CN111518066A - Bifunctional fluorescent probe for identifying hypochlorite and bisulfite and preparation method and application thereof - Google Patents
Bifunctional fluorescent probe for identifying hypochlorite and bisulfite and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a bifunctional fluorescent probe for identifying hypochlorite and hydrogen sulfite, a preparation method and application thereof. Under the excitation of 560nM, the probe has specific selectivity on hypochlorite, the lowest detection limit is 15 nM, the fluorescence emission wavelength is 654 nM, and the near infrared detection is realized; under 470nM excitation, the probe has specific selectivity on bisulfite, and the lowest detection limit is 16 nM. The fluorescent probe has extremely weak fluorescence in a buffer system, realizes double recognition of hypochlorite and bisulfite under the excitation of fluorescence with different wavelengths, has the advantages of strong anti-interference capability and low detection limit, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a bifunctional fluorescent probe for identifying hypochlorite and bisulfite, and a preparation method and application thereof.
Background
Sulfur dioxide (SO), a common environmental pollutant2) Mainly due to the combustion of minerals and the production of volcanic eruptions. Sulfites (SO)3 2-) With bisulfite (HSO)3 -) Is SO2In aqueous solution. Sulfur dioxide and its derivatives are now widely used as preservatives in foods and beverages to prevent food oxidation and bacterial growth, and to inhibit enzymatic and non-enzymatic browning during manufacture and storage. However, high doses of sulfite may cause adverse reactions and acute symptoms, and the world health organization dictates that threshold levels of sulfite in food products be tightly controlled. Recent pathological findings indicate high SO concentrations2Can induce many respiratory reactions, and is also closely related to hypertension, cardiovascular diseases and nervous system diseases. In addition, SO2Is involved in many physiological processes (such as decrease in vascular smooth muscle tone and blood pressure) as a gas signaling substance. Therefore, it is very important to develop a method for detecting sulfite with high sensitivity and selectivity.
Hypochlorite (ClO)-) And its protonated form, is widely used in daily life, as common bleaches, deodorants, and 84 disinfectants, among othersA strong nucleophilic non-free radical oxidant, hypochlorous acid, is a natural bactericide for the defense of various organisms in nature. However, excessive hypochlorite solution can be harmful to the health of the organism. On the other hand, hypochlorite participates in many physiological and pathological processes, and plays an important role in organisms. Hypochlorite ion (ClO)-) Is an important active oxygen species in organisms and plays a key role in the immune system. Studies have shown that hypochlorite levels in organisms are abnormal and cause a variety of diseases, such as arthritis, Parkinson's disease, Alzheimer's disease, multiple sclerosis and cancer. Therefore, the development of a method for rapidly, sensitively and specifically detecting hypochlorite ions has important research significance in the fields of environmental science, bioscience, food health and the like.
In recent years, organic small molecule fluorescent probes have attracted much attention for their application in the detection of specific target analytes. The fluorescence analysis method using the fluorescent probe has the advantages of high specificity selectivity, high sensitivity, fast response time, low detection limit and the like, and is widely applied to the fields of environmental science and biomedicine at present. Conventional fluorescent probes are single detection target analytes. However, biological studies have shown that there is some association of active small molecule species in the organism, and that changes in the concentration of one active small molecule species may result in changes in the concentration of the relevant active small molecule species that cannot be tracked by single recognition by conventional fluorescent probes. Therefore, the single recognition performance of the conventional fluorescent probe has certain limitations, and two or more active small molecules cannot be detected simultaneously. The development of the multifunctional fluorescent probe which can be used for identifying two or more kinds of active small molecule detection simultaneously has important scientific significance and practical application value.
Disclosure of Invention
The invention provides a bifunctional fluorescent probe for identifying hypochlorite and bisulfite and a preparation method and application thereof, and solves the technical problem that the conventional fluorescent probe in the prior art cannot simultaneously detect two active small molecules (hypochlorite and bisulfite) due to single identification performance.
