CN113072528B - Near-infrared ratio fluorescent probe for reversibly detecting bisulfite/formaldehyde, preparation method and application - Google Patents
Near-infrared ratio fluorescent probe for reversibly detecting bisulfite/formaldehyde, preparation method and application Download PDFInfo
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Abstract
The invention relates to a near-infrared bisulfite and formaldehyde reversible ratio fluorescent probe with high selectivity and high sensitivity. Specifically, the probe is a coumarin compound, can be used as a fluorescent probe with a reversible ratio for detecting bisulfite and formaldehyde, and can eliminate interference caused by an external environment and an instrument. Such probes can achieve at least one of the following technical effects: high selectivity recognition of bisulfite and formaldehyde; the response to formaldehyde can be quickly realized; can realize the high-sensitivity analysis of the peroxybisulfite and the paraformaldehyde; quantitative analysis of bisulfite and formaldehyde can be realized; and provides a powerful tool for understanding the processes of bisulphite and formaldehyde in the pathophysiology of organisms.
Description
Technical Field
The invention belongs to the field of fluorescent probes, and particularly relates to a coumarin compound-based near-infrared ratiometric fluorescent probe for reversibly detecting bisulfite/formaldehyde and application thereof in measuring, detecting or screening bisulfite/formaldehyde and live cell fluorescence imaging methods; the invention also provides a method for preparing the fluorescent probe.
Background
In living systems, formaldehyde, one of the simplest active carbonyl species, plays an extremely important role in maintaining carbon cycle metabolism. Endogenous production of formaldehyde is a normal physiological process mediated by an enzyme system, such as semicarbazide-sensitive amine oxidase, lysine-specific demethylase, amino-sensitive amine oxidase, and the like. The concentration level of formaldehyde in organisms is related to spatial memory and cognitive ability, however, abnormal accumulation of formaldehyde may cause protein and DNA damage, which further causes various diseases, and low dosage of formaldehyde may cause chronic respiratory diseases, pregnancy syndromes, neonatal constitutional reduction, chromosome abnormality and even nasopharyngeal carcinoma. High concentrations of formaldehyde are toxic to the nervous system, immune system, liver, etc. In addition, formaldehyde has teratogenic and carcinogenic effects. Formaldehyde is also reported to occur naturally in human cells and in different organisms, and under normal physiological conditions formaldehyde is present in relatively high concentrations in cells, up to 0.5mM in certain organelles, and normal levels of formaldehyde in human blood are about 0.06-0.08 mM. Therefore, in view of the hazard of formaldehyde and its importance in biological systems, a method capable of sensitively detecting formaldehyde is sought, so as to realize in-situ detection of formaldehyde in a living body.
On the other hand, in the organism, a delicate equilibrium relationship exists between bisulfite and formaldehyde for maintaining homeostasis. However, due to the lack of a method for monitoring the potential interaction of bisulfite and formaldehyde in real time, the specifics of the dynamic changes between the two are not known, and the interaction of bisulfite and formaldehyde in complex biological systems remains unknown. Therefore, it is very urgent to develop powerful chemical tools to study the possible dynamic changes between bisulfite and formaldehyde. In living systems, these molecular tools are extremely important for the pathophysiological study of the role and relationship of bisulfite and formaldehyde in organisms.
Fluorescent probes are widely used in the determination of analytes in biological imaging as a non-invasive tool due to their advantages of high sensitivity, high selectivity, significant spatial and temporal resolution, real-time monitoring, etc. So far, the reversible ratiometric fluorescent probes for detecting bisulfite and formaldehyde in organisms are relatively lacking, and it is necessary to develop a novel efficient fluorescent probe capable of rapidly detecting bisulfite and formaldehyde in environments and living cells. In addition, bisulfite/formaldehyde can be reversibly detected, and ratiometric fluorescent probes with good selectivity and high sensitivity are relatively few. Therefore, it is still crucial to develop a tool for reversible detection of bisulfite/formaldehyde in environmental and biological systems with high selectivity, high sensitivity and rapidity.
