M).
FIG. 6 is a graph showing the linear relationship between different concentrations of formaldehyde (0-50. mu.M) and the reaction solution of probe (5. mu.M) and sodium bisulfite (10. mu.M).
FIG. 7 is the fluorescence intensity of different ion analytes versus probe (5. mu.M). Analytes were blank, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, zinc nitrate, iron sulfate, nickel sulfate, copper sulfate, chromium sulfate, sodium bromide, potassium fluoride, sodium nitrate, sodium nitrite, cysteine (500 μ M), glutathione (1mM), hydrogen peroxide, t-butanol peroxide radical, potassium superoxide, singlet oxygen, hydroxyl radical, nitrite peroxide, sodium hydrosulfide, acetaldehyde (200 μ M), formaldehyde (200 μ M) and sodium bisulfite (5 μ M), respectively (except for the specific indication, the analyte concentrations were 100 μ M). The histogram represents the ratio of fluorescence intensity at F485/F685 nm for probes in the presence of different analytes.
FIG. 8 is a graph of the fluorescence intensity of solutions after reaction of different ion analytes to probes (5. mu.M) and sodium bisulfite (10. mu.M). Analyte blanks, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, zinc nitrate, iron sulfate, nickel sulfate, copper sulfate, chromium sulfate, sodium bromide, potassium fluoride, sodium nitrate, sodium nitrite, cysteine (500. mu.M), glutathione (1mM), hydrogen peroxide, t-butanol peroxide free radical, potassium superoxide, singlet oxygen, hydroxyl free radical, peroxynitrite, sodium hydrosulfide, acetaldehyde (200. mu.M) and formaldehyde (200. mu.M), respectively (other analyte concentrations were 100. mu.M, unless otherwise specified). The histogram represents the fluorescence intensity ratio of the probe at F685/F485 nm in the presence of different analytes.
FIG. 9 is the detection of formaldehyde with a probe (10. mu.M) successfully applied to zebrafish. a1-a2 is zebrafish incubated probe (10 μ M), b1-b2 is zebrafish incubated probe (10 μ M), followed by further incubation of sodium bisulfite (100 μ M), c1-c2 is zebrafish incubated probe (10 μ M), followed by further incubation of sodium bisulfite (100 μ M), and finally followed by further incubation of formaldehyde (200 μ M).
FIG. 10 shows the results of the colorimetric performance test of the fluorescent probe.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and should not be used to limit the scope of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, belong to the scope of the present invention.
Example 1: synthesis of Compounds of formula (II)
The synthetic design route is as follows:
embodiment 1: dissolving 204mg (1mmol) of the (V) compound and 193mg (1mmol) of 4- (diethylamino) salicylaldehyde in 5mL of methanesulfonic acid, refluxing at 90 ℃ for 6h, cooling to room temperature after the reaction is finished, pouring into ice water, adding 6mL of perchloric acid, and performing suction filtration to obtain a crude product; the crude product was further separated by column chromatography with dichloromethane as eluent to give 258mg of blue product in 56% yield.
Embodiment 2: dissolving 204mg (1mmol) of the (V) compound and 386mg (2mmol) of 4- (diethylamino) salicylaldehyde in 5mL of methanesulfonic acid, refluxing at 90 ℃ for 6h, cooling to room temperature after the reaction is finished, pouring into ice water, adding 6mL of perchloric acid, and performing suction filtration to obtain a crude product; the crude product was further separated by column chromatography using dichloromethane as eluent to give 295mg of blue product in 64% yield.
Embodiment 3: dissolving 204mg (1mmol) of (V) compound and 579mg (3mmol) of 4- (diethylamino) salicylaldehyde in 5mL of methanesulfonic acid, refluxing at 90 ℃ for 6 hours, cooling to room temperature after the reaction is finished, pouring into ice water, adding 6mL perchloric acid, and performing suction filtration to obtain a crude product; the crude product was further separated by column chromatography with dichloromethane as eluent to give 313mg of blue product in 68% yield.
Embodiment 4: dissolving 204mg (1mmol) of the (V) compound and 772mg (4mmol) of 4- (diethylamino) salicylaldehyde in 5mL of methanesulfonic acid, refluxing at 90 ℃ for 6 hours, cooling to room temperature after the reaction is finished, pouring into ice water, adding 6mL of perchloric acid, and performing suction filtration to obtain a crude product; the crude product was further separated by column chromatography using dichloromethane as eluent to give 368mg of blue product in 80% yield.
