Near-infrared frequency up-conversion fluorescent probe, benzopyran onium salt compound as well as preparation method and application of benzopyran onium salt compound

Abstract

The invention belongs to the field of molecular fluorescent probes, and particularly relates to a near-infrared frequency up-conversion fluorescent probe, a benzopyranium salt compound as well as a preparation method and application of the benzopyranium salt compound. According to the invention, benzopyranonium salt compounds derived from natural materials such as anthocyanin are used as a parent body, an electron-donating group is introduced as a donor, and then the small organic molecule with a D-pi-A structure is synthesized through a Knoevenagel condensation reaction, and the molecule has the effect of exciting and positioning mitochondria by down-conversion of 650 nm, and also has the effect of reducing mitochondria by down-conversion of 650 nm. And the fluorescent probe also has the characteristic of exciting up-conversion fluorescence targeting mitochondria at 760 nm, so that the fluorescent probe can be used as a fluorescent probe in cell imaging. The material has stronger penetrating power of biological tissues during fluorescence imaging, so that the material has a better fluorescence imaging effect. The scheme of the invention also provides a related process capable of stably synthesizing the fluorescent probe and the benzopyranium salt compound serving as a core raw material of the fluorescent probe, so that the fluorescent probe has higher practical value.

Description

Technical Field

[0001] The invention belongs to the field of molecular fluorescent probes, and specifically relates to a near-infrared frequency up-conversion fluorescent probe, a benzopyrylium salt compound, and preparation methods and applications of the two. Background Art

[0002] Mitochondria are very important organelles in cells, which play a vital role in maintaining cell homeostasis and completing cell life activities. Mitochondria are responsible for most of the energy supply in cells, and are also involved in a series of synthesis reactions and product metabolism. At present, most of the identification of mitochondria uses traditional Stokes luminescence (i.e., down-conversion fluorescence), that is, using a material that emits long-wavelength fluorescence after being excited by short-wavelength light to target mitochondria. However, such materials still have shortcomings such as large interference from their own fluorescence and easy damage to biological tissues when using ultraviolet or visible light excitation. In response to this problem, technicians have further developed anti-Stokes frequency up-conversion fluorescence (FUCL) dyes. FUCL dyes have high absorption rates and adjustable excitation and emission wavelengths. The structure is easy to modify, which can avoid photobleaching of the dye, reduce autofluorescence, deepen the penetration depth, and have good photostability.

[0003] Near-infrared fluorescent dyes have become a hot topic of research in recent years due to their unique properties and have been applied to various fields. Near-infrared dyes absorb energy in the near-infrared spectrum and release fluorescence in the visible light range. Therefore, they have a high tissue penetration depth and low autofluorescence. Benzopyranium salts are a class of compounds found in natural products such as anthocyanins. Because of their good photophysical properties and unique chemical structure, they are considered by technicians to be a key material for developing near-infrared fluorescent dyes with near-infrared frequency up-conversion luminescence properties. However, how to stably synthesize such materials and use them to develop safer fluorescent dyes that can be used commercially is becoming a technical problem that technicians in this field need to solve urgently. Summary of the invention

[0004] In order to solve the problem that the prior art lacks near-infrared frequency up-conversion fluorescent dyes that can be stably synthesized and applied, the present invention provides a near-infrared frequency up-conversion fluorescent probe, a benzopyrylium salt compound, and preparation methods and applications of the two.

[0005] The present invention is implemented by the following technical solutions: A near-infrared frequency up-conversion fluorescent probe has the following molecular structure expression: .

[0006] The near-infrared frequency up-conversion fluorescent probe provided by the invention has down-conversion fluorescence and frequency up-conversion fluorescence characteristics; the ultraviolet absorption spectrum includes 420-750nm.

[0007] Typically, under the condition of 650 nm light excitation, the fluorescence spectrum generated by the fluorescent probe is 660 nm to 900 nm; under the condition of 760 nm light excitation, the fluorescence spectrum generated is 620 nm to 750 nm.

[0008] The present invention also includes a benzopyrylium salt compound, which is used to synthesize the aforementioned near-infrared frequency up-conversion fluorescent probe; the chemical formula of the benzopyrylium salt compound is shown in Formula II: .