The technical scheme of the invention is realized as follows:
a bifunctional fluorescent probe for identifying hypochlorite and bisulfite, the structure of the bifunctional fluorescent probe is as follows:
。
the preparation method of the bifunctional fluorescent probe for identifying hypochlorite and bisulfite adopts the following technical route:
the method comprises the following steps:
(1) dissolving 2, 4-dihydroxy benzaldehyde in absolute ethyl alcohol, adding ethyl acetoacetate and piperidine, and heating and refluxing for a certain time A; cooling to room temperature after the reaction is finished, generating a large amount of yellow solid, performing suction filtration, and washing with glacial ethanol to obtain an intermediate 3-acetyl-7-hydroxycoumarin;
(2) dissolving an intermediate 3-acetyl-7-hydroxycoumarin and an intermediate 4-diethylamino keto acid in methanesulfonic acid, stirring and reacting for a certain time B at 90 ℃, cooling to room temperature after the reaction is finished, dropwise adding the reaction liquid into saturated salt water, separating out a large amount of solids, filtering, washing with ice water, and carrying out chromatographic separation on the obtained solid column to obtain an intermediate NIR-OH;
(3) and (2) dissolving the intermediate NIR-OH and triethylamine in anhydrous dichloromethane, dropwise adding the anhydrous dichloromethane solution dissolved in N, N-dimethylaminothioformyl chloride into the system solution at 0 ℃, dropwise adding the anhydrous dichloromethane solution for a time C, removing an ice bath after dropwise adding, stirring at room temperature for a certain time D, monitoring by thin-layer chromatography, removing the solvent under reduced pressure after the reaction is completed, and separating and purifying by column chromatography to obtain the bifunctional fluorescent probe NIR-P1 for identifying hypochlorite and bisulfite.
The molar ratio of the 2, 4-dihydroxy benzaldehyde, the ethyl acetoacetate and the piperidine in the step (1) is 1: (2-6): (1-5).
The reaction time A in the step (1) is 4-10 hours, and the yield is 55% -85%.
The molar ratio of the intermediate 3-acetyl-7-hydroxycoumarin to the intermediate 4-diethylaminoketoacid in the step (2) is 1: (1-6),
the reaction in the step (2) is carried out for a certain time B of 6-12 hours, eluent used for column chromatography separation is dichloromethane and methanol, the volume ratio is (10-30): 1, and the yield is 70% -90%.
The molar ratio of the compounds NIR-OH, triethylamine and N, N-dimethylamino thiocarbonyl chloride in the step (3) is 1: (2-8): (2-6).
In the step (3), the dripping time C is 20-50 minutes, the reaction time D is 1-6 hours, eluent used for column chromatography separation is dichloromethane and methanol, and the volume ratio is (30-60): 1, the yield is 55-75%.
The bifunctional fluorescent probe is applied to specific bifunctional recognition of hypochlorite and bisulfite.
The recognition mechanism of the bifunctional fluorescent probe on hypochlorite and bisulfite is as follows:
hypochlorite reacts specifically with a 'C = S' bond in a probe structure, so that hydroxyl is exposed, and fluorophore near infrared fluorescence is released (the fluorescence emission is 654 nm); the bisulfite reacts with the unsaturated bond in the probe to generate nucleophilic addition reaction to release fluorescence with the emission of 563 nm.
The invention has the following beneficial effects:
(1) the bifunctional fluorescent probe can simultaneously identify hypochlorite and bisulfite, and is novel in structure, simple in synthesis method and easy to separate and purify.
(2) The bifunctional fluorescent probe has high specificity on bifunctional identification of hypochlorite and bisulfite, the maximum fluorescence emission wavelengths (654 nm and 563nm respectively) are far away, and mutual interference cannot be generated; secondly, the probe of the invention can eliminate the interference of various metal ions and active small molecules and has high specific selectivity on hypochlorite and bisulfite.
(3) The bifunctional fluorescent probe has the advantages of high response speed and high sensitivity (the lowest detection limit of hypochlorite is 15 nM, and the lowest detection limit of bisulfite is 16 nM) for bifunctional identification of hypochlorite and bisulfite; the recognition of hypochlorite can reach near infrared region, and can effectively overcome the interference of self-fluorescence in some endogenous fluorophores in environment and organism and tissue.
(4) The utility model provides a difunctional fluorescence probe can reach the effect that the bore hole discerned to the discernment of hypochlorite root and bisulfite, and probe solution alone is pink, and solution color is blue-green behind the discernment hypochlorite root, and solution color is yellow behind the discernment bisulfite, has realized visual detection.
