CN113683604A

CN113683604A - Ratio type near-infrared fluorescent probe for detecting sulfur dioxide derivatives in crop mitochondria and preparation method and application thereof

Info

Publication number: CN113683604A
Application number: CN202110906243.1A
Authority: CN
Inventors: 龙凌亮; 李璐璐; 陈倩; 刘卫国; 陈晓东; 袁芳; 何丹
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2021-08-09
Filing date: 2021-08-09
Publication date: 2021-11-23
Anticipated expiration: 2041-08-09
Also published as: CN113683604B

Abstract

The invention provides a method for detecting SO in crop mitochondria₂The preparation method and the application of the derivative ratio type near-infrared fluorescent probe comprise the following steps: firstly, dissolving 4, 6-dimethyl pyrimidine and 1, 4-dibromomethylbenzene in acetonitrile, refluxing and heating the stirred mixture for reaction, and treating the mixture after cooling to room temperature to obtain a white solid, namely an intermediate product; then, adding 7- (diethylamino) coumarin aldehyde and the intermediate product into absolute ethyl alcohol, heating and stirring the mixture, refluxing for 5-6 hours, cooling the mixture to room temperature, and purifying to obtain a bluish purple solid for detecting SO in the mitochondria of the crops₂Derivative ratiometric near infrared fluorescent probe Q-1. The invention develops a novel high-performance ratio typeNear-infrared fluorescent probe and successful application thereof in detecting SO in crop mitochondria₂And (3) derivatives.

Description

Ratio type near-infrared fluorescent probe for detecting sulfur dioxide derivatives in crop mitochondria and preparation method and application thereof

Technical Field

The invention relates to the technical field of fluorescence detection, in particular to a ratiometric near-infrared fluorescent probe for detecting sulfur dioxide derivatives in crop mitochondria and a preparation method and application thereof.

Background

SO₂Is a well known atmospheric pollutant. With the development of industry and the combustion of coal and petroleum, SO₂Still remains a major problem of atmospheric pollution. The crop leaves absorb SO with higher concentration₂(above 12.2ppm) causes phytotoxicity, which can lead to photosynthesis inhibition, slow growth, change of crop morphology, decrease of yield, early abscission of leaves, and even death of crops.

Parsing SO₂The phytotoxicity mechanism to crops helps to prevent adverse effects on crops. There are SO references₂Research on nucleophilic attack of the derivative on crop protein functional groups. Formation of Reactive Oxygen Species (ROS) is also believed to be SO₂A factor in phytotoxicity. In addition, SO₂Interference of acidification with crop function may be another mechanism of phytotoxicity. Albeit SO₂The mechanism of action of (2) has been proposed, but its role in phytotoxicity is still unclear. Mitochondria are involved in a variety of important cellular processes in crops, including ATP production, apoptosis, stress, ROS production, and antimicrobial defenses. However, high concentrations of SO₂This leads to a loss of membrane potential in the mitochondria. Therefore, SO is presumed₂The interaction with the crop mitochondria may be SO₂Another route of phytotoxicity. Therefore, as a subject is subjected to SO₂Detecting SO in mitochondria during phytotoxicity₂Is very necessary.

Up to now, SO was detected₂The method of (2) is various, and there are various detection methods such as spectrophotometry, electrochemical analysis, chromatography, and chemiluminescence. These methods generally require treatment or disruption of sample tissue and are therefore not suitable for real-time detection in plant samples. The fluorescent probe has the advantages of specificity, high sensitivity, high resolution and the like, and particularly can be gathered in mitochondria in a non-invasive way by connecting a targeting group, so that the detection of the mitochondria is realized. Fluorescent probes have been recognized as highly effective molecular tools in numerous fields of biology, drug discovery, and clinical diagnostics. To date, few have been used to detect SO in mitochondria₂The amount of fluorescent probe is reported. Since the mitochondrial membrane potential is negative, it is known that all of the reported probes capable of accumulating to mitochondria have a positively charged group as a targeting group. Commercially available mitochondrial tracking agents include the mitogen series, rhodamine derivatives, carbocyanine derivatives, styryl derivatives, and arsine dyes, all of which are based on conventional cationic dyes with positively charged quaternary ammonium moieties. Due to high concentration of SO₂May result in a loss of mitochondrial membrane potential^[64]The reported probes diffuse out of the mitochondria and are therefore not suitable for tracking high concentrations of SO in crop mitochondria₂。