Disclosure of Invention
In view of this, the present invention aims to provide a near-infrared bisulfite/formaldehyde fluorescence probe with simple preparation, high sensitivity, high selectivity and reversible ratio, 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 bisulfite/formaldehyde under the condition of physiological level, especially can qualitatively and quantitatively analyze bisulfite/formaldehyde with colorimetric ratio.
Specifically, the invention provides a compound, which has a structure shown in a formula (I):
in the formula (I), R 1 ，R 2 ，R 3 ，R 4 ，R 5 ，R 6 ，R 7 ，R 8 ，R 9 ，R 10 ，R 11 And R 12 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 ，R 9 ，R 10 ，R 11 And R 12 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 a compound of formula (IV) to produce a compound of formula (I), wherein the reaction formula is as follows:
in formulae (I), (IV) and (III): r 1 ，R 2 ，R 3 ，R 4 ，R 5 ，R 6 ，R 7 ，R 8 ，R 9 ， R 10 ，R 11 And R 12 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 ， R 9 ，R 10 ，R 11 And R 12 May be the same or different.
Specifically, the compound of formula (III) and the compound of formula (IV) are dissolved in acetic acid, then heated under reflux, and after the reaction is completed, the solvent is removed by rotary evaporation under reduced pressure to obtain a crude product. The crude product is further separated by chromatography to give the pure compound of formula (I).
In some embodiments of the invention, the molar ratio of the compound of formula (III) to the compound of formula (iv) is from 1:1 to 5: 1.
In some embodiments of the invention, the reaction time is from 1 to 12 hours.
The invention also provides a fluorescent probe composition for measuring, detecting or screening bisulfite/formaldehyde, which comprises the compound of formula (I) of 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 bisulfite/formaldehyde in a sample or determining the bisulfite/formaldehyde content of 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 bisulfite/formaldehyde in a sample or determining the bisulfite/formaldehyde content of a sample, comprising the 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 bisulfite/formaldehyde
The bisulfite/formaldehyde fluorescence probe of the invention can effectively detect the content of bisulfite/formaldehyde at the same time, overcomes the defect that the traditional fluorescence probe can only be used for one-time detection, and can be used for real-time reversible detection of bisulfite/formaldehyde; meanwhile, the bisulfite/formaldehyde ratio can be detected, and the background interference is effectively reduced.
(2) Colorimetric detection bisulfite/formaldehyde fluorescent probe
The bisulfite/formaldehyde fluorescent probe can detect bisulfite/formaldehyde by colorimetry, is convenient for detecting sulfur dioxide/formaldehyde by naked eyes and is convenient for qualitative analysis.
(3) High selectivity and high anti-interference ability
The bisulfite/formaldehyde fluorescent probe can selectively and specifically react with bisulfite/formaldehyde to generate a product with fluorescence change, and compared with other common metal ions and other substances in a living body, the bisulfite/formaldehyde fluorescent probe has higher selectivity and strong anti-interference capability.
(4) High sensitivity and quick response
The bisulfite/formaldehyde fluorescent probe of the invention reacts with bisulfite/formaldehyde very sensitively and responds quickly, thereby being beneficial to detecting bisulfite/formaldehyde.
(5) Can be applied under physiological level condition
The bisulfite/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 bisulfite/formaldehyde fluorescent probe, so the bisulfite/formaldehyde fluorescent probe can be applied to living cell fluorescence imaging; in addition, the probe belongs to a near infrared fluorescent probe, and has obvious superiority when being applied to analysis of biological samples.
(6) Good stability
The bisulfite/formaldehyde fluorescent probe has good stability and can be stored and used for a long time.
(7) Simple synthesis
The bisulfite/formaldehyde fluorescent probe of the invention has simple synthesis and is 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 description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1a is the response time at 515nm after addition of bisulfite (50. mu.M) to the probe (5. mu.M).
FIG. 1b is the response time at 788nm after addition of the probe (5. mu.M) to bisulfite (50. mu.M).
FIG. 2a shows the response time at 515nm after adding formaldehyde (100. mu.M) to the solution after the probe (5. mu.M) was reacted with bisulfite (50. mu.M).