Embodiment 5: dissolving 204mg (1mmol) of the (V) compound and 772mg (4mmol) of 4- (diethylamino) salicylaldehyde in 5mL of methanesulfonic acid, refluxing at 90 ℃ for 2h, cooling to room temperature after the reaction is finished, pouring into ice water, adding 6mL of perchloric acid, and performing suction filtration to obtain a crude product; the crude product was further separated by column chromatography with dichloromethane as eluent to give 239mg of blue product in 52% yield.
Example 2: testing the time dynamics of fluorescent probes for sodium sulfite
25 μ L of the probe stock solution was taken out and put in a 5mL test system, and then 10 μ M of sodium bisulfite was added to the test system, and the change in fluorescence intensity was measured by a fluorescence spectrometer immediately after shaking uniformly. The above assay was performed in PBS buffer (20mM PBS, pH 7.4, 10% EtOH), the probe used was the probe prepared in example 1, and all spectroscopic measurements were performed at 25 ℃.
The test results are shown in fig. 1a and 1b, and the fluorescence intensity values at about 30s, 485nm and 685nm respectively reach the maximum value and the minimum value and are kept unchanged, which indicates that the probe reacts with sodium bisulfite quickly, and can provide a quick analysis method for the determination of the sodium bisulfite.
Example 3: the time kinetics of the solution after reaction of the fluorescent probe with sodium bisulfite (10. mu.M) for formaldehyde were tested
And (3) placing the solution obtained after the reaction of the probe and the sodium bisulfite into a 5mL test system, then adding 200 mu M of formaldehyde into the test system, and immediately testing the change of the fluorescence intensity of the test system by using a fluorescence spectrometer after shaking uniformly. The above assay was performed in PBS buffer (20mM PBS, pH 7.4, 10% EtOH), the probe used was the probe prepared in example 1, and all spectroscopic measurements were performed at 25 ℃.
The test results are shown in fig. 2a and 2b, when formaldehyde is counted in, the fluorescence intensity values at 485nm and 685nm respectively reach the minimum value and the maximum value and are kept unchanged after 3min, which indicates that the probe and formaldehyde react rapidly, and a rapid analysis method can be provided for the measurement of methylene aldehyde.
Example 4: testing the concentration gradient of fluorescent probes to sodium bisulfite
A plurality of parallel samples with the probe concentration of 5 mu M are arranged in a 10mL colorimetric tube, then sodium bisulfite with different concentrations is added into the test system, and the test system is shaken uniformly and then is kept stand for 1 minute. The above assay was performed in PBS buffer (20mM PBS, pH 7.4, 10% EtOH), the probe used was the probe prepared in example 1, and all spectroscopic measurements were performed at 25 ℃.
The change of fluorescence intensity was measured by a fluorescence spectrometer, and it is clear from FIG. 3 that the fluorescence intensity at 485nm gradually increased and the fluorescence intensity at 685nm gradually decreased with the increase of the concentration of sodium bisulfite. Also, as can be seen from FIG. 4, at the ratio F485/F685, the fluorescence intensity of the fluorescent probe (5. mu.M) after the addition of sodium bisulfite (0-5. mu.M) shows a good linear relationship, which demonstrates that sodium bisulfite can be quantitatively analyzed with the aid of the fluorescent probe.
Example 4: test the concentration gradient of formaldehyde added to the solution after reaction of the fluorescent probe with sodium bisulfite (10. mu.M)
Prepare multiple parallel samples of reacted sodium bisulfite (10 μ M) and probe (5 μ M) in 10mL colorimetric tubes, add different concentrations of formaldehyde to the test system, shake uniformly and then stand for 3 minutes. The above assay was performed in PBS buffer (20mM PBS, pH 7.4, 10% EtOH), the probe used was the probe prepared in example 1, and all spectroscopic measurements were performed at 25 ℃.
The fluorescence intensity change of the sample is tested by a fluorescence spectrometer, and as can be clearly seen from figure 5, the fluorescence intensity at 485nm is gradually reduced along with the increase of the concentration of the added formaldehyde, the fluorescence intensity at 685nm is gradually increased, and the fluorescence intensity is recovered. Also, as can be seen from FIG. 6, at the ratio F685/F485, the fluorescence intensity of the reaction solution of the fluorescent probe (5. mu.M) and sodium bisulfite (10. mu.M) after adding formaldehyde (0-50. mu.M) showed a good linear relationship, which demonstrates that formaldehyde can be quantitatively analyzed by means of the fluorescent probe.