[0009] The preparation method of the benzopyrylium salt compound as described above provided by the present invention comprises: (1) 8-hydroxyjulolide is subjected to a condensation reaction with bis(2,4,6-trichlorophenyl)malonate to obtain an intermediate 1 as shown in Formula IV; .

[0010] (2) The intermediate 1 is subjected to an acidification reaction to obtain the intermediate 2 represented by the chemical formula V: .

[0011] (3) The intermediate 2 is subjected to a condensation reaction with acetophenone to obtain the benzopyrylium salt compound.

[0012] As a further improvement of the present invention, in the condensation reaction of step (1), toluene is used as the reaction solvent, the reaction temperature is 105-115° C., and the reaction time is 3-5 h.

[0013] As a further improvement of the present invention, in the acidification reaction of step (2), 30% sulfuric acid is used as the acidifying agent, the reaction temperature is 120-130° C., and the reaction time is 4-6 h.

[0014] As a further improvement of the present invention, in the condensation reaction of step (3), glacial acetic acid is used as the reaction solvent, and a perchloric acid solution is added to the reaction solvent; during the reaction, the reaction is first carried out at a temperature of 55 to 65° C. for 5 to 7 hours, and then at a temperature of 105 to 115° C. for 11 to 13 hours.

[0015] The present invention also includes a method for preparing a fluorescent probe, which is used to prepare the aforementioned near-infrared frequency up-conversion fluorescent probe. The preparation method comprises: The aforementioned benzopyrylium salt compound and p-dimethylaminobenzaldehyde of formula III are added to an organic solvent in proportion, and a Knoevenagel condensation reaction is carried out in a protective atmosphere to obtain a target product of formula I; .

[0016] As a further improvement of the present invention, the organic solvent used in the Knoevenagel condensation reaction is n-butanol and toluene in a volume ratio of 13:7; nitrogen is used as the protective atmosphere; the reaction temperature is 115° C. to 125° C.; and the condensation reaction time under stirring conditions is 9 to 11 hours.

[0017] The present invention also includes an application of the aforementioned near-infrared frequency up-converted fluorescent probe in cell imaging, specifically comprising: co-incubating the cells with the fluorescent probe to achieve targeting of the mitochondria in the cells.

[0018] The technical solution provided by the present invention has the following beneficial effects: The present invention provides a new material as a near-infrared frequency up-conversion fluorescent probe, which has both the effect of down-converting 650 nm excitation to identify mitochondria and the characteristic of up-converting fluorescence targeting mitochondria at 760 nm excitation. The fluorescent probe provided by the present invention has stronger biological tissue penetration during fluorescence imaging, and thus has a better fluorescence imaging effect.

[0019] The present invention also provides a method for stably synthesizing materials used as fluorescent probes and their precursors, which makes the synthesis of the material no longer dependent on the extraction of natural materials, can greatly improve the yield of the product and reduce production costs, thereby enhancing the practical value and commercial prospects of the material. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the nuclear magnetic hydrogen spectrum of the BZPN material prepared by the present invention in the test experiment.

[0021] Figure 2 This is the ultraviolet absorption spectrum of the BZPN material provided by the present invention in the test experiment.

[0022] Figure 3 This is a down-conversion emission spectrum diagram of the BZPN material provided by the present invention under 650nm laser excitation in a test experiment.

[0023] Figure 4 This is an up-conversion emission spectrum diagram of the BZPN material provided by the present invention under 760nm laser excitation in a test experiment.

[0024] Figure 5 The graph shows the variation of the up-conversion luminescence intensity of the BZPN provided by the present invention with the excitation power.

[0025] Figure 6 It is a bar graph showing the survival rate of Hela cells in different concentrations of BZPN.

[0026] Figure 7This is a comparison diagram of the down-conversion fluorescence imaging and up-conversion fluorescence imaging effects of BZPN provided by the present invention and the imaging effects of traditional commercial mitochondrial dyes. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] Example 1 This embodiment provides a near-infrared frequency up-conversion fluorescent probe, which is an organic small molecule with a D-π-A structure developed by using a benzopyrylium salt compound as a parent (A) and introducing an electron-donating group as a donor (D). This small molecule material has both down-conversion fluorescence and frequency up-conversion fluorescence characteristics. In practical applications, the excitation light of the down-conversion fluorescence and frequency up-conversion fluorescence with high absorption rate is preferably 650nm and 760nm, respectively. For example, under the condition of 650nm light excitation, the fluorescence spectrum generated by the fluorescent probe is 660nm~900nm. Under the condition of 760nm light excitation, the fluorescence spectrum generated is 620nm~750nm.