(5) The identification mechanism of the bifunctional fluorescent probe for hypochlorite and bisulfite is confirmed by high resolution mass spectrometry. The probe recognizes hypochlorite and then becomes a fluorophore containing hydroxyl, the theoretical molecular weight of the probe is 482.1598 (positive ion), and the high-resolution test data is 482.1592 (positive ion mode, FIG. 13); the theoretical molecular weight of the probe after specific nucleophilic addition with bisulfite is 649.1320 (negative ion), and the high resolution test data is 649.1327 (negative ion mode, FIG. 14). High-resolution mass spectrum data verifies the recognition mechanism of the bifunctional probe for recognizing hypochlorite and bisulfite, and provides theoretical support for further developing more related fluorescent probes.
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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. 1 shows the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe NIR-P1 (the solvent is CDCl)3）。
FIG. 2 shows the nuclear magnetic resonance carbon spectrum of the fluorescent probe NIR-P1 (the solvent is CDCl)3）。
FIG. 3 shows the NIR-P1 high resolution mass spectrum of the fluorescence probe (solvent is CH)3OH）。
FIG. 4 is a graph of the UV selectivity of the fluorescent probe NIR-P1 of the invention to common metal ions and reactive small molecules.
FIG. 5 is a graph showing fluorescence selectivity of the fluorescent probe NIR-P1 of the invention for identifying hypochlorite, wherein the excitation wavelength is 560nm, and the emission wavelength is 654 nm.
FIG. 6 is a graph showing the fluorescence interference resistance of the fluorescent probe NIR-P1 for identifying hypochlorite according to the invention, wherein the excitation wavelength is 560nm, and the emission wavelength is 654 nm.
FIG. 7 is a fluorescence titration chart of hypochlorite recognized by the fluorescent probe NIR-P1 of the invention, wherein the inset is a lowest detection limit chart, an excitation wavelength is 560nm, and an emission wavelength is 654 nm.
FIG. 8 is a graph showing fluorescence selectivity of the fluorescent probe NIR-P1 for identifying bisulfite according to the invention, wherein the excitation wavelength is 470nm, and the emission wavelength is 563 nm.
FIG. 9 is a graph showing the fluorescence interference resistance of the fluorescent probe NIR-P1 for identifying bisulfite according to the invention, wherein the excitation wavelength is 470nm, and the emission wavelength is 563 nm.
FIG. 10 is a graph of fluorescence titration of bisulfite identification by the fluorescent probe NIR-P1 of the present invention, with the lowest detection limit at 470nm for excitation and 563nm for emission.
FIG. 11 is a pH adaptation graph for bisulfite identification by the fluorescent probe NIR-P1 of the present invention, with an excitation wavelength of 470nm and an emission wavelength of 563 nm.
FIG. 12 is a fluorescence kinetic diagram of the fluorescent probe NIR-P1 of the invention for identifying bisulfite, with an excitation wavelength of 470nm and an emission wavelength of 563 nm.
FIG. 13 is a high-resolution mechanism verification diagram of the fluorescent probe NIR-P1 for identifying hypochlorite (solvent is CH) in the invention3OH）。
FIG. 14 shows the present inventionHigh-resolution mechanism verification diagram for identifying bisulfite by fluorescent probe NIR-P1 (solvent is CH)3OH）。
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following 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 not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The preparation method of the bifunctional fluorescent probe for identifying hypochlorite and bisulfite of the embodiment comprises the following steps:
(1) preparation of intermediate 3-acetyl-7-hydroxycoumarin
2, 4-dihydroxybenzaldehyde (1.38 g, 10 mmol) was dissolved in 30 mL of anhydrous ethanol, ethyl acetoacetate (3.9 g, 30 mmol) and piperidine (1 mL, 11 mmol) were added thereto, and the reaction was refluxed for 6 hours. After the reaction is finished, the reaction product is cooled to room temperature, a large amount of yellow solid is generated, the reaction product is filtered, and the filtrate is washed by using glacial ethanol, so that 1.53g of an intermediate 3-acetyl-7-hydroxycoumarin is obtained, and the yield is 71%.