Therefore, a novel mitochondrial targeting and immobilized near-infrared ratiometric fluorescent probe Q-1 is reasonably constructed and used for detecting SO in crop mitochondria₂And (3) derivatives. The probe Q-1 itself showed strong fluorescence in the near infrared region. The probe pair SO₂The derivatives showed ratiometric fluorescent responses. Fluorescence imaging shows that the probe can not only target mitochondria, but also can be remained in mitochondria to continuously detect SO after mitochondrial membrane potential disappears₂And (3) derivatives. Importantly, for SO in mitochondria of crop tissues₂The derivatives were subjected to fluorescence imaging. Proves high concentration of SO₂Can cause the abnormity of the mitochondrial membrane potential of the crop tissue, which is in parallel with SO₂Is closely related to phytotoxicity of (A).

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for detecting SO in crop mitochondria₂A derivative ratio type near-infrared fluorescent probe and a preparation method and application thereof. The method has the advantages of good selectivity, high sensitivity, simple preparation method and good application to crop linesSO in the mitochondria₂And (4) detecting the derivative.

The invention is realized by the following technical scheme:

method for detecting SO in crop mitochondria₂The structure of the derivative ratio type near infrared fluorescent probe is as follows:

detection of SO in crop mitochondria₂The preparation method of the derivative ratio type near-infrared fluorescent probe comprises the following steps:

step 1, dissolving 4, 6-dimethyl pyrimidine and 1, 4-dibromomethylbenzene in acetonitrile, placing the mixture in a round-bottom flask, and refluxing and heating the stirred mixture for reaction; after cooling to room temperature, excess acetonitrile was removed from the mixture by suction filtration under reduced pressure; the intermediate product can be obtained by column chromatography oil ether and dichloro separation and purification.

Step 2, adding the intermediate product obtained in the step 1 and 7- (diethylamino) coumarin aldehyde into absolute ethyl alcohol, and heating, refluxing and stirring the mixture for reaction; after the mixture was cooled to room temperature, the excess solvent was removed; purifying by column chromatography to obtain SO in mitochondria of crops₂Derivative ratio type near infrared fluorescent probe.

In the step 1, the dosage ratio of the 4, 6-dimethylpyrimidine, the 1, 4-dibromomethylbenzene and the acetonitrile is 100-110 μ L: 178 to 180 μ L: 5mL to 6 mL.

In the step 1, the temperature of the reflux heating reaction is 80-85 ℃, and the time is 23-24 h.

In the step 2, the dosage ratio of the 7- (diethylamino) coumarin aldehyde, the intermediate product and the acetonitrile is 168 mg-170 mg: 100 mg-110 mg: 10mL to 11 mL.

In the step 2, the heating and stirring reaction is carried out at the temperature of 75-80 ℃ for 5-6 h, and the solvent removal method is rotary evaporation.

The ratio type near-infrared fluorescent probe prepared by the invention is used for detecting SO in crop mitochondria₂DerivatisationThe specific application method is as follows:

(1) the fluorescent probe Q-1 provided by the invention can be used for targeting and positioning mitochondria in crops, and the specific method comprises the following steps:

co-localization experiments: live colon cancer cells (SW480) were plated on 35 mm glass-bottomed plates and incubated with 1. mu.M probe Q-1 for 25 minutes. Staining was then performed for 25 min with 0.5 μ M rhodamine 123 (commercial mitochondrial localization reagent). Fluorescence images were obtained on a lycra TCS SP5 II laser confocal scanning microscope using a 40-fold objective lens. The near infrared channel of probe Q-1 involves emission in the 650-700nm spectral window under 543nm excitation. Under the excitation of 514nm, the green channel of rhodamine 123 is recorded on an emission window of 495-545 nm.