FIG. 2b is the response time at 788nm after addition of formaldehyde (100. mu.M) to the solution after the probe (5. mu.M) was reacted with bisulfite (50. mu.M).
FIG. 3 is a graph showing UV absorption spectra of fluorescent probes (5. mu.M) alternately added with bisulfite (10. mu.M), formaldehyde (200. mu.M), bisulfite (50. mu.M) and formaldehyde (300. mu.M).
FIG. 4 is a graph showing the color change of a solution after bisulfite (10. mu.M) and formaldehyde (200. mu.M) were added to a solution of a fluorescent probe (5. mu.M) in a circulating manner (at intervals of 2 minutes).
FIG. 5 is a graph showing the change in the absorbance ratio during the addition of bisulfite (10. mu.M) and formaldehyde (200. mu.M) cyclically (at 2-minute intervals) in a solution of fluorescent probe (5. mu.M).
FIG. 6 is a graph showing the change in fluorescence spectrum between the fluorescent probe (5. mu.M) and bisulfite (0 to 10. mu.M).
FIG. 7 is a linear graph of the ratio of fluorescence intensity (F515/F788 nm) measured with the fluorescent probe (5. mu.M) added to different concentrations of bisulfite (0-10. mu.M) versus bisulfite concentration.
FIG. 8 is a graph showing the change in fluorescence spectrum after the reaction of the fluorescent probe (5. mu.M) with bisulfite and the addition of formaldehyde (0 to 30. mu.M).
FIG. 9 is a linear relationship graph of the measured fluorescence intensity ratio (F788/F515 nm) and formaldehyde concentration after reacting the fluorescent probe (5. mu.M) with bisulfite and adding formaldehyde (0-30. mu.M) at different concentrations.
FIG. 10 is a graph of the fluorescent response of a fluorescent probe (5. mu.M) to different analytes (100. mu.M concentration, except for label); the analytes are blank, K respectively + 、Ca 2+ 、Na + 、Mg 2+ 、Zn 2+ 、Fe 3+ 、Fe 2+ 、Cu 2+ 、 NO 3- 、NO 2- 、F - 、CO 3 2- 、Br - 、Cys(500μM)、GSH(1mM)、H 2 S、 1 O 2 、·O 2 - 、·O t Bu、·OH、TBHP、H 2 O 2 、NO、ONOO - 、HOCl、HSO 3 - (20. mu.M). The histogram represents the fluorescence intensity ratio of the probe at F515/F788 nm in the presence of different analytes.
FIG. 11 is the fluorescent response of the solution after reaction of fluorescent probe (5. mu.M) and bisulfite (10. mu.M) to different analytes (100. mu.M concentration, except for label); the analytes are blank, K respectively + 、Ca 2+ 、 Na + 、Mg 2+ 、Zn 2+ 、Cu 2+ 、NO 3 - 、NO 2 - 、Cl - 、F - 、CO 3 2 -、Br - 、Cys(500μM)、Hcy(500 μM)、GSH(1mM)、H 2 S、·O 2 - 、·O t Bu、·OH、TBHP、H 2 O 2 、NO、ONOO - Acetaldehyde (200. mu.M), FA (200. mu.M). The histogram represents the fluorescence intensity ratio of the probe at F788/F515 nm in the presence of different analytes.
FIG. 12 is fluorescence microscopy imaging of bisulfite and formaldehyde in HeLa cells.
FIG. 13 is fluorescence microscopy imaging of endogenous bisulfite and formaldehyde in HeLa cells.
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 the compound (355mg,1mmol) of the formula (III) and the compound (245mg, 1mmol) of the formula (IV) in acetic acid (30ml), refluxing at 110 ℃ for 3h, and after the reaction is finished, removing the solvent by evaporation under reduced pressure to obtain a crude product; after drying, purification by column chromatography (CH2Cl 2: CH3OH ═ 60:1) gave 326mg of a dark purple solid in 56% yield.
