A dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide, and preparation method and use thereof

Abstract

The present invention belongs to the field of fluorescent probe detection technology, and specifically relates to a dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide, and its preparation method and use. The fluorescent probe has a structure as shown in formula (I), and detects changes in the concentrations of fluoride ions and sulfur dioxide through a fluorescence spectrometer. It has the characteristics of high sensitivity and good selectivity, and realizes the response to dual analytes. At the same time, it also realizes the fluorescence imaging of exogenous fluoride ions and endogenous SO2 in medicinal plants. The probe has simple synthesis steps, easy-to-obtain raw materials, and high yield, laying a foundation for subsequent plant research. #imgabs0# Formula (I)

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probe detection, and in particular relates to a dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide, and a preparation method and application thereof. Background Art

[0002] When plants absorb nutrients and water, fluorine enters the body through the roots or enters the plant through the stomata of the leaves. It has a certain impact on the photosynthesis, respiration and enzyme activity of the plant, causing the plant to eventually produce an oxidative stress state. Similarly, when plants are stressed by a certain concentration of sulfur dioxide, it not only causes certain harm to the leaves of the plant, but also affects the permeability of the plant's cell membrane, photosynthesis, chlorophyll and other aspects, which may cause oxidative damage. Therefore, medicinal plants are no exception. It is imperative to detect the accumulation of fluoride ions and sulfur dioxide in medicinal plants. The Chinese Pharmacopoeia limits the sulfur dioxide residue in Chinese medicinal materials to 11 traditional Chinese medicinal materials and their decoction pieces that are fumigated with sulfur. The sulfur dioxide residue shall not exceed 400 mg / kg; the sulfur dioxide residue in other Chinese medicinal materials and their decoction pieces shall not exceed 150 mg / kg.

[0003] At present, the methods for detecting fluoride ions and sulfur dioxide in plants include electrode method, ion chromatography, etc. However, these methods have the disadvantages of complex operation, troublesome pretreatment, and expensive instruments. We choose to use fluorescent probe detection technology to detect the accumulation of fluoride ions and sulfur dioxide in medicinal plants. This method has the advantages of simple operation and high sensitivity. However, in the current literature, there are only single-point detection of fluoride ions or sulfur dioxide, or fluorescent probes that are not composed of these two. There is no fluorescent probe that can dual-detect fluoride ions and sulfur dioxide, such as Chemical Engineering Journal (Chemical Engineering Journal, 448 (2022) 137631, YP Zhang, Y. Qu , YQ Zhang , Y. Gao, L. Wang, Development of a fluorescent strategy for quantification of fluoride ions infods and toothpaste.), Sensors & Actuators: B.Chemical, 375 (2023) 132959, YL Cui, J. Ning, LZ Duan, YH Zhou, YM Zhou, Rational Therefore, it is necessary to develop a dual-site fluorescent probe with simple operation, low cost and high sensitivity. Summary of the invention

[0004] In view of the limitations of the prior art, the present invention provides a dual-site fluorescent probe that can be used to detect fluoride ions and sulfur dioxide, and a preparation method and use thereof, to visually detect the accumulation of fluoride and sulfur dioxide in traditional Chinese medicine plants. The probe is simple to synthesize, easy to obtain, has high sensitivity for detecting fluoride ions and sulfur dioxide, and can dually detect two substances, which is of great significance for the visual detection of fluoride ions and sulfur dioxide in traditional Chinese medicines.

[0005] In a first aspect of the present invention, a dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide is provided, which has a structure as shown in formula (I):

[0006] ;

[0007] Formula (I)

[0008] Wherein, X is a halogen.

[0009] According to a specific embodiment of the present invention, X is iodine, chlorine or bromine, preferably iodine.