(2) The fluorescent probe Q-1 provided by the invention can be used for detecting SO by fixing in mitochondria after mitochondrial membrane potential disappears₂The specific method of the derivative is as follows:

mitochondrial fixation: colon cancer cells (SW480) were plated on 35 mm glass-bottom plates and incubated with 1. mu.M probe Q-1 for 25 minutes. Then further treated with CCCP (mitochondrial membrane potential eliminator) at a concentration of 10. mu.M for 30 minutes. Fluorescence imaging was performed in an uninterrupted fashion from 0 to 90 minutes in the near infrared channel (650-700 nm).

SO in mitochondria of living cells₂Fluorescence imaging of derivatives: live SW480 cells were plated on 35 mm glass-bottomed dishes, incubated with Probe Q-1 (1. mu.M) for 25 minutes, washed 3 times with PBS buffer (pH 7.4), and then plated with HSO at various concentrations^3-Further incubation was carried out for 25 minutes. Fluorescence images were collected using a 40-fold objective lens lycra TCS SP5 II laser confocal scanning microscope. The near infrared channel is recorded at 650-700nm, and the excitation wavelength is 543 nm. The green channel was recorded at 495-545nm with an excitation wavelength of 514 nm.

(3) The fluorescent probe Q-1 provided by the invention is used for HSO in living cells₃ ^-There is a rate and dose dependent response. The specific method comprises the following steps:

cell culture: colon cancer cells (SW480) were incubated in a 35 mm glass-bottomed culture dish in DMEM containing 10% fetal bovine serum in an incubator containing 5% carbon dioxide and thermostatted at 37 ℃.

Cell staining: in colon cancer cell (SW480) staining experiments, colon cancer cells were stained with probe Q-1 for 25 minutes in 24-well plates. Then, the mixture was washed 3 times with a pH 7.4 potassium phosphate buffer solution to remove extra probes outside the cells, and then HSO was added to the 24-well plates at concentrations of 0, 300 and 600. mu.M, respectively₃ ^-Incubated for 30 minutes.

(4) The fluorescent probe Q-1 provided by the invention can be used for detecting SO suffered by crops₂Tracking SO in plant tissue mitochondria after phytotoxicity₂The specific method of the derivative is as follows:

culturing orychophragmus violaceus: the orychophragmus violaceus is taken as a research object, seeds of the orychophragmus violaceus are placed on wet gauze for accelerating germination, the seeds are kept in a wet state, and the seeds are transferred into perlite for water culture after sprouting after 2-3 days. During the period, 25% of Hoagland nutrient solution is added from time to time, and after one month, the orychophragmus violaceus grows into seedlings which are transplanted into nutrient soil for testing.

Dyeing orychophragmus violaceus: plants were planted in nutrient soil and incubated with 5 μ M Q-1 daily for 30 minutes. The residual dye on the leaves was washed off with water. Then using HSO of different concentration every day₃ ^-(0, 400. mu.M, 800. mu.M) plant leaves were sprayed and the orychophragmus violaceus leaves were cut into 0.5-1 mm transparent thin tissue and subjected to fluorescence imaging on a confocal laser scanning microscope. The near infrared channel (650-700nm) and the green channel (495-545nm) are respectively arranged.

The invention has the following beneficial effects:

(1) the invention provides a brand new SO₂The fluorescent probe Q-1 has simple synthesis method and is used for SO analysis and detection₂The derivatives have good selectivity S₂O₃ ^2-、HS^-、HSO_4-、SO₄ ^2-、Na⁺、NO^2-、ACO^-Related interferents such as biomolecules (GSH, Cys and Hcy) have no influence on detection; the fluorescence emission peak (654nm) of the probe is in the near infrared region, which is helpful to eliminate the interference of background fluorescence, reduce the detection error and improve the analysis and detectionThe accuracy of the measurement; ratio of fluorescence intensity of Probe solution (I)₅₃₀/I₆₅₄) And HSO^3-The concentration is in a good linear relationship in the range of 0-400. mu.M, and extremely high sensitivity (lower detection limit of 0.512. mu.M) is exhibited.