[0029] In particular, the fluorescent probe provided in this embodiment has the following molecular structure expression: .

[0030] The preparation method of the specific fluorescent probe provided in this embodiment includes: A benzopyrylium salt compound of formula II and p-dimethylaminobenzaldehyde of formula III are added to an organic solvent in proportion, and a Knoevenagel condensation reaction is carried out in a protective atmosphere to obtain a target product of formula I.

[0031] , .

[0032] Among them, the product of formula I is generated by condensation of formula II and formula III at a molar ratio of 1:1. In order to ensure the yield, in the reaction system of the Knoevenagel condensation reaction, according to the molar ratio of the substance, the amount of the two reactants can be set to 1: (1-1.5), which can increase the amount of benzopyrylium salt compounds or the amount of dimethylaminobenzaldehyde. In the preparation method of the fluorescent probe provided in this embodiment, the organic solvent can use various commonly used organic solvents, such as various alcohol solvents and benzene solvents. In the typical scheme of this embodiment, a mixed solvent of n-butanol and toluene configured at a volume ratio of 13:7 is used. The Knoevenagel condensation reaction of this embodiment needs to be carried out under a protective atmosphere. In practical applications, the type of gas used in the protective atmosphere is not limited. Various protective gases familiar to those skilled in the art can be used, such as nitrogen and inert gases. In this embodiment, the reaction temperature is set to 115°C to 125°C; the condensation reaction time under stirring conditions is 9-11h. The final product purified after the reaction is a dark blue solid.

[0033] The typical application scenario of the fluorescent probe provided in this embodiment is to realize cell imaging. The fluorescent probe can specifically bind to the mitochondria in the cell. For example, after the cells are co-incubated with the fluorescent probe, the mitochondria in the cell can be targeted. In this state, after the fluorescent probe is excited to produce fluorescence based on a specific light field, the cell can be fluorescently imaged and the mitochondria in the cell can be located.

[0034] Example 2 The fluorescent probe provided in Example 1 is mainly synthesized by condensation reaction of two substances, benzopyrylium salt compounds and p-dimethylaminobenzaldehyde. Wherein, p-dimethylaminobenzaldehyde belongs to a commercial raw material that can be stably supplied, and benzopyrylium salt compounds belong to a special new material, which can be separated from natural substances such as anthocyanidins, but the purity and yield of the benzopyrylium salt compounds obtained by this separation and purification method are insufficient, and the separation cost is high, and it is difficult to meet market demand. In view of this situation, the present embodiment further provides a preparation method of a benzopyrylium salt compound, which generates a benzopyrylium salt compound by chemical synthesis, thereby effectively ensuring the purity and output rate of the product, and reducing manufacturing costs.

[0035] Specifically, the preparation method of the benzopyrylium salt compound provided in this embodiment includes: (1) 8-hydroxyjulolide is subjected to a condensation reaction with bis(2,4,6-trichlorophenyl)malonate to obtain an intermediate 1 as shown in Formula IV; .

[0036] In detail, in the condensation reaction of this step, toluene can be selected as the reaction solvent, 8-hydroxyljuloridine and bis(2,4,6-trichlorophenyl)malonate are added to the reaction system at a molar ratio of 1:(1-1.5), the reaction temperature is 105-115°C, and the reaction time is 3-5h. After the reaction is completed, the solid is purified to obtain the intermediate 1.

[0037] (2) The intermediate 1 is subjected to an acidification reaction to obtain the intermediate 2 represented by the chemical formula V: .

[0038] In detail, the acidification reaction in this step can select 30% sulfuric acid as the acidifying agent, the reaction temperature is 120-130° C., and the reaction time is 4-6 hours.

[0039] (3) The intermediate 2 is reacted with acetophenone to obtain a benzopyrylium salt compound.

[0040] In detail, the condensation reaction in this step uses glacial acetic acid as the reaction solvent, and a perchloric acid solution is added to the reaction solvent; during the reaction, the temperature is first 55-65° C. for 5-7 hours, and then the temperature is 105-115° C. for 11-13 hours. After the reaction is completed, the purified dark red solid is the desired benzopyranium salt compound.