[0010] In a second aspect of the present invention, a method for preparing the aforementioned dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide is provided: reacting a compound represented by formula (a) with a compound represented by formula (b) to obtain a compound represented by formula (I), selecting tert-butyldiphenylchlorosilane as the reaction site for fluoride ions, selecting a suitable acid-binding agent triethylamine, adding an excess of the compound represented by formula (b), and reacting all the raw materials to obtain a compound represented by formula (I) with a higher yield, the reaction formula is as follows:

[0011] ;

[0012] Formula (a) Formula (b) Formula (I)

[0013] Wherein, X in formula (a) and formula (I), X in formula (b) ’ are the same or different halogens; X ’ Preferably, X is chlorine, preferably iodine, which is easy to form salt and simple to synthesize.

[0014] According to a specific embodiment of the present invention, the molar ratio of the compound represented by formula (a) to the compound represented by formula (b) is 1:2-4 (preferably 1:3); preferably, the reaction conditions include: 1) reacting in the presence of a base and an organic solvent; 2) the reaction temperature is 20-30°C (preferably 25°C room temperature), and the reaction time is 15-60min (preferably 30min); the base is any one of triethylamine, potassium carbonate, and cesium carbonate, preferably triethylamine, and the organic solvent is any one of dichloromethane and anhydrous acetonitrile, preferably dichloromethane.

[0015] The third aspect of the present invention provides the use of the aforementioned dual-site fluorescent probe for visually detecting the accumulation of fluoride ions and sulfur dioxide in a traditional Chinese medicinal plant; preferably, the traditional Chinese medicinal plant is Atractylodes lancea.

[0016] A fourth aspect of the present invention provides a reagent for simultaneously detecting fluoride ions and sulfur dioxide, which contains the aforementioned dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide.

[0017] The beneficial effects of the present invention are:

[0018] 1. Most fluorescent probes are targeted at single fluoride ions or sulfur dioxide, and there is no dual-site fluorescent probe for simultaneously detecting fluoride ions and sulfur dioxide. The present invention uses naphthalene indole hemicyanine as a fluorophore, lengthens the carbon chain to enhance its water solubility, conjugates it, and uses the carbon-carbon double bond as the reaction site of sulfur dioxide. When sulfur dioxide attacks C=C, a nucleophilic addition reaction occurs. The detection limit is 9nM, the sensitivity is high, and there is a good linear relationship between the concentration of 0-10μM. Based on the ICT mechanism, tert-butyldiphenylchlorosilane is connected as the reaction site of fluoride ions to detect F - When the detection limit is 1.17 μM, there is a good linear relationship between the concentrations of 0-640 μM. The combination of the two has simple synthesis operation, is easy to obtain, and has a high yield. It can realize the rapid detection of fluoride ions and sulfur dioxide, and has good selectivity and anti-interference effects in metal ions, active oxygen, etc.

[0019] 2. The present invention realizes F in the root of Atractylodes lancea for the first time - Fluorescence imaging of SO2 and F - When exogenously cultivating Atractylodes lancea, it was found that the signal of the red channel gradually increased with the increase of different concentrations. When exogenously cultivating Atractylodes lancea with SO2, the data showed that the signal of the blue channel gradually increased with the increase of different SO2 concentrations. At the same time, when Cys was incubated in Atractylodes lancea, SO2 could be endogenously generated. However, when only the roots of Atractylodes lancea were incubated with the probe, there was no strong fluorescence signal. This dual-site fluorescent probe was used for the first time to detect F in the roots of Atractylodes lancea. - and SO2, which provides a basis and direction for future research. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the synthetic route of the fluorescent probe SYT-F;

[0021] Figure 2 This is the H NMR spectrum of the fluorescent probe SYT-F;

[0022] Figure 3 This is the carbon NMR spectrum of the fluorescent probe SYT-F;

[0023] Figure 4 The following are schematic diagrams of the results when different concentrations of fluoride ions (0-1000 μM) are added to the fluorescent probe (10 μM): a. UV spectra of different concentrations of fluoride ions (0-1000 μM) added to the fluorescent probe (10 μM); b. Fluorescence spectra of different concentrations of fluoride ions (0-1000 μM) added to the fluorescent probe (10 μM); c. Linear relationship between fluorescence intensity and fluoride ion concentration when different concentrations of fluoride ions are added to the fluorescent probe (10 μM);

