CN110563609A - Preparation method and application of near-infrared fluorescent probe for detecting selenious acid roots - Google Patents
Preparation method and application of near-infrared fluorescent probe for detecting selenious acid roots Download PDFInfo
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Abstract
The present invention belongs to selenious acid heel (SeO)3 2‑) The detection field relates to a preparation method and application of a near-infrared fluorescent probe for detecting selenious acid roots. Activating levulinic acid by catalysts EDC and DMAP, and reacting the activated product with malononitrile isophorone intermediate 1 (2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene)]Malononitrile) to obtain a compound based on malononitrile isophorone which can be used as a high choice for selenite in ethanol-water solutionThe fluorescence emission wavelength of the sex fluorescent probe is above 650 nm, and near-infrared fluorescence spectrum recognition is realized. The results show that: the probe SeP1 has high-efficiency and specific selectivity on selenite and strong anti-interference capability. The lowest detection limit of the probe to selenite is 0.068 mu M, and the probe has practical value of being applied to detection of selenite in the environment.
Description
Technical Field
The present invention belongs to selenious acid heel (SeO)3 2-) The detection field relates to a preparation method and application of a near-infrared fluorescent probe for detecting selenious acid roots.
Background
Selenium is one of essential trace elements in human body, participates in the synthesis of various selenium-containing enzymes and selenium-containing proteins in human body, and has various physiological functions of resisting oxidation, resisting tumor and the like. Due to the important function of selenium, the proper amount of selenium supplement for human bodies can play roles in preventing organ aging and pathological changes, delaying aging, enhancing immunity and resisting harmful heavy metals.
Selenium exists in nature in two forms of organic selenium and inorganic selenium. The organic selenium is formed by combining selenium with amino acid through biotransformation, mainly exists in the form of selenomethionine, participates in the synthesis of protein in a living body, and plays important functions of resisting oxidation and enhancing the immunity of the human body. The inorganic selenium mainly refers to sodium selenate and sodium selenite, and has low biological effectiveness, high toxicity and easy environmental pollution. The toxicity of inorganic selenium is obviously higher than that of organic selenium, and the toxicity of selenite is slightly higher than that of sodium selenate. Therefore, the method has important significance for detecting and evaluating selenium with different forms, particularly inorganic selenium.
At present, the inorganic selenium form analysis method mainly adopts a method of combining chromatographic separation and atomic fluorescence technology, but the method has high requirements on instruments and equipment, has strict requirements on the technical level of operators, needs expensive reagents and has high cost, and is not beneficial to wide popularization.
The organic fluorescent molecular sensing technology developed in recent years is applied to the detection of various ions due to its advantages of high sensitivity, high selectivity, no need of separation, easy observation, etc., and also becomes a popular research field. Therefore, it becomes very significant to design fluorescent probes for rapid detection of selenous acid heel. The fluorescent probe has the advantages of good selectivity, high sensitivity, simple and rapid operation, less damage to a detected object and the like, and is widely applied to the aspects of detecting metal cations, anions, active small molecules in organisms and the like in environments and biological systems. The fluorescent probe detection method not only makes up the defects of large sample reagent amount requirement, difficult real-time online analysis, complicated steps, unsuitability for biological and toxicological researches and the like of the traditional molecular and ion analysis method, but also has more attractive advantages compared with the traditional detection method. The research on fluorescent probes has grown considerably over the past decades; patent 201510178114.X discloses a malononitrile isophorone copper ion fluorescent probe and a preparation method thereof, the probe can selectively identify copper ions in a buffer solution, and the copper ions are represented by low detection limit (only 0.2 mu M), large Stokes displacement, fluorescence enhancement, long-wavelength emission and color change under visible light, and the copper ions can be detected by naked eyes by using the fluorescence enhancement and the color change under the visible light; the fluorescence enhancement response