(2) After the synthesis method is improved, the side reaction can be effectively prevented, impurities which are difficult to separate in the reaction process are greatly reduced, and a high-purity target product can be obtained.

(3) The invention develops a novel mitochondria targeting and immobilization ratio type near-infrared fluorescent probe. The first application is to detect SO in mitochondria of crop leaf tissue₂Derivatives, and more importantly, SO at high concentrations₂When the derivative causes the mitochondrial membrane potential to disappear, the probe can be kept in mitochondria to continuously detect SO₂And (3) derivatives. The results show that SO is present in the mitochondria of the crop₂With SO₂Is related to phytotoxicity of (a). The relation between the mitochondrial damage and the toxicity of the crops is proved, and data support and theoretical basis are provided for further preventing and controlling the crops.

Drawings

FIG. 1 shows the detection of SO in crop mitochondria in example 1 of the present invention₂Derivative (HSO)₃ ^-) Synthetic route maps of ratiometric near-infrared fluorescent probes of (1);

FIG. 2 is a diagram of the detection mechanism of the sulfur dioxide derivative detected by the ratiometric near-infrared fluorescent probe of the present invention;

FIG. 3a shows the fluorescent probe detecting SO of different concentrations in example 1 of the present invention₂Derivative (HSO)^3-) A fluorescence titration map of (a); FIG. 3b shows the fluorescent probe used in example 1 to detect SO at different concentrations₂Derivative (HSO)^3-) Spectral data of (a) to (b) to (d₅₃₀/I₆₅₄) With SO₂Derivative (HSO)^3-) A linear plot of concentration; abscissa is SO₂Derivative (HSO)^3-) The concentration of (c); the ordinate is the fluorescence intensity ratio;

FIG. 4a is a histogram of competition for fluorescent probes of example 1 of the present invention; the abscissa is the addition condition of different ions or molecules, and the ordinate is the ratio value of the fluorescence intensity; FIG. 4b is a kinetic study of the fluorescent probe in example 1 of the present invention; the abscissa is the reaction time and the ordinate is the fluorescence intensity;

FIG. 5 is a bar graph showing the selectivity of the fluorescent probe in example 1 of the present invention; the abscissa is the addition condition of different ions or molecules, and the ordinate is the ratio value of the fluorescence intensity;

FIG. 6 shows the pH value of the solution versus the fluorescent probe and SO in example 1 of the present invention₂Derivative (HSO)₃ ^-) Influence of the ratio of fluorescence intensity values before and after the action; the abscissa is pH, and the ordinate is the ratio value of fluorescence intensity;

FIG. 7 is a fluorescence imaging diagram of co-localization of the fluorescent probe and rhodamine 123 in colon cancer cells (SW480) in example 1 of the present invention.

FIG. 8 is a confocal fluorescence imaging diagram of colon cancer cells (SW480) and CCCP (10 μ M) stained with fluorescent probes at different time intervals in example 1 of the present invention.

FIG. 9 shows the staining of colon cancer cells (SW480) with fluorescent probe in example 1 of the present invention, and recording of different SO concentrations₂Derivative (HSO)^3-) Fluorescence imaging of (1).

FIG. 10 is a graph of fluorescent images of orychophragmus violaceus stained with fluorescent probe in example 1 of the present invention.

Detailed Description

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1:

and (3) synthesizing a fluorescent probe.

The preparation steps are summarized as follows:

step 1, dissolving 4, 6-dimethyl pyrimidine and 1, 4-dibromomethylbenzene in acetonitrile, placing the mixture in a round-bottom flask, and heating the stirred mixture for 24 hours under reflux; after cooling to room temperature, excess acetonitrile was removed from the mixture; and separating and purifying by column chromatography to obtain an intermediate product.

Step 2, adding 7- (diethylamino) coumarin aldehyde and the intermediate product obtained in the step 1 into absolute ethyl alcohol, heating, refluxing and stirring the mixtureStirring and reacting; after the mixture was cooled to room temperature, the excess solvent was removed; purifying by column chromatography to obtain SO in mitochondria of crops₂Derivative ratio type near infrared fluorescent probe.