Embodiment 2: dissolving the compound (710mg,2mmol) of the formula (III) and the compound (245mg, 1mmol) of the formula (IV) in acetic acid (30ml), refluxing for 3h at 110 ℃, and after the reaction is finished, removing the solvent by reduced pressure evaporation to obtain a crude product; after drying, purification by column chromatography (CH2Cl 2: CH3OH ═ 60:1) gives 372mg of a dark purple solid in 64% yield.
Embodiment 3: dissolving the compound (1065mg,3mmol) of the formula (III) and the compound (IV) (245mg, 1mmol) in acetic acid (30ml), refluxing at 110 ℃ for 3h, and after the reaction is completed, removing the solvent by evaporation under reduced pressure to obtain a crude product; after drying, purification by column chromatography (CH2Cl 2: CH3OH ═ 60:1) gave 396mg of a dark purple solid in 68% yield.
Embodiment 4: dissolving the compound (1420mg,4mmol) of the formula (III) and the compound (IV) (245mg, 1mmol) in acetic acid (30ml), refluxing at 110 ℃ for 3h, and after completion of the reaction, removing the solvent by evaporation under reduced pressure to obtain a crude product; after drying, purification by column chromatography (CH2Cl 2: CH3OH ═ 60:1) gave 442mg of a dark purple solid in 76% yield.
Embodiment 5: dissolving the compound of formula (III) (1420mg,4mmol) and the compound of formula (IV) (245mg, 1mmol) in acetic acid (30ml), refluxing at 110 deg.C for 1.5h, after the reaction is completed, evaporating the solvent under reduced pressure to obtain crude product; after drying, purification by column chromatography (CH2Cl 2: CH3OH ═ 60:1) gave 303mg of a dark purple solid in 52% yield.
Example 2: testing the time kinetics of fluorescent probes for sodium sulfite
Preparing 4mL of a 5 mu M fluorescent probe concentration test system, adding 50 mu M bisulfite into the test system, shaking up, and immediately testing the change of the fluorescence intensity by using a fluorescence spectrometer. 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 FIGS. 1a and 1b, respectively, and the fluorescence intensity values at about 30s and 515nm and 788nm reach the maximum and minimum values respectively and remain unchanged, which indicates that the probe reacts with bisulfite rapidly, and can provide a rapid analysis method for measuring bisulfite.
Example 3: testing the time kinetics of the solution of the fluorescent probe reacted with bisulfite for formaldehyde
After the reaction of the fluorescent probe (5. mu.M) and bisulfite (50. mu.M), 100. mu.M of formaldehyde was added to the solution, and the change in fluorescence intensity was measured by a fluorescence spectrometer immediately after shaking the solution 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, respectively, and the fluorescence intensity values at 515nm and 788nm reach minimum and maximum values and remain unchanged around 2min, which indicates that the probe exhibits a reversible fast response to bisulfite and formaldehyde.
Example 4: testing ultraviolet absorption spectrum change after the alternative reaction of the fluorescent probe and the bisulfite/formaldehyde
Two 4mL test systems (20mM PBS, pH 7.4, 10% EtOH) were prepared, labeled A and B, and test system B was used as a control, without any manipulation, and after adding probe (5. mu.M) to test system A, measurement was performed using UV absorption spectroscopy, followed by the addition of bisulfite (10. mu.M), formaldehyde (200. mu.M), bisulfite (50. mu.M), and formaldehyde (300. mu.M), and each addition of analyte was measured using UV absorption spectroscopy, and the results are shown in FIG. 3.
Example 5: testing the absorbance ratio and color change of the fluorescent probe and the bisulfite/formaldehyde alternation process
A4 mL probe (5 μ M) solution is prepared and added into an ultraviolet cuvette, bisulfite (10 μ M) and formaldehyde (200 μ M) are added cyclically at intervals of 2 minutes, 5 times of reversible experiments are carried out, the solution color changes obviously, the solution becomes yellow after the bisulfite is added, the solution returns to blue after the formaldehyde is added, and the color change is shown in figure 4, which shows that the bisulfite and the formaldehyde can be detected by the probe with naked eyes.