of the fluorescent probe to the copper ions can be used for carrying out qualitative or quantitative analysis on the copper ions; but the method is mainly used for researching the quantitative and qualitative analysis of cation-copper ions, the lowest detection limit of the probe is higher, and a recognition group in the chemical structure of the probe cannot be used for detecting anions, so that the probe cannot be applied to detecting the anion selenite; patent 201310125465.5 discloses a method for detecting selenate ion content and reducing to selenite ion online, which is used to reduce hexavalent selenate ion online to tetravalent selenite ion, thereby realizing liquid chromatography/ion chromatography and atomic fluorescence combined morphological analysis method of selenate ion high-sensitivity steam generation-atomic fluorescence detection, the patent detects selenite ion by chromatography and atomic fluorescence combined analysis, the detection limit of the method can not reach the requirement of detecting selenite in the environment, and the detection method is complex, not beneficial to real-time rapid detection, the fluorescence probe analysis method adopted by the invention directly detects tetravalent selenite ion, and is essentially different from the patent methods; therefore, the development prospect of developing fluorescent probes for recognizing inorganic selenium with high sensitivity and high selectivity is still a huge challenge and brings huge returns.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method and application of a near-infrared fluorescent probe for detecting selenite, levulinic acid is activated through catalysts EDC and DMAP, the activated product and malononitrile isophorone intermediate 1 (2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene ] malononitrile) are subjected to condensation acylation reaction to obtain a compound based on malononitrile isophorone, the compound can be used as a high-selectivity fluorescent probe for selenite in ethanol-water solution, the fluorescence emission wavelength of the compound is above 650 nm, and near-infrared fluorescence spectrum identification is realized.
The technical scheme of the invention is realized as follows:
A near-infrared fluorescent probe molecule for detecting selenite roots is disclosed, wherein the structural formula of the fluorescent probe is as follows:
。
the method for preparing the near-infrared fluorescent probe molecule for detecting selenite roots has the following synthetic route:
。
The preparation steps are as follows:
(1) dissolving levulinic acid, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine) in an anhydrous dichloromethane solution, and stirring at room temperature for reaction for a period of time to obtain a solution I;
(2) Dissolving the intermediate 1 (2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene ] malononitrile) in an anhydrous dichloromethane solution to obtain an intermediate 1 solution, then dropwise adding the intermediate 1 solution into the solution I obtained in the step (1) at room temperature, and reacting for a period of time after dropwise adding to obtain a solution II;
(3) And (3) washing the solution II obtained in the step (2) with a saturated sodium chloride solution, drying the obtained organic phase with anhydrous sodium sulfate, filtering, removing the solvent under reduced pressure, and separating by silica gel column chromatography to obtain a product SeP1, namely the fluorescent probe molecule.
in the step (1), the molar ratio of the levulinic acid to the EDC to the DMAP is 1 (0.5-4) to 0.1-2, and the reaction is carried out for 10-60 minutes under stirring at room temperature.
In the step (2), the intermediate 1 is 2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene ] malononitrile, the molar ratio of the levulinic acid in the solution I to the intermediate 1 is 1 (0.5-2), and the reaction time is 8-24 hours after the dropwise addition is finished.
The silica gel column separation in the step (3) adopts ethyl acetate and petroleum ether eluent with the volume ratio of 1 (3-12), and the yield is 40-80%.
The near-infrared fluorescent probe molecule is applied to the field of high-sensitivity and specific detection of selenious acid roots.
The Latin reagent company, such as chemical reagent, solvent, and metal ion, used in the process of preparing the fluorescent probe SeP1 according to the present invention. A DTX-400 nuclear magnetic resonance spectrometer of Bruke company is adopted in the process of confirming and testing the performance of the fluorescent probe SeP1, the solvent is deuterated chloroform, and a hydrogen spectrum and a carbon spectrum of nuclear magnetic resonance are recorded by taking TMS as an internal standard. High resolution mass spectral data were recorded using a Q-exact HR-MS mass spectrometer from Thermo. The fluorescence spectrum was recorded using a F-7000 fluorescence spectrometer from Hitachi, Japan.