(2) Preparation of intermediate NIR-OH
The intermediate 3-acetyl-7-hydroxycoumarin (204 mg, 1 mmol) and the intermediate 4-diethylaminoketo acid (313 mg, 1 mmol) were dissolved in methanesulfonic acid (10 mL) and reacted with stirring at 90 ℃ for 6 hours. After the reaction, cooling to room temperature, adding the reaction solution dropwise into saturated saline solution to precipitate a large amount of solid, filtering, washing with ice water, and performing column Chromatography (CH)2Cl2/CH3OH =20:1, v/v) yielded 337 mg of intermediate NIR-OH in 70% yield.
(3) Preparation of Probe NIR-P1
The compound NIR-OH (577 mg, 1 mmol) and triethylamine (0.3 mL, 2 mmol) were dissolved in anhydrous dichloromethane (25 mL) and the solution was N, N-dimethyl at 0 deg.CA solution of aminothiocarbonyl chloride (244 mg, 2 mmol) in anhydrous methylene chloride (15 mL) was added dropwise to the above system solution over a period of 20 minutes. After the completion of the dropwise addition, the ice bath was removed, and the reaction was stirred at room temperature for 2 hours. Monitoring by thin layer chromatography, removing solvent under reduced pressure after reaction is completed, and performing column Chromatography (CH)2Cl2/CH3OH =35:1, v/v) isolation and purification gave 365.2 mg of dark yellow solid in 55% yield.
Nuclear magnetic resonance measurement:1H NMR (CDCl3, 400 MHz) 1.19 (t,J= 6.0 Hz, 6 H), 3.37(t,J= 8.0 Hz, 7 H), 3.48 (s, 3 H), 1.19 (t,J= 6.0 Hz, 6 H), 6.23 (d,J=8.0 Hz, 1 H), 6.41 (d,J= 8.0 Hz, 1 H), 6.48 (s, 1 H), 6.76 (s, 1 H), 7.08(d,J= 8.0 Hz, 2 H), 7.29 (d,J= 8.0 Hz, 1 H), 7.65 (m, 3 H), 7.99 (d,J=8.0 Hz, 1 H), 8.50 (s, 1 H);13C NMR (CDCl 3100 MHz) 12.6, 39.0, 43.4,44.5, 97.4, 103.4, 104.6, 109.3, 111.1, 116.8, 117.9, 120.2, 124.0, 125.0,126.6, 128.5, 129.4, 129.5, 134.6, 139.5, 146.0, 149.4, 152.6, 153.6, 153.7,156.8, 157.7, 169.9, 186.6. The hydrogen spectrum and carbon spectrum of nuclear magnetic resonance are shown in FIG. 1 and 2, respectively.
High-resolution mass spectrometry: HR-ESI-MS calcd for C32H29N2O6S+569.1741 and found 569.1737. The high resolution mass spectrum is shown in figure 3.
Example 2
The preparation method of the bifunctional fluorescent probe for identifying hypochlorite and bisulfite of the embodiment comprises the following steps:
(1) preparation of intermediate 3-acetyl-7-hydroxycoumarin
2, 4-dihydroxybenzaldehyde (1.38 g, 10 mmol) was dissolved in 30 mL of anhydrous ethanol, ethyl acetoacetate (2.6 g, 20 mmol) and piperidine (2 mL, 22 mmol) were added thereto, and the reaction was refluxed for 4 hours. After the reaction is finished, the reaction product is cooled to room temperature, a large amount of yellow solid is generated, the reaction product is filtered, and the filtrate is washed by using glacial ethanol, so that 1.21 g of the intermediate 3-acetyl-7-hydroxycoumarin is obtained, and the yield is 56%.
(2) Preparation of intermediate NIR-OH
The intermediate 3-acetyl-7-hydroxycoumarin (204 mg, 1 mmol) and the intermediate 4-diethylamino-keto acid (626 mg, 2 mmol) were dissolved in methanesulfonic acid (10 mL) and reacted with stirring at 90 ℃ for 8 hours. After the reaction, cooling to room temperature, adding the reaction solution dropwise into saturated saline solution to precipitate a large amount of solid, filtering, washing with ice water, and performing column Chromatography (CH)2Cl2/CH3OH =25:1, v/v) gave 390 mg of intermediate NIR-OH in 81% yield.