Meanwhile, changes in absorbance ratio were measured by an ultraviolet absorption spectrometer every time color was changed during the whole cycle, the measurement was performed in a PBS buffer solution (20mM PBS, pH 7.4, 10% EtOH), the probe used was the probe prepared in example 1, and all the spectroscopic measurements were performed at 25 ℃, and the results of the measurements are shown in FIG. 5.
As is clear from FIGS. 4 and 5, the absorbance of the probe solution and the color of the probe solution are reversibly changed when bisulfite and formaldehyde are added, indicating the potential of the probe for real-time monitoring of bisulfite and formaldehyde in cell samples.
Example 5: testing the concentration gradient of fluorescent probes to bisulfite
A plurality of parallel samples with the probe concentration of 5 mu M are configured in a 10mL colorimetric tube, and then bisulfite with different concentrations is added into the configured test system, and the test system is shaken uniformly and then is stood for 1 minute. The change in fluorescence intensity was measured by fluorescence spectroscopy. 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 ℃ with the results shown in FIG. 6 and FIG. 7.
As is clear from FIG. 6, after the probe was reacted with bisulfite, the fluorescence intensity at 515nm gradually increased, and the fluorescence intensity at 788nm gradually decreased; furthermore, as can be seen from FIG. 7, the ratio of the fluorescence intensity of the probe between 515 and 788nm (F515/F788) is a function related to the bisulfite concentration, and the ratio of the fluorescence intensity between 515 and 788nm (F515/F788) is in a good linear relationship with the equation y of 4.9008x-2.5472, R is between 0 and 10. mu.M in the bisulfite concentration range 2 0.9912, wherein y is the ratio of the fluorescence intensity of the reaction solution, and x is the concentration of the bisulfite, the result shows that the probe has better sensitivity for detecting the bisulfite, and can provide an effective tool for detecting the bisulfite in the living body.
Example 6: testing the concentration gradient of the solution after the reaction of the fluorescent probe and the bisulfite added with formaldehyde
Adding formaldehyde with different concentrations into the colorimetric tube after the probe reacts with the bisulfite radical, shaking uniformly, standing for 3 minutes, and testing the change of the fluorescence intensity by using a fluorescence spectrometer. 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 ℃ with the results shown in FIGS. 8 and 9.
As is clear from FIG. 8, after formaldehyde was added to the probe solution after the reaction with bisulfite, the fluorescence intensity at 515nm gradually decreased, while the fluorescence intensity at 788nm gradually increased; furthermore, as can be seen from FIG. 9, the ratio of the fluorescence intensity of the probe between 788 and 515nm (F788/F515) is a function related to the formaldehyde concentration, and in the formaldehyde concentration range of 0-10. mu.M, the ratio of the fluorescence intensity between 515 and 788nm (F515/F788) has a good linear relationship, where y is 0.0004x +0.0036, and R is 0.0004x +0.0036 2 0.9979, wherein y is the ratio of fluorescence intensity of the reaction solution, x is the concentration of formaldehyde, the result shows that the probe has better sensitivity and reversibility for detecting the bisulfite and the formaldehyde, and provides a reagent for real-time monitoring for detecting the bisulfite and the formaldehyde in the life bodyAn efficient tool.
Example 7: testing selectivity of fluorescent probes for bisulfite recognition
A plurality of parallel samples with the probe concentration of 5 mu M are arranged in a 10mL colorimetric tube, then various analytes are respectively added, the mixture is shaken up, and after 1 minute, a fluorescence spectrometer is used for testing the fluorescence intensity change in each colorimetric tube. 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 results are shown in FIG. 10, from which it can be seen that bisulfite alone causes a significant change in the ratio of the fluorescence intensities of the probes (F515/F788), while the effect of other analytes on the probes is almost negligible. The above results indicate that the probe has high selectivity for bisulfite, and that even other high concentrations of analyte cannot cause a change in the fluorescence intensity of the probe. The high selectivity of the probe is beneficial to the application of the probe in cell detection.