In the step 1, the heating and stirring reaction is carried out at the temperature of 80-85 ℃ for 23-24 hours.

In the step 1, the method for removing the solvent is vacuum filtration.

The method comprises the following specific steps:

(1) 4, 6-dimethylpyrimidine (100. mu.L to 110. mu.L) and 1, 4-dibromomethylbenzene (178. mu.L to 180. mu.L) were dissolved in 5mL of acetonitrile and placed in a 25mL round-bottomed flask. And refluxing and heating the stirred mixture at 80-85 ℃ for 24 hours. After cooling to room temperature, the excess acetonitrile was removed from the mixture. Column chromatography on silica gel (petroleum ether: dichloromethane ═ 1:3, v/v) gave a white solid, i.e., intermediate (82mg, yield: 31.3%).

(2) 7- (diethylamino) coumarin aldehyde (168 mg-170 mg) and intermediate (100 mg-110 mg) were added to 10mL of absolute ethanol. The mixture is heated and stirred at 75-80 ℃ and refluxed for 5-6 hours, and after the mixture is cooled to room temperature, the excess solvent is removed. Purifying with silica gel (petroleum ether: dichloromethane ═ 1:3, v/v) by column chromatography to obtain bluish purple solid for detecting SO in crop mitochondria₂Ratiometric near-infrared fluorescent probe of the derivative 0.051g (55mg, yield: 21.70%),

example 2:

test SO obtained in example 1₂Derivative fluorescent probe Q-1 to HSO^3-The fluorescence spectral response of (a).

The spectroscopic properties were tested in 20mM potassium phosphate buffer/DMSO (4:1v/v,) at pH 7.4. By HSO₃ ^-As SO₂The derivative is represented by the fluorescence enhancement at 654nm after adding the fluorescent probe, and the fluorescence of the region belongs to near infrared fluorescence, as shown in FIG. 3 a. With HSO₃ ^-(0-600 mu M) concentration is increased continuously, the fluorescence intensity of the fluorescent probe at 654nm is slowly reduced, an absorption band appears at 530nm and is correspondingly gradually enhanced, and HSO is increased at 0-400 mu M₃ ^-The fluorescence probe obtained in example 1 showed a good linear response over the concentration range (FIG. 3b), thus for different concentrations of HSO^3-All had excellent fluorescence response.

Example 3:

detection of SO on the fluorescent Probe Q-1 obtained in example 1 by other common ions or molecules₂Interference experiments of derivatives.

Adding other possible interferents into 10 μ M fluorescent probe molecule test solution, including S₂O₃ ^2-、HS^-、HSO₄ ^-、SO₄ ^2-、Na⁺、NO₂ ^-、ACO^-And biomolecules (GSH, Cys, Hcy), and adding HSO to the solutions₃ ^-. After mixing for 2 minutes, fluorescence spectroscopy was performed under the same conditions with excitation at 493nm to obtain fluorescence spectra of the respective solutions, and the results are shown in FIG. 4 a.

As shown in FIG. 4a, under the experimental conditions, only HSO was present₃ ^-Can remarkably improve the fluorescence emission ratio (I)₅₃₀/I₆₅₄) And S is₂O₃ ^2-、HS^-、HSO₄ ^-、SO₄ ^2-、Na⁺、NO₂ ^-、ACO^-And biomolecules (GSH, Cys, Hcy) did not elicit a significant response. In addition, the fluorescent probe obtained in example 1 has a very obvious change in the color of fluorescence generated by different substances, and can be observed by naked eyes only when HSO is added₃ ^-When the fluorescence color of the test solution changes from rose redThe color of the solution becomes yellow-green, and the color of the solution added with other ions still keeps rose-red. Thus, the fluorescent probe obtained in example 1 was directed to HSO₃ ^-Is superior to other anions, and is suitable for HSO in complex samples₃ ^-Accurate detection of.

Example 4:

test SO obtained in example 1₂Kinetic study of derivative fluorescent Probe Q-1.