(3) Preparation of Probe NIR-P1
The compound NIR-OH (577 mg, 1 mmol) and triethylamine (0.6 mL, 4 mmol) were dissolved in anhydrous dichloromethane (25 mL), and a solution of N, N-dimethylaminothiocarbonyl chloride (366 mg, 3 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to the above system solution at 0 ℃ over 30 minutes. After the completion of the dropwise addition, the ice bath was removed, and the reaction was stirred at room temperature for 4 hours. Monitoring by thin layer chromatography, removing solvent under reduced pressure after reaction is completed, and performing column Chromatography (CH)2Cl2/CH3OH =40:1, v/v) isolation and purification gave 431.6 mg of dark yellow solid in 65% yield.
Nuclear magnetic resonance measurement:1H NMR (CDCl3, 400 MHz) 1.19 (t,J= 6.0 Hz, 6 H), 3.37(t,J= 8.0 Hz, 7 H), 3.48 (s, 3 H), 1.19 (t,J= 6.0 Hz, 6 H), 6.23 (d,J=8.0 Hz, 1 H), 6.41 (d,J= 8.0 Hz, 1 H), 6.48 (s, 1 H), 6.76 (s, 1 H), 7.08(d,J= 8.0 Hz, 2 H), 7.29 (d,J= 8.0 Hz, 1 H), 7.65 (m, 3 H), 7.99 (d,J=8.0 Hz, 1 H), 8.50 (s, 1 H);13C NMR (CDCl 3100 MHz) 12.6, 39.0, 43.4,44.5, 97.4, 103.4, 104.6, 109.3, 111.1, 116.8, 117.9, 120.2, 124.0, 125.0,126.6, 128.5, 129.4, 129.5, 134.6, 139.5, 146.0, 149.4, 152.6, 153.6, 153.7,156.8, 157.7, 169.9, 186.6. The hydrogen spectrum and carbon spectrum of nuclear magnetic resonance are shown in FIG. 1 and 2, respectively.
High-resolution mass spectrometry: HR-ESI-MS calcd for C32H29N2O6S+569.1741 and found 569.1737. The high resolution mass spectrum is shown in figure 3.
Example 3
The preparation method of the bifunctional fluorescent probe for identifying hypochlorite and bisulfite of the embodiment comprises the following steps:
(1) preparation of intermediate 3-acetyl-7-hydroxycoumarin
2, 4-dihydroxybenzaldehyde (1.38 g, 10 mmol) was dissolved in 30 mL of anhydrous ethanol, ethyl acetoacetate (7.8 g, 60 mmol) and piperidine (4.5 mL, 55 mmol) were added thereto, and the reaction was refluxed for 10 hours. After the reaction is finished, the reaction product is cooled to room temperature, a large amount of yellow solid is generated, the reaction product is filtered, and the filtrate is washed by using glacial ethanol, so that 1.83 g of the intermediate 3-acetyl-7-hydroxycoumarin is obtained, and the yield is 85%.
(2) Preparation of intermediate NIR-OH
The intermediate 3-acetyl-7-hydroxycoumarin (204 mg, 1 mmol) and the intermediate 4-diethylaminoketoacid (1252 mg, 4 mmol) were dissolved in methanesulfonic acid (10 mL) and reacted with stirring at 90 ℃ for 10 hours. After the reaction, cooling to room temperature, adding the reaction solution dropwise into saturated saline solution to precipitate a large amount of solid, filtering, washing with ice water, and performing column Chromatography (CH)2Cl2/CH3OH =28:1, v/v) gave 433 mg of intermediate NIR-OH in 90% yield.
(3) Preparation of Probe NIR-P1
The compound NIR-OH (577 mg, 1 mmol) and triethylamine (0.75 mL, 5 mmol) were dissolved in anhydrous dichloromethane (25 mL), and a solution of N, N-dimethylaminothiocarbonyl chloride (610 mg, 5 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to the above system solution at 0 ℃ over 40 minutes. After the completion of the dropwise addition, the ice bath was removed, and the reaction was stirred at room temperature for 6 hours. Monitoring by thin layer chromatography, removing solvent under reduced pressure after reaction is completed, and performing column Chromatography (CH)2Cl2/CH3OH =45:1, v/v) isolation and purification gave 464.8 mg of dark yellow solid in 70% yield.