The invention has the following beneficial effects:
(1) The fluorescent probe is a compound based on malononitrile isophorone, namely an intermediate 1 (2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene)]Malononitrile) has strong fluorescence when the hydroxyl group is exposed, and hardly has fluorescence when the hydroxyl group is protected. According to the invention, an acetyl propyl group is adopted to protect the hydroxyl in the structure of the intermediate 1 to obtain the fluorescent probe SeP1, and when a selenite ion and the acetyl propyl group perform a specific reaction, the intermediate 2 is generated and separated, so that the hydroxyl in the structure is deprotected to regenerate the intermediate 1 and release strong fluorescence (a specific mechanism diagram is shown in figure 8). The reaction mechanism is verified by means of high-resolution mass spectrometry (as shown in figure 9), and the result of the high-resolution mass spectrometry test of the solution after the fluorescent probe SeP1 recognizes selenite is 289.1347 ([ M-H ]+]-) The theoretical calculation of anion for intermediate 1 was 289.1341. This data corroborates the mechanism of action shown in FIG. 8.
(2) The invention researches the situation that the probe SeP1 is in CH by a fluorescence spectrometer3CH2OH-H2The identification performance of the O solution and related analytes is that the fluorescence intensity (657 nm) obtained by adding selenite in the presence of metal cations and common anions is basically the same as that obtained by adding selenite alone, as shown in FIG. 5, and the result shows that the probe SeP1 has stronger anti-metal cation and common anion interference performance on the detection of selenite and can overcome the interference of complex background matrix fluorescence in organisms and environments.
(3) The invention adopts a fluorescence spectrometer to detect the selenite root of the probe SeP1Minimum detection limit in CH3CH2OH-H2In O (1:1, v/v) solution, the concentration of the immobilized probe SeP1 is 16.7 mu M, the response intensity of the immobilized probe to selenite with different concentrations is measured, the system fluorescence intensity is continuously enhanced along with the increase of the selenite concentration (figure 6), and the research shows that the solution fluorescence intensity value is linear (R is between 0 and 2.5 equivalent of the selenite concentration)2= 0.985), according to IUPAC rules, the detection limit of the probe molecule to the bisulfite is 0.068 mu M through calculation (3 sigma/k), and the test result shows that the probe SeP1 has practical value for detecting selenite in the environment, and has wide application prospect in the detection field of environment and biological systems.
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. 1 shows the NMR spectrum of the fluorescent probe SeP1 of the present invention.
FIG. 2 is a nuclear magnetic resonance carbon spectrum of the fluorescent probe SeP1 of the present invention.
FIG. 3 is a high resolution mass spectrum of the fluorescent probe SeP1 of the present invention.
FIG. 4 is a graph showing fluorescence selectivity of the fluorescent probe SeP1 of the present invention, with an excitation wavelength of 550 nm.
FIG. 5 shows that the fluorescent probe SeP1 of the present invention recognizes SeO3 2-The excitation wavelength is 550 nm, and the emission wavelength is 657 nm.
FIG. 6 shows that the fluorescent probe SeP1 of the present invention recognizes SeO3 2-Fluorescence titration graph of (1), excitation wavelength 550 nm.
FIG. 7 shows that the fluorescent probe SeP1 of the present invention recognizes SeO3 2-The excitation wavelength is 550 nm, and the emission wavelength is 657 nm.
FIG. 8 shows a fluorescent probe of the present inventionSeP1 identifying SeO3 2-the reaction mechanism diagram of (1).
FIG. 9 shows SeO recognition by fluorescent probe SeP1 according to the present invention3 2-High resolution verification of the reaction mechanism (2).
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.
the preparation method of the intermediate 1 comprises the following steps:
。
Commercial starting material (3,5, 5-trimethylcyclohex-2-enylidene) malononitrile (compound A, 186 mg, 1 mmol) and p-hydroxybenzaldehyde (244 mg, 2 mmol) were dissolved in 6 mL of an ethanol solution, one to two drops of piperidine and acetic acid were added dropwise to the above system, respectively, and the reaction was heated under reflux for 4 hours, and cooled to room temperature after TLC monitoring completion of the reaction. The reaction solution was poured into 15ml of an ice-water mixture, and a precipitate was formed. The precipitate was filtered off and redissolved in dichloromethane and isolated by column chromatography to give intermediate 1 (203 mg) as a yellow solid in 70% yield.