[0024] Figure 5The diagram is a schematic diagram of the results when the fluorescent probe (10 μM) is added with different concentrations of sodium bisulfite (0-20 μM), a. UV spectrum of the fluorescent probe (10 μM) when it is added with different concentrations of sodium bisulfite (0-20 μM); b. Fluorescence spectrum of the fluorescent probe (10 μM) when it is added with different concentrations of sodium bisulfite (0-20 μM); c. Linear relationship between fluorescence intensity and concentration of the fluorescent probe (10 μM) when different concentrations of sodium bisulfite are added;

[0025] Figure 6 The fluorescent probe (10 μM) is added with different ions and small molecules: from left to right: probe, Co 2+ , Cu 2+ 、Ag 2+ , Fe 2+ , Fe 3+ , K + , Li + , Hg 2+ NH4 + 、Zn 2+ , Cl - Br - ,I - 、HCO3 - 、NO2 - 、NO3 - 、S2O3 2- 、SO4 2- 、CH3COO - 、CO3 2- , Cys, GSH, Hcy, H2O2, •OH, F - , about F - Selectivity and anti-interference fluorescence spectrum change diagram;

[0026] Figure 7 The fluorescent probe (10 μM) is added with different ions and small molecules: from left to right: probe, Ag 2+ , Fe 2+ , Fe 3+ , K + , Li + Mg 2+ 、Na + 、Zn 2+ , Cl - Br - ,I - 、CH3COO - 、CO3 2- 、HCO3 - , HS - 、NO3 - 、S2O3 2- 、SO42- , ClO - 、H2O2、•OH、ONOO - 、Cys、GSH、Hcy、HSO3 - , about the selectivity and anti-interference fluorescence spectrum change of SO2;

[0027] Figure 8 The roots of Atractylodes lancea were treated with different concentrations of fluoride ions (0, 200, 300, 500 μM) for 30 minutes, and the fluorescence imaging was observed; the concentration of the fluorescent probe was 10 μM;

[0028] Fig. 9 The roots of Atractylodes lancea were incubated with different concentrations of sodium bisulfite (0, 40, 80, 100 μM) for 30 minutes to observe their fluorescence imaging; the concentration of the fluorescent probe was 10 μM;

[0029] Fig.10 The roots of Atractylodes lancea were incubated with different concentrations of cysteine ​​(0, 2, 5, 10 mM) for 24 h, and their fluorescence imaging was observed; the concentration of the fluorescent probe was 10 μM. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Comparative Example 1 Preparation of probe SYT-1

[0032]

[0033] The intermediate SYT-OH (0.05 g, 0.15 mmol, i.e., the compound of formula (a)) was dissolved in dichloromethane, triethylamine (1.5 g, 15 mmol) was added, and stirred for 30 min. Then, b, i.e., tert-butyldimethylsilyl chloride (0.05 g, 0.30 mmol) was added at room temperature and reacted for 30 min. After the reaction, the compound SYT-1 was evaporated under reduced pressure and subjected to silica gel column chromatography. It was found that the compound SYT-1 had poor stability, and the silicon-oxygen bond was easily broken and reduced to a phenol product, which was not conducive to the experiment. It was decided not to use SYT-1.

[0034] Comparative Example 2 Preparation of probe SYT-2

[0035]

[0036] The silicon-oxygen bond in silica gel is easily broken, making it unstable and releasing a quinone structure. In order to improve the stability of the probe, we hope to introduce a transition system between the naphthylindole fluorophore and the reaction site, while ensuring that the probe responds well to fluoride ions, so we introduced a p-hydroxybenzyl group.

[0037] The intermediate SYT-OH (0.01 g, 0.03 mmol, i.e., compound of formula (a)) was dissolved in DMF, K2CO3 (0.008 g, 0.06 mmol) was added, and stirred for 30 min. Then, b (0.03 g, 0.06 mmol) was added thereto at room temperature for 1 h. After the reaction, the mixture was evaporated under reduced pressure and chromatographed on a silica gel column (CH2Cl2:MeOH=10:1) to obtain a powder. However, it was found after testing that the mixture did not respond to fluoride ions, and SYT-2 was invalid, so it was not used.