Fluorescence probe detection of HSO^3-The kinetic study of (1) takes 493nm as the excitation wavelength and detects the HSO of the fluorescent probe pair^3-Fluorescence spectrum over time. As shown in FIG. 4b, no HSO was added^3-The fluorescence intensity of the fluorescent probe test solution at 519nm hardly changed. Adding HSO^3-Thereafter, the fluorescence intensity of the probe solution at 519nm increased sharply and leveled off within 5s, indicating that the probe was directed to SO₂The derivative can quickly respond, SO that SO can be detected in real time₂And (3) derivatives.

Example 5:

fluorescent probes Q-1 vs SO obtained in example 1₂Selectivity of derivative fluorescence detection.

A10. mu.M fluorescent probe test solution was prepared with 20mM potassium phosphate buffer/DMF (4:1v/v, pH 7.4) and was ready for use. Adding other possible interferents into 10 μ M fluorescent probe molecule test solution, including S₂O₃ ^2-、HS^-、HSO₄ ^-、SO₄ ^2-、Na⁺、NO₂ ^-、ACO^-And biomolecules (GSH, Cys, Hcy), and adding HSO to the solutions^3-. After mixing for 2 minutes, fluorescence spectroscopy was performed under the same conditions with excitation at 493nm to obtain fluorescence spectra of the respective solutions, and the results are shown in FIG. 5.

From the results of FIG. 5, it can be found that when S is added to the system₂O₃ ^2-、HS^-、HSO₄ ^-、SO₄ ^2-、Na⁺、NO₂ ^-、ACO^-And biomolecules (GSH, Cys, Hcy), etcAfter interferent, fluorescence intensity of each group was compared with HSO alone^3-There was no significant difference in fluorescence intensity of the blank solution. The results show that: the fluorescent probe pair of the invention is HSO^3-Has high selectivity and is not interfered by other coexisting ions or molecules, which makes them suitable for SO in complex samples₂Accurate detection of the derivative.

Example 6:

pH detection of SO on fluorescent Probe Q-1 obtained in example 1₂Influence of the derivative.

For detecting SO under different pH conditions of probe molecules₂The responses of the derivatives were measured by preparing phosphate buffers at different pH values (2.0-10.4). As shown in FIG. 6, the fluorescence intensity ratio of the fluorescent probe was almost constant between pH 2.0 and 10.4, indicating that the probe was stable over a wide pH range. With HSO₃ ^-When the pH value is more than 4.0, the fluorescence intensity ratio (I) of the fluorescent probe₅₃₀/I₆₅₄) And increases sharply. Therefore, the fluorescent probe Q-1 of the invention can well detect SO in a physiological range₂And (3) derivatives.

Example 7:

practical application of the fluorescent probe Q-1 obtained in example 1.

(1) Co-localized fluorescence imaging of the fluorescent probe Q-1 obtained in example 1.

Live colon cancer cells (SW480) were plated on 35 mm glass-bottomed plates and incubated with 1. mu.M probe Q-1 for 25 minutes. Staining was then performed for 25 min with 0.5 μ M rhodamine 123 (commercial mitochondrial localization reagent). Fluorescence images were obtained on a lycra TCS SP5 II laser confocal scanning microscope using a 40-fold objective lens. The near infrared channel of probe Q-1 involves emission in the 650-700nm spectral window under 543nm excitation. Under the excitation of 514nm, the green channel of rhodamine 123 is recorded on an emission window of 495-545 nm.

FIG. 7 shows a confocal fluorescence image of live colon cancer cells (SW480) co-stained with rhodamine 123 and probe Q-1. (a) The image (b) of the green channel is the image of the red channel, (c) is the image obtained by superimposing (a) and (b), and (d) is the intensity distribution of ROIs in the co-stained living colon cancer cells (SW480) at the positions indicated by line segments in the image (c). The results indicate that probe Q-1 can be localized in the mitochondria of the cell.

(2) Confocal fluorescence imaging of colon cancer cells (SW480) and CCCP (10. mu.M) stained with the fluorescent probes obtained in example 1 at different time intervals.