Nuclear magnetic resonance measurement:1H NMR (CDCl3, 400 MHz) 1.19 (t,J= 6.0 Hz, 6 H), 3.37(t,J= 8.0 Hz, 7 H), 3.48 (s, 3 H), 1.19 (t,J= 6.0 Hz, 6 H), 6.23 (d,J=8.0 Hz, 1 H), 6.41 (d,J= 8.0 Hz, 1 H), 6.48 (s, 1 H), 6.76 (s, 1 H), 7.08(d,J= 8.0 Hz, 2 H), 7.29 (d,J= 8.0 Hz, 1 H), 7.65 (m, 3 H), 7.99 (d,J=8.0 Hz, 1 H), 8.50 (s, 1 H);13C NMR (CDCl 3100 MHz) 12.6, 39.0, 43.4,44.5, 97.4, 103.4, 104.6, 109.3, 111.1, 116.8, 117.9, 120.2, 124.0, 125.0,126.6, 128.5, 129.4, 129.5, 134.6, 139.5, 146.0, 149.4, 152.6, 153.6, 153.7,156.8, 157.7, 169.9, 186.6. The hydrogen spectrum and carbon spectrum of nuclear magnetic resonance are shown in FIG. 1 and 2, respectively.
High-resolution mass spectrometry: HR-ESI-MS calcd for C32H29N2O6S+569.1741 and found 569.1737. The high resolution mass spectrum is shown in figure 3.
Examples of the effects of the invention
1 mM probe solution preparation: the probe (NIR-P1) prepared in example 1 was weighed accurately and NIR-P1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM solution for use.
Ultraviolet selectivity experiment:
specific selectivity is an important criterion for determining whether a fluorescent probe molecule is efficient. Firstly, ultraviolet-visible spectrophotometer is adopted to investigate the ultraviolet selectivity of the probe to metal ions and active small molecules. As shown in FIG. 4, the probe has strong UV absorption at 630 nm for hypochlorite and at 490 nm for sulfite and bisulfite. It is worth noting that the probe does not show obvious ultraviolet absorption to other common metal ions and active small molecules, so that specific identification of different ultraviolet absorption wavelengths of hypochlorite and sulfite/bisulfite by the probe can be realized through an ultraviolet selectivity experiment.
Fluorescence selectivity experiments:
ultraviolet selectivity experiments prove that the probe has specific selectivity on hypochlorite and sulfite/bisulfite at different ultraviolet absorption wavelengths. Application ofFluorescence spectrometer examine the fluorescence selectivity of probe NIR-P1 for hypochlorite and bisulfite under different excitation conditions. As shown in figure 5, under the excitation condition at 560nm, the NIR-P1(10 μ M) probe alone has weak fluorescence emission intensity at 654 nm in Tris-HCl buffer solution, and when hypochlorite is added (10 eq.), the fluorescence emission intensity at 654 nm is obviously enhanced, but when 10 equivalents of other substances (common metal ions and active small molecules) are added, the fluorescence emission intensity (F654) of the solution system is not obviously changed compared with the fluorescence emission intensity (F654) of the probe system alone. As shown in FIG. 8, under the excitation condition at 470nm, the NIR-P1(10 μ M) probe alone has weak fluorescence emission intensity at 563nm in Tris-HCl buffer solution, and after adding bisulfite (10 eq.), the fluorescence emission intensity at 563nm is obviously enhanced, but after adding 10 equivalents of other substances (common metal ions and active small molecules), the fluorescence emission intensity (F563) of the solution system is not obviously changed compared with the fluorescence emission (F563) intensity of the probe system alone, and only the potassium superoxide (KO) is changed2) There was a slight fluorescence enhancement but not enough to affect the bisulfite recognition by the probe. The experimental results show that the probe has good fluorescence specificity selectivity on hypochlorite under the 560nm excitation condition; under the excitation condition of 470nm, the probe has better fluorescence specificity selectivity to bisulfite.