Example 8: testing the selectivity of the solution after the reaction of the fluorescent probe and the bisulfite to the formaldehyde recognition
Preparing a plurality of parallel samples with the probe concentration of 5 mu M into a 10mL colorimetric tube, then respectively adding various analytes, shaking up, and testing the fluorescence intensity change in each colorimetric tube by using a fluorescence spectrometer after 5 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 test results are shown in FIG. 11, and it can be seen from FIG. 11 that only formaldehyde can significantly change the ratio of the fluorescence intensities of the probes (F788/F515). The result shows that the probe has higher selectivity on formaldehyde, and can be applied to reversible detection of formaldehyde.
Example 9: fluorescence imaging of fluorescent probes for detection of bisulfite and formaldehyde in Hela cells
a1-a 2: cells were incubated with probe (10 μ M) for 30 min; b1-b 2: adding bisulfite- (100. mu.M) to group a cells and incubating for 30 min; c1-c 2: then adding formaldehyde (200 mu M) into the cells in the group b for incubation for 30 min; d: the green channel and red channel fluorescence intensity ratios (Fred/Fgreen) for images a-c.
Due to the characteristic of low toxicity of the probe, the probe can be applied to real-time detection of the probe for a long time. First, the ability of the probe to image exogenous bisulfite and formaldehyde in living cells was evaluated. For control cells incubated with probe only, the fluorescence signal was weaker in the green channel and stronger in the red channel (FIG. 12 a1-a 3). The cells were then imaged by continued treatment with bisulfite and found to have increased fluorescence signal in the green channel and decreased fluorescence signal in the red channel (FIG. 12 b1-b 3), indicating that it is possible to image intracellular bisulfite at a specific rate. Finally, formaldehyde was added thereto and whether the probe could detect formaldehyde at a reversible rate in the cells was observed, and it was found that the fluorescence signal was decreased in the green channel and increased in the red channel (FIG. 12 c1-c 3). The fluorescence intensity value of the green channel at 434nm excitation light was divided by the fluorescence intensity value at 607nm red channel (Fred/Fgreen) to obtain a ratio image of the cells (fig. 12 d). The ratio imaging result shows that the probe can detect the bisulfite and formaldehyde in a reversible ratio in cells, so the probe has potential biological application value.
Example 10: fluorescence imaging of fluorescent probes for detection of endogenous bisulfite and formaldehyde in Hela cells
a1-a 2: cells were incubated for 30min with probe (10 μ M); b1-b 2: cys (key factor for intracellular SO2 production) (400. mu.M) was added to group a cells and incubated for 30 min; c1-c 2: then adding Tet (an endogenous source of formaldehyde) (400. mu.M) to the cells of group b and incubating for 30 min; d: green and red channel fluorescence intensity ratios (Fred/Fgreen) for images a-c.
The ability of the probes to image endogenous bisulfite and formaldehyde in living cells was evaluated. For control cells incubated with probe only, the fluorescence signal was weaker in the green channel and stronger in the red channel (FIG. 13 a1-a 3). The cells were then further treated with Cys and then imaged and found to have increased fluorescence signal in the green channel and decreased fluorescence signal in the red channel (FIG. 13 b1-b 3), indicating that they can image endogenous bisulfite in the cell at an intracellular rate. Finally, Tet was added thereto, and the fluorescence signal was found to decrease in the green channel and increase in the red channel (FIG. 13 c1-c 3), indicating that the probe can detect endogenous formaldehyde at a reversible rate in the cell. Fred/Fgreen obtains an image of the ratio of endogenous bisulfite and formaldehyde of the cell (FIG. 13d), and the results indicate that the probe can detect endogenous bisulfite and formaldehyde at a reversible ratio in the cell.
Claims (3)
1. A method for detecting the presence of bisulfite/formaldehyde in a sample or determining the bisulfite/formaldehyde content of a sample, comprising:
a) contacting a compound of formula (i) with a sample to form a compound that changes fluorescence;
wherein: r is 1 ，R 2 ，R 3 ，R 4 ，R 5 ，R 6 ，R 7 ，R 8 ，R 9 ，R 10 ，R 11 And R 12 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 ，R 9 ，R 10 ，R 11 And R 12 May be the same or different;
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.
3. Use of a compound of formula (i) according to claim 1 in cellular fluorescence imaging for detecting, measuring or screening for bisulfite/formaldehyde.
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