example 1
The preparation method of the near-infrared fluorescent probe for detecting selenite roots in the embodiment comprises the following steps:
Levulinic acid (116.0 mg, 1.0 mmol) was dissolved in 10 mL of anhydrous dichloromethane, and EDC (96 mg, 0.5 mmol) and DMAP (12.2 mg, 0.1 mmol) were added and stirred at room temperature for 10 minutes. Dissolving the intermediate 1 (145 mg, 0.5 mmol) in 10 mL of anhydrous dichloromethane solution, dropwise adding the mixture into the mixed solution at room temperature, reacting for 8 hours after dropwise adding, washing the reaction solution for 3 times by using 10 mL of saturated sodium chloride solution, drying an organic phase by using anhydrous sodium sulfate, filtering, removing the solvent under reduced pressure, and separating by using ethyl acetate and petroleum ether as detergents (the volume ratio is 1: 3) through silica gel column chromatography to obtain 77.6 mg of a product SeP1 with the yield of 40%.
Nuclear magnetic resonance measurement:1H NMR (CDCl3, 400 MHz) δ 1.10 (s, 6 H), 2.25 (s, 3 H), 2.48 (s, 2 H), 2.62 (s, 2 H), 2.88 (m, 4 H), 6.86 (s, 1 H), 7.00 (q,J = 18.2 Hz, 2 H), 7.15 (d,J = 8.4 Hz, 2 H), 7.53 (d, J = 8.4 Hz, 2 H); 13C NMR (CDCl3100 MHz) delta 206.4, 171.3, 169.3, 153.6, 151.7, 135.9, 133.4, 129.3, 128.6, 123.7, 122.2, 113.5, 112.7, 78.9, 43.0, 39.2, 37.9, 32.1, 29.9,29.7, 28.2, 28.0. 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 C24H24N2O3：388.1787, found 387.1709 [M-H+]-. The high resolution mass spectrum is shown in figure 3.
Example 2
the preparation method of the near-infrared fluorescent probe for detecting selenite roots in the embodiment comprises the following steps:
levulinic acid (116.0 mg, 1.0 mmol) was dissolved in 10 mL of anhydrous dichloromethane, and EDC (192 mg, 1 mmol) and DMAP (61 mg, 0.5 mmol) were added and stirred at room temperature for 20 minutes. Dissolving the intermediate 1 (290 mg, 1 mmol) in 15mL of anhydrous dichloromethane solution, dropwise adding the mixture into the mixed solution at room temperature, reacting for 10 hours after dropwise adding, washing the reaction solution for 3 times by using 10 mL of saturated sodium chloride solution, drying an organic phase by using anhydrous sodium sulfate, filtering, removing the solvent under reduced pressure, using ethyl acetate and petroleum ether as detergents (the volume ratio is 1: 6), and separating by silica gel column chromatography to obtain 213.4 mg of a product SeP1, namely a fluorescent probe, wherein the yield is 55%.
Nuclear magnetic resonance measurement of fluorescent probe:1H NMR (CDCl3, 400 MHz) δ 1.10 (s, 6 H), 2.25 (s, 3 H), 2.48 (s, 2 H), 2.62 (s, 2 H), 2.88 (m, 4 H), 6.86 (s, 1 H), 7.00(q,J = 18.2 Hz, 2 H), 7.15 (d, J = 8.4 Hz, 2 H), 7.53 (d, J = 8.4 Hz, 2 H); 13C NMR (CDCl3100 MHz) delta 206.4, 171.3, 169.3, 153.6, 151.7, 135.9, 133.4, 129.3, 128.6, 123.7, 122.2, 113.5, 112.7, 78.9, 43.0, 39.237.9, 32.1, 29.9,29.7, 28.2, 28.0; 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 C24H24N2O3：388.1787, found 387.1709 [M-H+]-(ii) a The high resolution mass spectrum is shown in figure 3.