[0041] In order to verify the properties and performance of the material provided in this embodiment, technicians verified the schemes of Examples 1 and 2 in subsequent test examples and experiments, experimentally synthesized relevant samples, analyzed the physical and chemical properties of the synthesized substances in subsequent experiments, and tested the various properties of the material when used as a fluorescent probe.

[0042] Test Example 1 (1) Preparation of intermediate 1 According to the proportion, 5.68g, 0.03mol of 8-hydroxyjulolide, 13.89g, 0.03mol of bis(2,4,6-trichlorophenyl)malonate and 70mL of toluene were added to a 250mL flask to form a reactant system; the reactant system was heated to reflux at 110°C for 12h. After the reaction was completed, it was cooled to room temperature, filtered, washed with petroleum ether and anhydrous ether, and dried to obtain intermediate 1.

[0043] (2) Preparation of intermediate 2 According to the proportion, 2.2 g, 0.008 mol of intermediate 1 was added to a 100 mL flask, and then 34 mL of 30% sulfuric acid solution was added to the flask to form a reactant system. The above reactant system was heated to reflux at 125 ° C for 5 h, and cooled to room temperature after the reaction. Next, NH3 ·H 2 O The solution was adjusted to neutral, and the product system was transferred to a separatory funnel, and extracted with dichloromethane and deionized water for three times. After the extraction, the organic phase was dried with anhydrous sodium sulfate and then filtered. The filtrate was subjected to vacuum rotary evaporation to remove the solvent to obtain a crude product; the crude product was separated by column chromatography using a mixture of petroleum ether and ethyl acetate (the volume ratio of petroleum ether to ethyl acetate was 50:1) as an eluent, and the eluent was subjected to vacuum rotary evaporation to remove the eluent to obtain intermediate 2.

[0044] (3) Preparation of benzopyrylium salt compounds According to the proportion, 1.08g, 0.005mol of intermediate 2 and 2.5ml of acetophenone were dissolved in 15ml of glacial acetic acid, then the reaction system was stirred at room temperature, and 8.4ml of 70% perchloric acid solution was added dropwise, and heated to reflux for 6h under the reaction condition of 60°C, and then heated to reflux for 12h under the condition of 110°C. After the reaction was completed, it was cooled to room temperature and 100ml of deionized water was added. At this time, a dark red precipitate was generated, which was filtered under reduced pressure and washed with ethyl acetate, and then dried in a vacuum drying oven. The obtained dark red solid was a benzopyranium salt compound.

[0045] (4) Synthetic fluorescent probe materials According to the proportion, 291 mg, 0.7 mmol of benzopyrylium salt compound, 104 mg, 0.7 mmol of p-dimethylaminobenzaldehyde, 13 mL of n-butanol and 7 mL of toluene were added to a round-bottom flask, heated to 120°C under nitrogen protection, and reacted for 10 hours under stirring. After the reaction was completed, it was cooled to room temperature, filtered under reduced pressure, and the filter cake was washed with petroleum ether and anhydrous ether, and then dried in a vacuum drying oven to obtain a dark blue solid product.

[0046] It should be noted that: although the above test examples only provide one scheme for the reaction conditions such as raw material ratio, reaction solvent, reaction time, reaction temperature, etc., it is well known to those skilled in the art that based on the same reaction principle, the reaction can still be ensured by fine-tuning the above parameters, and the only difference is that there are differences in the yield and purity of the product. These differences can be compensated by purifying the product.

[0047] Test experiment 1. Product composition analysis 1.1. Benzopyrylium salt compounds In this experiment, the red solid obtained in step (3) of test example 1 was first subjected to nuclear magnetic resonance hydrogen spectrum ( 1 HNMR) analysis, the analytical results obtained are as follows: 1H NMR (600 MHz, DMSO- d 6 ) δ 8.20 (d, J = 7.7 Hz, 2H), 7.99 (s, 1H), 7.81 (s, 1H), 7.67 (d, J = 6.9 Hz, 1H), 7.64 (t, J = 7.5 Hz, 2H), 3.55 (t, J = 5.8 Hz, 4H), 3.01 (t, J = 6.3 Hz, 2H), 2.89 (t, J = 6.1 Hz, 2H), 2.74 (s,3H), 2.01 – 1.96 (m, 2H), 1.92 (p, J = 6.3 Hz, 2H).