[0038] It can be seen from Comparative Examples 1 and 2 that it is technically difficult to prepare a dual-site fluorescent probe for simultaneously detecting fluoride ions and sulfur dioxide while ensuring the stability of the structure.

[0039] Example 1: Synthesis of fluorescent probe SYT-F

[0040] Synthesis route diagram Figure 1 As shown, the synthesis of fluorescent probe molecules is carried out according to the synthesis route:

[0041] Preparation of probe SYT-F:

[0042] The intermediate SYT-OH (0.05 g, 0.15 mmol, i.e., compound of formula (a), prepared according to the reference A novel colorimetric and fluorescent probe for simultaneous detection of SO3 2- / HSO3 - and HSO4 - by different emission channels and its bioimaging in living cells》) was dissolved in dichloromethane, triethylamine (1.5 g, 15 mmol) was added, and after stirring for 30 min, tert-butyldiphenylsilyl chloride (0.12 g, 0.45 mmol, i.e., compound of formula (b)) was added thereto at room temperature for 30 min. After the reaction, the mixture was evaporated under reduced pressure and subjected to silica gel column chromatography (CH2Cl2:MeOH=10:1) to obtain an orange-yellow solid (0.02 g, yield 23%). The hydrogen and carbon NMR spectra of SYT-F are shown as follows: Figure 2~Figure 3 shown.

[0043] 1 H NMR (600 MHz, CD3CN) δ 8.36 (d, J = 8.5 Hz, 1H), 8.29 (d, J = 16.3Hz, 1H), 8.20 (d, J = 9.0 Hz, 1H), 8.14 (d, J = 8.3 Hz, 1H), 7.87 (dd, J =13.6, 8.3 Hz, 3H), 7.77 (d, J = 7.9 Hz, 5H), 7.70 (t, J = 7.5 Hz, 1H), 7.52(t, J = 7.4 Hz, 2H), 7.45 (t, J = 7.6 Hz, 4H), 7.27 (d, J = 16.3 Hz, 1H),6.95 (d, J = 7.4 Hz, 2H), 4.62 (q, J = 7.2 Hz, 2H), 1.99 (s, 6H), 1.55 (t, J= 7.4 Hz, 3H), 1.11 (s, 9H). 13 C NMR (151 MHz, CD3CN) δ 182.81, 161.30, 153.37,139.37, 138.72, 135.93, 134.20, 133.06, 132.28, 131.88, 131.07, 130.65,128.97, 128.66, 128.35, 127.95, 127.75, 123.76, 121.39, 113.12, 110.11,54.61, 43.31, 26.25, 25.90, 19.49, 13.72.

[0044] The present invention is compared with a document reported before (Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 211 (2019) 125–131, X. Wang, YM Zhou, CG Xu, HH Song, XB Pang, XQ Liu, A dual-responsive fluorescent probe for detection of fluoride ion and hydrazine based on test strips): both are dual-responsive fluorescent probes, and the document reports a dual-responsive probe for fluoride ion and hydrazine hydrate, but the emission wavelength of fluoride ion is short and the emission wavelengths of the two designated analytes are almost the same, which is easy to cause interference and difficult to distinguish. In contrast, the emission wavelengths of the fluorescent probe fluoride ion and sulfur dioxide of the present invention are very easy to distinguish, the Stocks shift is large, and it is not easy to cause crosstalk. In addition, in the present invention, the naphthylindole fluorophore is conjugated with the benzene ring, and sulfur dioxide attacks C=C, the reaction is fast, and it can react with SO2 quickly, the sensitivity is very high, and the detection limit is only 9nM.

[0045] In summary, the present invention introduces a benzene ring between the naphthylindole fluorophore and the reaction site to increase its conjugation. Naphthylindole itself carries blue light, and by introducing the benzene ring, it is red-shifted from blue light to orange-red light. The test shows that it has good stability, and SYT-F can be used as a fluorescent probe in this experiment.