Example 3
The preparation method of the near-infrared fluorescent probe for detecting selenite roots in the embodiment comprises the following steps:
levulinic acid (116.0 mg, 1.0 mmol) was dissolved in 10 mL of anhydrous dichloromethane, and EDC (384 mg, 2 mmol) and DMAP (122 mg, 1 mmol) were added and stirred at room temperature for 20 minutes. Dissolving the intermediate 1 (435 mg, 1.5 mmol) in 15mL of anhydrous dichloromethane solution, dropwise adding the mixture into the mixed solution at room temperature, reacting for 15 hours after dropwise adding, washing the reaction solution for 3 times by using 10 mL of saturated sodium chloride solution, drying an organic phase by using anhydrous sodium sulfate, filtering, removing the solvent under reduced pressure, using ethyl acetate and petroleum ether as detergents (the volume ratio is 1: 10), and separating by silica gel column chromatography to obtain 252.2 mg of product SeP1, namely a fluorescent probe, wherein the yield is 65%.
nuclear magnetic resonance measurement:1H NMR (CDCl3, 400 MHz) δ 1.10 (s, 6 H), 2.25 (s, 3 H), 2.48 (s, 2 H), 2.62 (s, 2 H), 2.88 (m, 4 H), 6.86 (s, 1 H), 7.00 (q,J = 18.2 Hz, 2 H), 7.15 (d,J = 8.4 Hz, 2 H), 7.53 (d, J = 8.4 Hz, 2 H); 13C NMR (CDCl3100 MHz) delta 206.4, 171.3, 169.3, 153.6, 151.7, 135.9, 133.4, 129.3, 128.6, 123.7, 122.2, 113.5, 112.7, 78.9, 43.0, 39.237.9, 32.1, 29.9,29.7, 28.2, 28.0. 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 C24H24N2O3：388.1787, found 387.1709 [M-H+]-. High resolution qualityThe spectrum is shown in FIG. 3.
Example 4
The preparation method of the near-infrared fluorescent probe for detecting selenite roots in the embodiment comprises the following steps:
levulinic acid (116.0 mg, 1.0 mmol) was dissolved in 10 mL of anhydrous dichloromethane, and EDC (768 mg, 4 mmol) and DMAP (244 mg, 2 mmol) were added and stirred at room temperature for 60 minutes. Dissolving the intermediate 1 (580 mg, 2 mmol) in 15mL of anhydrous dichloromethane solution, dropwise adding the mixture into the mixed solution at room temperature, reacting for 24 hours after dropwise adding, washing the reaction solution for 3 times by using 10 mL of saturated sodium chloride solution, drying an organic phase by using anhydrous sodium sulfate, filtering, removing the solvent under reduced pressure, using ethyl acetate and petroleum ether as detergents (the volume ratio is 1: 12), and separating by silica gel column chromatography to obtain 310 mg of product SeP1, namely a fluorescent probe, wherein the yield is 80%.
Nuclear magnetic resonance measurement:1H NMR (CDCl3, 400 MHz) δ 1.10 (s, 6 H), 2.25 (s, 3 H), 2.48 (s, 2 H), 2.62 (s, 2 H), 2.88 (m, 4 H), 6.86 (s, 1 H), 7.00 (q,J = 18.2 Hz, 2 H), 7.15 (d,J = 8.4 Hz, 2 H), 7.53 (d, J = 8.4 Hz, 2 H); 13C NMR (CDCl3100 MHz) delta 206.4, 171.3, 169.3, 153.6, 151.7, 135.9, 133.4, 129.3, 128.6, 123.7, 122.2, 113.5, 112.7, 78.9, 43.0, 39.237.9, 32.1, 29.9,29.7, 28.2, 28.0. 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 C24H24N2O3：388.1787, found 387.1709 [M-H+]-. The high resolution mass spectrum is shown in figure 3.