Fixation of mitochondria: colon cancer cells (SW480) were plated on 35 mm glass-bottom plates and incubated with 1. mu.M probe Q-1 for 25 minutes. Then further treated with CCCP (mitochondrial membrane potential eliminator) at a concentration of 10. mu.M for 30 minutes. Fluorescence imaging was performed in an uninterrupted fashion from 0 to 90 minutes in the near infrared channel (650-700 nm).

As can be seen from FIGS. 8a to 8c, probe Q-1 was preserved in mitochondria after incubation with CCCP for 90 minutes and had a distinct mitochondrial filamentous structure. These results indicate that bromobenzyl group in Q-1 can immobilize probe in mitochondria even in the case of loss of mitochondrial membrane potential

(3) Fluorescent probe Q-1 obtained in example 1 stained colon cancer cells (SW480), and SO was recorded at various concentrations₂Derivative (HSO)^3-) Fluorescence imaging of (1).

Cell staining: in colon cancer cell (SW480) staining experiments, colon cancer cells were stained with probe Q-1 for 25 minutes in 24-well plates. Then, the mixture was washed 3 times with a pH 7.4 potassium phosphate buffer solution to remove extra probes outside the cells, and then HSO was added to the 24-well plates at concentrations of 0, 300 and 600. mu.M, respectively^3-Incubated for 30 minutes.

As shown in fig. 9, the near infrared channel fluorescence intensity is strong (fig. 9b), but the green channel has little fluorescence (9 a). It is found from FIG. 9d and FIG. 9g that HSO is added in different concentrations₃ ^-After 30 minutes of incubation, the fluorescence intensity of the green channel gradually increased. While the fluorescence intensity of the near infrared channel gradually decreased (fig. 9e and 9 f). In addition, the emission ratio (green/near infrared) and the addition of HSO₃ ^-Is linearly related to the concentration of (c). Therefore, the results indicate that the probe is directed to HSO in living cells₃ ^-There is a rate and dose dependent response.

the orychophragmus violaceus was planted in fertile soil, incubated with 5 μ M probe Q-1 for 30min each day, and the residual dye on the leaves was washed off with water. Then using HSO of different concentration every day₃ ^-(0, 400. mu.M, 800. mu.M) plant leaves. Spraying 800 mu M HSO after 3 days₃ ^-The typical SO appears on the blade₂Yellowing phenomenon (FIG. 10 h). 400 μ M HSO₃ ^-The leaves of the plants have yellowish appearance, and 0 mu M HSO is sprayed₃ ^-The blade of (2) did not show such phenomena (FIGS. 10d, h), which indicates SO₂Causing toxicity to crops. When the leaf of orychophragmus violaceus was cut into a transparent thin tissue of 0.5-1 mm, it can be seen from FIG. 10b that the fluorescence intensity in the near infrared channel was high, whereas there was almost no fluorescence in the green channel, indicating that the probe Q-1 could enter the leaf tissue and mitochondria of orychophragmus violaceus and no SO was found in either₂And (3) derivatives. While the fluorescence of the near infrared channel in 9f is weakened, and the fluorescence appears in the green channel, which indicates that HSO is sprayed at 400 mu M₃ ^-After 3 days, the orychophragmus violaceus had been affected. Especially, spraying 800 mu M HSO on leaves₃ ^-The leaf tissue of (1) is visible with significant filiform fluorescence in the green channel and little fluorescence in the near infrared channel.

High SO concentrations in mitochondria when oxidative stress occurs₂Derivative, leaf exposure to SO₂The effects of phytotoxicity. In addition, when SO₂When the derivative attacks orychophragmus violaceus, the combination of 7-diethylcoumarin groups and pyrimidine groups is broken by destroying the vinyl groups on both sides in the probe Q-1, and the ICT process is interrupted. Thus, probe Q-1 will be directed to SO₂The derivatives exhibit a ratiometric fluorescent response. Thereby achieving the aim of controlling SO in the mitochondria of the orychophragmus violaceus leaves₂Process for preparing derivativesAnd (6) detecting. According to this experiment, when HSO is used₃ ^-When the concentration reaches 400 mu M, the crops should be treated, the further diffusion of the phytotoxicity is avoided, and the yellowing and even death of the crops are effectively reduced.