[0046] Example 2: Spectral response of fluorescent probe to fluoride ion / sulfur dioxide

[0047] The detection method steps are as follows:

[0048] 1. Probe SYT-F to F - / Changes in the UV / fluorescence spectrum of SO2

[0049] The fluorescent probe prepared in Example 1 was used to prepare a probe mother solution with a concentration of 1 mM using CH3CN; NaF was dissolved in deionized water to prepare a fluoride ion stock solution with a concentration of 50 mM; NaHSO3 was dissolved in deionized water to prepare a HSO3 with a concentration of 3 mM; - Stock solution; prepare spectral solution in CH3CN / PBS (pH = 7.4, v / v = 6:4).

[0050] Accurately pipette 3 mL of spectral solution into the cuvette, take 30 μL of probe stock solution into the cuvette, make the probe detection concentration 10 μM, and draw 0, 2, 5, 10, 20, 30, 40, 50, 60 μL of fluoride ion stock solution in turn, react for 20 min, and obtain concentration spectra from UV spectrum and fluorescence spectrum respectively. Same as the above operation, draw 0, 2, 4, 6, 8, 10, 14, 16, 18, 20 μL of sodium bisulfite stock solution in turn, react for 5 min, and obtain concentration spectra from UV spectrum and fluorescence spectrum respectively.

[0051] from Figure 4 It can be seen that in the UV spectrum, as the fluoride ion concentration increases, the original UV absorption peak of 450nm red-shifts to 550nm, and the absorption value gradually increases; at the same time, it can be seen from the fluorescence spectrum that as the fluoride ion concentration increases, under the excitation of 520nm, the fluorescence at 575nm gradually increases, showing concentration dependence. Through data fitting analysis, the fluorescence intensity and fluoride ion concentration show a good linear relationship in the range of 0-640 μM, and the fitting constant R 2 = 0.95, k = 0.12127. The data show that the probe can effectively detect fluoride ions. The calculated detection limit of fluoride ion is 1.17 μM. is the standard deviation of ten blank solutions, and k is the slope of the linear equation.

[0052] from Figure 5 It can be seen that in the UV spectrum, as the concentration of bisulfite ions increases, the original UV absorption peak of 450nm blue shifts to 320nm, and the absorption value gradually increases; at the same time, it can be seen from the fluorescence spectrum that as the concentration of bisulfite ions increases, under the excitation at 330nm, the fluorescence at 475nm gradually increases, showing concentration dependence. Through data fitting analysis, the fluorescence intensity and the concentration of bisulfite ions show a good linear relationship in the range of 0-10μM, and the fitting constant R 2 = 0.9973, k = 16.65999. The data show that the probe can detect bisulfite ions very well. In summary, the fluorescent probe can sensitively detect fluoride ions and bisulfite ions. The detection limit of sulfur dioxide was calculated to be 9 nM. is the standard deviation of ten blank solutions, and k is the slope of the linear equation. Compared with the fluorescent probe for detecting sulfur dioxide in the prior art, the dual-site fluorescent probe with the chemical structure of the present invention has significantly improved detection sensitivity for sulfur dioxide.

[0053] 2. Selectivity and anti-interference experiments

[0054] Accurately pipette 3 mL of the spectral solution into the cuvette, take 30 μL of the probe stock solution into the cuvette, and then add other cations and anions and other small molecules (Co 2+ , Cu 2+ 、Ag 2+ , Fe 2+ , Fe 3+ , K + , Li + , Hg 2+ NH4 + 、Zn 2+ , Cl - Br - ,I - 、HCO3 - 、NO2 - 、NO3 - 、S2O3 2- 、SO4 2- 、CH3COO - 、CO3 2- , Cys, GSH, Hcy, H2O2, •OH, F - ) were measured, and the selectivity data were recorded by the fluorescence spectrometer. At the same time, 3 mL of the spectral solution was accurately transferred to the cuvette, and 30 μL of the probe mother solution was added to the cuvette, and other cations and anions and other small molecules (Co 2+ , Cu 2+ 、Ag 2 + , Fe 2+ , Fe 3+ , K + , Li + , Hg 2+ NH4 + 、Zn 2+ , Cl - Br - ,I - 、HCO3 - 、NO2 - 、NO3 - 、S2O3 2- 、SO4 2- 、CH3COO - 、CO3 2- , Cys, GSH, Hcy, H2O2, •OH, F - ) and then add 60 μL of each F - (60 μL) for determination, F -The concentration in the system is 1000 μM, the concentration of other cations and anions and other small molecules in the system is 1000 μM, the concentration of the probe in the system is 10 μM, the probe is excited at a wavelength of 520 nm, and its anti-interference data is recorded by a fluorescence spectrometer.