Application Effect examples
1 mM probe solution preparation: the corresponding probe (SeP1) was weighed accurately, and SeP1 was dissolved in ethanol to prepare a 1 mM solution for use.
selective experiments:
Specific selectivity is an important criterion for determining whether a fluorescent probe molecule is efficient. Fluorescence spectroscopy examined the specific selectivity of probe SeP1 for selenite. As shown in the attached figure 4 of the drawings,Probe SeP1 alone (16.7. mu.M) in CH with excitation at 550 nm3CH2OH-H2The O (1:1, v/v) solution has weak fluorescence emission intensity at 657nm, when selenite (10 eq.) is added, the fluorescence emission intensity at 657nm is obviously enhanced, but when other substances (common metal ions and anions are 10 equivalents) are added, the fluorescence emission intensity (F657) intensity of the solution system is not obviously changed compared with the fluorescence emission (F657) intensity of a single probe system. The experimental results show that the probe has good specific selectivity on selenious acid root and can realize ratio detection.
Fluorescence interference experiment:
In order to test the anti-interference capability of the probe molecules on the detection of selenite, the anti-interference capability of the probe molecules on metal cations and common anions is tested in a fluorescence emission spectrum. As shown in FIG. 5, at SeP1(16.7 μ M) in CH3CH2OH-H2in the O (1:1, v/v) solution, the tested various metal cations and common anions (10 equivalents) are added respectively to test the fluorescence emission intensity (657 nm), then 10 equivalents of selenite solution is added to the solution, as can be seen from the attached figure 5, the fluorescence intensity (657 nm) obtained by adding selenite in the presence of the metal cations and common anions is basically the same as that obtained by adding selenite alone, and the result shows that the probe SeP1 has stronger anti-metal cation and common anion interference capability on the detection of selenite.
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 probe SeP1 for selenite was determined by fluorescence spectroscopy at CH3CH2OH-H2In O (1:1, v/v) solution, the concentration of the immobilized probe SeP1 is 16.7 mu M, the response intensity of the immobilized probe to selenite with different concentrations is measured, the system fluorescence intensity is continuously enhanced along with the increase of the selenite concentration (figure 6), and the research shows that the solution fluorescence intensity value is linear (R is between 0 and 2.5 equivalent of the selenite concentration)2= 0.985), the probe was calculated (3 σ/k) according to the IUPAC rulesthe detection limit of the molecule for bisulfite is 0.068. mu.M. The test result shows that the probe SeP1 has practical value in the detection of selenious acid roots in the environment.
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 (6)
1. A near-infrared fluorescent probe molecule for detecting selenite roots is characterized in that the structural formula of the fluorescent probe is as follows:
。
2. The method for preparing the near-infrared fluorescent probe molecule for detecting selenite root as claimed in claim 1, which comprises the following steps:
(1) Dissolving levulinic acid, EDC and DMAP in an anhydrous dichloromethane solution, and stirring and reacting at room temperature for a period of time to obtain a solution I;
(2) Dissolving the intermediate 1 in an anhydrous dichloromethane solution to obtain an intermediate 1 solution, then dropwise adding the intermediate 1 solution into the solution I obtained in the step (1) at room temperature, and reacting for a period of time after dropwise adding to obtain a solution II;
(3) and (3) washing the solution II obtained in the step (2) with a saturated sodium chloride solution, drying the obtained organic phase with anhydrous sodium sulfate, filtering, removing the solvent under reduced pressure, and separating by silica gel column chromatography to obtain a product SeP1, namely the fluorescent probe molecule.
3. The method of claim 2, wherein: in the step (1), the molar ratio of the levulinic acid to the EDC to the DMAP is 1 (0.5-4) to 0.1-2, and the reaction is carried out for 10-60 minutes under stirring at room temperature.
4. the method of claim 2, wherein: in the step (2), the intermediate 1 is 2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene ] malononitrile, the molar ratio of the levulinic acid in the solution I to the intermediate 1 is 1 (0.5-2), and the reaction time is 8-24 hours after the dropwise addition is finished.
5. The method of claim 2, wherein: the silica gel column chromatography separation in the step (3) adopts ethyl acetate and petroleum ether eluent with the volume ratio of 1 (3-12), and the yield is 40-80%.
6. The near-infrared fluorescent probe molecule of claim 1, applied to the field of high-sensitivity and specific detection of selenious acid root.
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