In conclusion, a novel fluorescent probe Q-1 for detecting SO in mitochondria of crop leaf tissue is reasonably developed₂And (3) derivatives. The probe has good selectivity and sensitivity, and is HSO₃ ^-Capable of specific response, probe Q-1 to HSO₃ ^-The detection limit of (2) was 0.512. mu.M. Probe Q-1 has near infrared fluorescence and is directed to HSO₃ ^-The kit has a ratio response, effectively reduces background fluorescence and experimental errors, and has deeper penetrating power for tissues.

Fluorescence imaging experiments show that the probe Q-1 can not only enter mitochondria in a targeted manner, but also enter mitochondria when SO is in high concentration₂When the derivative enables the mitochondrial membrane potential to disappear, the probe Q-1 can be kept in mitochondria to continuously detect SO₂And (3) derivatives. As a substance exposed to SO₂When phytotoxic, probe Q-1 has been successfully used to track SO in mitochondria in crop tissues₂The concentration of the derivative. The results show that SO is present in the mitochondria of the crop₂With SO₂Is related to phytotoxicity of (a). The relation between the mitochondrial damage and the toxicity of the crops is proved, and data support and theoretical basis are provided for further preventing and controlling the crops.

Claims

1. Method for detecting SO in crop mitochondria₂The derivative ratio type near infrared fluorescent probe is characterized by having the following structure:

2. method for detecting SO in crop mitochondria₂The preparation method of the derivative ratio type near-infrared fluorescent probe is characterized by comprising the following steps:

step 1, dissolving 4, 6-dimethyl pyrimidine and 1, 4-dibromomethylbenzene in acetonitrile, placing the mixture in a round-bottom flask, and refluxing and heating the stirred mixture for reaction; after cooling to room temperature, excess acetonitrile was removed from the mixture; separating and purifying by column chromatography to obtain intermediate product;

step 2, adding 7- (diethylamino) coumarin aldehyde and the intermediate product obtained in the step 1 into absolute ethyl alcohol, and heating, refluxing and stirring the mixture for reaction; after the mixture was cooled to room temperature, the excess solvent was removed; purifying by column chromatography to obtain detected SO₂Derivative ratio type near infrared fluorescent probe.

3. The method according to claim 2, wherein in step 1, the 4, 6-dimethylpyrimidine, 1, 4-dibromomethylbenzene, and acetonitrile are used in a ratio of 100 to 110. mu.L: 178 to 180 μ L: 5mL to 6 mL.

4. The preparation method according to claim 2, wherein in the step 1, the temperature of the reflux heating reaction is 80-85 ℃ and the time is 23-24 hours.

5. The method according to claim 2, wherein the excess acetonitrile is removed by suction filtration under reduced pressure in step 1.

6. The preparation method according to claim 2, wherein in the step 2, the 7- (diethylamino) coumarin aldehyde, the intermediate product and the acetonitrile are used in a ratio of 168mg to 170 mg: 100 mg-110 mg: 10mL to 11 mL.

7. The preparation method according to claim 2, wherein in the step 2, the temperature of the heating and stirring reaction is 75-80 ℃ for 5-6 h, and the solvent removal method is rotary evaporation.

8. The method of claim 1 for detecting SO in mitochondria in a crop₂Of ratiometric near-infrared fluorescent probes of derivativesUse of the detection SO₂Ratio type near-infrared fluorescent probe of derivative is used for targeting and positioning in crop mitochondria and carrying out mitochondria SO₂And (4) detecting the derivative.

9. The method of claim 1 for detecting SO in mitochondria in a crop₂Use of ratiometric near-infrared fluorescent probes of derivatives, characterized in that said detection of SO is carried out₂When the ratio type near-infrared fluorescent probe of the derivative is used for detecting SO continuously in mitochondria after mitochondrial membrane potential disappears₂And (3) derivatives.