[0055] like Figure 6 It shows that after adding ions or small molecules that may interfere, there is no obvious change in fluorescence. However, after adding fluoride ions, the fluorescence intensity increases significantly, and the anti-interference property is the same, indicating that the probe has good selectivity and anti-interference ability.

[0056] Accurately pipette 3 mL of the spectral solution into the cuvette, take 30 μL of the probe stock solution into the cuvette, and then add other cations and anions and other small molecules (Ag 2+ , Fe 2+ , Fe 3+ , K + , Li + Mg 2+ 、Na + 、Zn 2+ , Cl - Br - ,I - 、CH3COO - 、CO3 2- 、HCO3 - , HS - 、NO3 - 、S2O3 2- 、SO4 2- , ClO - 、H2O2、•OH、ONOO - 、Cys、GSH、Hcy、HSO3 - ) were measured, and the selectivity data were recorded by the fluorescence spectrometer. At the same time, 3 mL of the spectral solution was accurately transferred to the cuvette, and 30 μL of the probe mother solution was added to the cuvette, and other cations and anions and other small molecules (Ag 2+ , Fe 2+ , Fe 3+ , K + , Li + Mg 2+ 、Na + 、Zn 2+ , Cl - Br - ,I - 、CH3COO - 、CO3 2- 、HCO3 - , HS - 、NO3- 、S2O3 2- 、SO4 2- , ClO - 、H2O2、•OH、ONOO - 、Cys、GSH、Hcy、HSO3 - ) 20 μL each, then continue to add HSO3 - (20 μL) for determination, HSO3 - The concentration in the system is 20 μM, the concentration of the probe in the system is 10 μM, the concentration of other cations and anions and other small molecules in the system is 20 μM, and a wavelength of 330 nm is selected for excitation, and the anti-interference data is recorded by a fluorescence spectrometer.

[0057] like Figure 7 It shows that after adding ions or small molecules that may interfere, there is no obvious change in fluorescence. However, after adding bisulfite ions, the fluorescence intensity increases significantly, and the anti-interference property is the same, which does not affect the probe's response to bisulfite ions. In summary, the probe has a certain selectivity and anti-interference ability for fluoride ions / bisulfite ions.

[0058] Example 3: Fluorescence probes for the detection of fluoride ions and sulfur dioxide in the roots of Atractylodes lancea

[0059] 1. Exogenous F - Confocal fluorescence imaging of roots of Atractylodes lancea seedlings

[0060] The fluorescent probe prepared in Example 1 was used for detection, the detection object was Atractylodes lancea seedlings, and the incubation conditions were all carried out at room temperature.

[0061] The detection imaging method is as follows: Take an appropriate amount of Atractylodes lancea seeds and sow and culture them under sterile, suitable temperature, humidity and light conditions. When the seeds develop into seedlings, transplant them into solutions containing different fluoride ion concentrations (0, 200, 300, 500 μM) for incubation, and incubate them for 30 minutes at each concentration. After that, wash them three times with deionized water and transfer them to a 10 μM probe solution for further incubation. The treatment time is 30 minutes, and then wash them three times with deionized water. Then put the roots on a slide and perform fluorescence imaging using a laser confocal microscope.

[0062] from Figure 8 It can be seen that with the increase of fluoride ion concentration, the red channel fluorescence signal distributed in the root seedlings of Atractylodes lancea gradually increases.