Example 5: testing the selectivity of fluorescent probes for sodium bisulfite identification
The analytes were blank, sodium chloride, potassium chloride, calcium chloride, formaldehyde, magnesium sulfate, zinc nitrate, iron sulfate, nickel sulfate, copper sulfate, chromium sulfate, sodium bromide, potassium fluoride, sodium nitrate, sodium nitrite, cysteine (500. mu.M), glutathione (1mM), hydrogen peroxide, t-butanol peroxide radical, potassium superoxide, singlet oxygen, hydroxyl radical, peroxynitrite, sodium hydrosulfide, acetaldehyde (200. mu.M), formaldehyde (200. mu.M) and sodium bisulfite (10. mu.M), respectively (except for the specific indication, the analyte concentration was 100. mu.M). The histogram represents the fluorescence intensity ratio of the probe at F485/F685 nm in the presence of different analytes. The above assay was performed in pure water (20mM PBS, pH 7.4, 10% EtOH), the probe used was the probe prepared in example 1, and all spectroscopic measurements were performed at 25 ℃. Specifically, a plurality of parallel samples with a probe concentration of 5 μ M were placed in a 10mL cuvette, and then a certain amount of analyte was added, shaken up, and measured after 1 minute.
As can be seen from FIG. 7, the common ions existing in the organism do not significantly interfere with the fluorescence intensity of the probe to sodium bisulfite, so the probe has good selectivity.
Example 6: test the selectivity of the solution after reaction of the fluorescent probe with sodium bisulfite (10. mu.M) for formaldehyde recognition
The analytes were blank, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, zinc nitrate, iron sulfate, nickel sulfate, copper sulfate, chromium sulfate, sodium bromide, potassium fluoride, sodium nitrate, sodium nitrite, cysteine (500. mu.M), glutathione (1mM), hydrogen peroxide, t-butanol peroxide radical, potassium superoxide, singlet oxygen, hydroxyl radical, peroxynitrite, sodium hydrosulfide, acetaldehyde (200. mu.M) and formaldehyde (200. mu.M), respectively (other analyte concentrations were 100. mu.M, unless otherwise specified). The histogram represents the fluorescence intensity ratio of the probe at F685/F485 nm in the presence of different analytes. The above assay was performed in pure water (20mM PBS, pH 7.4, 10% EtOH), the probe used was the probe prepared in example 1, and all spectroscopic measurements were performed at 25 ℃. Specifically, a plurality of parallel samples of reacted sodium bisulfite (10 μ M) and probe (5 μ M) were placed in a 10mL cuvette, then a certain amount of analyte was added, shaken up, and measured after 5 minutes.
As can be seen from FIG. 8, the fluorescence intensity of formaldehyde by the probe is not significantly interfered by common ions existing in organisms, so that the probe has good selectivity.
Example 7: fluorescence imaging of fluorescent probe for detecting formaldehyde in zebra fish cells
The probe (10 μ M) was first incubated in zebrafish for 20 minutes, the culture broth removed and washed 3 times with fish culture water, and the fluorescence of the blue and red channels was collected. In addition, sodium bisulfite (100. mu.M) is added into the zebrafish culture solution after the probe incubation to incubate the zebrafish for 30 minutes, so that the red fluorescence is weakened, and the blue fluorescence is strengthened. On the other hand, formaldehyde (200. mu.M) was added to the incubated zebrafish culture solution after the probe and sodium bisulfite were incubated for 30 minutes, and the fluorescence of the probe was recovered, i.e., the red fluorescence was increased and the blue fluorescence was decreased. And finally, carrying out fluorescence imaging on the cells by using a confocal fluorescence microscope. The results are shown in FIG. 9.
Example 8: colorimetric Performance test with fluorescent Probe
After 25. mu.L of the probe stock solution was taken out and placed in a 5mL test system, the solution appeared blue in color, 10. mu.M sodium bisulfite was added to the test system, and the solution turned yellow after shaking uniformly. The results are shown in FIG. 10.
Although the invention has been described with respect to the above embodiments, it will be understood that the invention is capable of further modifications and variations without departing from the spirit of the invention and these modifications and variations are within the scope of the invention.
Claims (2)
1. A method for detecting the presence of or determining the sulfur dioxide/formaldehyde content in a sample comprising:
a) contacting a compound of formula (ii) with a sample to form a compound that changes fluorescence;
b) determining the fluorescent properties of the fluorescence-altered compound.
2. The method of claim 1, wherein the sample is a chemical sample or a biological sample.
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| CN106544007B (en) * | 2015-09-23 | 2018-07-31 | 济南大学 | Hypochlorous fluorescence probe and its application in a kind of detection biosystem |
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