Fluorescence interference experiment:
in order to test the anti-interference capability of the probe molecules on hypochlorite and bisulfite detection, the anti-interference performance of the probe molecules on common active small molecules is tested in a fluorescence emission spectrum. As shown in figure 6, under the excitation condition of 560nm, 10 equivalents of various common active small molecules are respectively added into a single probe NIR-P1(10 mu M) Tris-HCl buffer solution, the fluorescence emission intensity (654 nm) of the various common active small molecules is respectively tested, then 10 equivalents of hypochlorite solution is respectively added into the solution, and as can be seen from figure 6, the fluorescence intensity (654 nm) obtained when hypochlorite is added in the presence of the common active small molecules is basically the same as that obtained when hypochlorite is added in the single probe NIR-P1, and the result shows that the probe NIR-P1 has stronger capacity for resisting the interference of the common active small molecules in the detection of the hypochlorite. As shown in figure 9, under the excitation condition of 470nm, 10 equivalents of each common active small molecule to be tested are respectively added into a single probe NIR-P1(10 mu M) Tris-HCl buffer solution, the fluorescence emission intensity (563nm) is respectively tested, then 10 equivalents of bisulfite solution is respectively added into the solution, and as can be seen from figure 9, the fluorescence intensity (563nm) obtained when bisulfite is added in the presence of the common active small molecule is basically the same as that obtained when bisulfite is added separately, and the result shows that the probe NIR-P1 has stronger anti-interference capability to the detection of the bisulfite.
Minimum detection limit experiment:
the good detection limit is one of the criteria for checking whether a probe molecule has an application value. The lowest detection limit of the probe NIR-P1 on hypochlorite and hydrogen sulfite is determined by adopting a fluorescence spectrometer, the concentration of the fixed probe NIR-P1 is 10 mu M in Tris-HCl buffer solution, the response intensity of the probe NIR-P1 on hypochlorite with different concentrations is determined under the excitation condition of 560nm, the fluorescence intensity of the system is continuously enhanced along with the increase of the concentration of the hypochlorite (figure 7), and the research shows that the fluorescence intensity (654 nm) value of the solution is linear (R) within the range of 0-0.5 mu M of the concentration of the hypochlorite2= 0.998), the detection limit of this probe molecule for hypochlorite was found to be 15 nM by calculation (3 σ/k) according to the IUPAC rules. As shown in the attached figure 10, in a Tris-HCl buffer solution, the concentration of an immobilized probe NIR-P1 is 10 mu M, under the excitation condition of 470nm, the response intensity of the immobilized probe NIR-P1 to bisulfite with different concentrations is measured, the fluorescence intensity of the system is continuously enhanced along with the increase of the concentration of the bisulfite, and the research shows that the value of the fluorescence intensity (563nm) of the solution is linear (R) between 0 and 5 mu M of the concentration of the bisulfite2= 0.997), the detection limit of bisulfite by this probe molecule was found to be 16nM by calculation (3 σ/k) according to the IUPAC rules. The test result shows that the probe NIR-P1 has higher practical value for detecting hypochlorite and bisulfite.
Effect of pH on Probe recognition ability
In order to test that the probe can recognize bisulfite under different pH values (pH), the influence of different pH values (pH) on the recognition of bisulfite by the probe is examined. Tris-HCl buffer solutions with pH values of 4, 5, 6, 7, 8, 9 and 10 mM were prepared, respectively. In the Tris-HCl buffer solution system, the fluorescence emission (563nm) of a single probe (10 mu L) is not greatly changed by changing the pH value of the buffer system (4-10), but the fluorescence emission intensity (563nm) of the system is obviously enhanced between pH 5 and 10 in the presence of bisulfite (the concentration is 100 mu L) (figure 11), and the result shows that the probe can be applied to identify the bisulfite under physiological conditions.