[0063] 2. Exogenous HSO3 - Confocal fluorescence imaging of roots of Atractylodes lancea seedlings

[0064] The seedlings of Atractylodes lancea were transplanted into solutions containing different bisulfite ion concentrations (0, 40, 80, 100 μM) for incubation, and each concentration was incubated for 30 minutes. After that, they were washed three times with deionized water and transferred to 10 μM probe solution for further incubation for 30 minutes. After that, they were washed three times with deionized water, and then the roots were placed on a slide for fluorescence imaging using a laser confocal microscope.

[0065] from Fig. 9 It can be seen that with the increase of bisulfite ion concentration, the blue channel fluorescence signal distributed in the root seedlings of Atractylodes lancea gradually increases.

[0066] 3. Endogenous HSO3 - Confocal fluorescence imaging of roots of Atractylodes lancea seedlings

[0067] The seedlings of Atractylodes lancea were transplanted into solutions containing different cysteine ​​concentrations (0, 2, 5, 10 mM) for incubation, and each concentration was incubated for 24 hours. After that, they were washed three times with deionized water and transferred to 10 μM probe solution for further incubation for 30 minutes. After that, they were washed three times with deionized water, and then the roots were placed on a slide for fluorescence imaging using a laser confocal microscope.

[0068] from Fig.10 It can be seen that with the increase of cysteine ​​concentration, the blue channel fluorescence signal distributed in the root seedlings of Atractylodes lancea gradually increases.

[0069] In summary, the fluorescent probe provided by the present invention has high selectivity and sensitivity to fluoride ions and sulfur dioxide, can visualize the detection of fluoride ions and sulfur dioxide in the roots of medicinal plants, and can intuitively see the fluorescent signals distributed in the roots of medicinal plants, laying the foundation for subsequent research and providing thinking and direction.

[0070] The dual-site fluorescent probe of the present invention has a structure as shown in formula (I), and detects changes in the concentrations of fluoride ions and sulfur dioxide by a fluorescence spectrometer. It has the characteristics of high sensitivity and good selectivity, and realizes the response to dual analytes. At the same time, it also realizes the fluorescence imaging of exogenous fluoride ions and endogenous and exogenous SO2 in medicinal plants. The synthesis steps of the probe are simple, the raw materials are easy to obtain, and the yield is high, which lays a foundation for subsequent plant research.

[0071] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide, characterized in that: It has a structure as shown in formula (I): Wherein, X is iodine.

2. The method for preparing a dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide according to claim 1, characterized in that: The preparation method comprises: reacting the compound represented by formula (a) with the compound represented by formula (b) to obtain the compound represented by formula (I), and the reaction formula is as follows: Wherein, X is iodine, X ’ For chlorine.

3. The method for preparing a dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide according to claim 2, characterized in that: The molar ratio of the compound represented by formula (a) to the compound represented by formula (b) is 1:2-4.

4. The method for preparing a dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide according to claim 2, characterized in that: The reaction conditions include: reacting in the presence of a base and an organic solvent; the reaction temperature is 20-30° C., and the reaction time is 15-60 min.

5. The method for preparing a dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide according to claim 4, characterized in that: The base is triethylamine, the organic solvent is dichloromethane; the reaction temperature is 25° C., the reaction time is 30 min; the molar ratio of the compound represented by formula (a) to the compound represented by formula (b) is 1:

3.

6. Use of the dual-site fluorescent probe according to claim 1 for visually detecting the accumulation of fluoride ions and sulfur dioxide in traditional Chinese medicinal plants.

7. Use of the dual-site fluorescent probe according to claim 6 for visually detecting the accumulation of fluoride ions and sulfur dioxide in traditional Chinese medicinal plants, characterized in that: The Chinese medicinal plant is Atractylodes lancea.

8. A reagent for simultaneously detecting fluoride ions and sulfur dioxide, characterized in that: It contains the dual-site fluorescent probe for detecting fluoride ions and sulfur dioxide as described in claim 1.

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