Experiment of dynamics
Good recognition speed is one of the important indicators for probe investigation. A fluorescence spectrometer is adopted to investigate the kinetic experiment of the probe on the bisulfite. The change of fluorescence emission (563nm) of the probe alone and the probe bisulfite was measured with time under excitation conditions at 470nm with an immobilized probe NIR-P1 concentration of 10. mu.M in Tris-HCl buffer solution. As shown in FIG. 12, the fluorescence intensity of the individual probes did not fluctuate much with time; when bisulfite was added, the fluorescence emission intensity of the solution system increased instantaneously and reached a response plateau at 10 s. The results show that the probe is quick and sensitive in recognition response to the bisulfite, and the effect of real-time detection is achieved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. The bifunctional fluorescent probe for identifying hypochlorite and bisulfite is characterized by having the following structure:
。
2. the method for preparing a bifunctional fluorescent probe for identifying hypochlorite and bisulfite according to claim 1, characterized by comprising the steps of:
(1) dissolving 2, 4-dihydroxy benzaldehyde in absolute ethyl alcohol, adding ethyl acetoacetate and piperidine, and heating and refluxing for a certain time A; cooling to room temperature after the reaction is finished, generating a large amount of yellow solid, performing suction filtration, and washing with glacial ethanol to obtain an intermediate 3-acetyl-7-hydroxycoumarin;
(2) dissolving an intermediate 3-acetyl-7-hydroxycoumarin and an intermediate 4-diethylamino keto acid in methanesulfonic acid, stirring and reacting for a certain time B at 90 ℃, cooling to room temperature after the reaction is finished, dropwise adding the reaction liquid into saturated salt water, separating out a large amount of solids, filtering, washing with ice water, and carrying out chromatographic separation on the obtained solid column to obtain an intermediate NIR-OH;
(3) and (2) dissolving the intermediate NIR-OH and triethylamine in anhydrous dichloromethane, dropwise adding the anhydrous dichloromethane solution dissolved in N, N-dimethylaminothioformyl chloride into the system solution at 0 ℃, dropwise adding the anhydrous dichloromethane solution for a time C, removing an ice bath after dropwise adding, stirring at room temperature for a certain time D, monitoring by thin-layer chromatography, removing the solvent under reduced pressure after the reaction is completed, and separating and purifying by column chromatography to obtain the bifunctional fluorescent probe NIR-P1 for identifying hypochlorite and bisulfite.
3. The method of claim 2, wherein: the molar ratio of the 2, 4-dihydroxy benzaldehyde, the ethyl acetoacetate and the piperidine in the step (1) is 1: (2-6): (1-5).
4. The method of claim 2, wherein: the reaction time A in the step (1) is 4-10 hours, and the yield is 55% -85%.
5. The method of claim 2, wherein: the molar ratio of the intermediate 3-acetyl-7-hydroxycoumarin to the intermediate 4-diethylaminoketoacid in the step (2) is 1: (1-6),
the method of claim 2, wherein: the reaction in the step (2) is carried out for a certain time B of 6-12 hours, eluent used for column chromatography separation is dichloromethane and methanol, the volume ratio is (10-30): 1, and the yield is 70% -90%.
6. The method of claim 2, wherein: the molar ratio of the compounds NIR-OH, triethylamine and N, N-dimethylamino thiocarbonyl chloride in the step (3) is 1: (2-8): (2-6).
7. The method of claim 2, wherein: in the step (3), the dripping time C is 20-50 minutes, the reaction time D is 1-6 hours, eluent used for column chromatography separation is dichloromethane and methanol, and the volume ratio is (30-60): 1, the yield is 55-75%.
8. The bifunctional fluorescent probe of claim 1, for specific bifunctional recognition of hypochlorite and bisulfite.
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| CN113072528A (en) * | 2021-04-14 | 2021-07-06 | 济南大学 | Near-infrared ratiometric fluorescent probe for reversibly detecting bisulfite/formaldehyde, preparation method and application |
| CN115010691A (en) * | 2022-05-26 | 2022-09-06 | 山西大学 | Fluorescent probe and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113072528A (en) * | 2021-04-14 | 2021-07-06 | 济南大学 | Near-infrared ratiometric fluorescent probe for reversibly detecting bisulfite/formaldehyde, preparation method and application |
| CN115010691A (en) * | 2022-05-26 | 2022-09-06 | 山西大学 | Fluorescent probe and preparation method and application thereof |
| CN115010691B (en) * | 2022-05-26 | 2023-12-26 | 山西大学 | Fluorescent probe and preparation method and application thereof |
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