[0048] Combined with the above characterization data, it can be seen that the dark red solid synthesized in Test 1 is a benzopyrylium salt compound, and its chemical structure is as follows: .

[0049] 1.2 Final product This experiment further performed H NMR spectrum analysis on the dark blue solid obtained in step (4) of Test Example 1. The H NMR spectrum obtained is as follows: Figure 1 The analysis results are as follows: 1 H NMR (600 MHz, DMSO- d 6 ) δ 8.30 (d, J = 15.2 Hz, 1H), 8.19 – 8.15 (m,2H), 8.12 (s, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.81 (d, J = 8.5 Hz, 2H), 7.67 –7.60 (m, 2H), 7.59 (t, J = 7.1 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 3.42 (q, J = 5.8 Hz, 4H), 3.08 (s, 6H), 2.89 (d, J = 6.6 Hz, 2H), 2.83 (t,J = 6.3 Hz,2H), 1.95 (q, J = 6.1 Hz, 2H), 1.90 (t, J = 6.0 Hz, 2H).

[0050] Combined with the above characterization data, the chemical structure of the dark blue solid synthesized in Test 1 is as follows: .

[0051] 2. Fluorescence characteristics test 2.1. UV absorption performance In this experiment, the dark blue solid product finally synthesized in Test Example 1 was used as a fluorescent probe (BZPN), and the ultraviolet absorption spectrum of the fluorescent probe was tested. The specific experimental process includes: The fluorescent probe BZPN was dissolved in dimethyl sulfoxide (DMSO) solvent to prepare a mother solution with a concentration of 10mM. 20μL of the mother solution was diluted with DMSO to a working concentration of 10μM. The solution was placed in a 1.0cm quartz cell to measure the ultraviolet absorption performance. The following is drawn based on the experimental results: Figure 2 The UV absorption spectrum is shown.

[0052] analyze Figure 2 It can be found from the experimental data that the ultraviolet absorption spectrum of the product synthesized in this test example is between 420nm and 750nm.

[0053] 2.2 Fluorescence spectrum test This experiment further tested the fluorescence spectrum of the 10 μM solution prepared in the aforementioned quartz cell. In the experiment, the emission spectrum of BZPN produced under the excitation of 650 nm laser was first tested. The results are as follows: Figure 3 The emission spectrum of BZPN under the excitation of 760nm laser was tested again, and the results are as follows Figure 4 shown.

[0054] right Figure 3 and Figure 4 After comprehensive analysis, it was found that under the condition of 650nm light excitation, the fluorescence spectrum produced by the fluorescent probe is 660nm~900nm. Under the condition of 760nm light excitation, the fluorescence spectrum produced is 620nm~750nm. Figure 3 The spectrum belongs to down-conversion fluorescence, Figure 4 This is frequency up-conversion fluorescence, or FUCL.

[0055] 2.3 Laser power and frequency up-conversion fluorescence performance Combined with the frequency up-conversion fluorescence characteristics of BZPN in the previous test, this experiment further studied the effect of the emission power of the laser used for excitation on the emission peak intensity of BZPN fluorescence. The experimental process includes: First, the fluorescent probe BZPN was dissolved in dimethyl sulfoxide solvent to prepare a mother solution with a concentration of 10 mM. 20 μL of the mother solution was diluted with DMSO to a working concentration of 10 μM, and the solution was placed in a 1.0 cm quartz cell for upconversion emission spectroscopy testing.

[0056] In the up-conversion emission spectrum test of each group, the excitation wavelength was fixed at 760 nm, and the laser power of the laser light source was gradually increased. The up-conversion emission peak of the fluorescence generated by BZPN under different laser power conditions was tested and recorded. The experimental data obtained from the test were fitted as follows: Figure 5 The curve of BZPN upconversion luminescence intensity changing with excitation power is shown.

[0057] analyze Figure 5 The experimental data in the figure show that under the condition of a fixed laser wavelength of 760 nm, the upconversion emission peak intensity of BZPN is positively correlated with the excitation light power, and the two show an obvious linear relationship with a slope of 0.83. This also proves that the upconversion emission process of BZPN provided by this experiment is a single-photon absorption process.

[0058] 3. Biological toxicity and cell imaging effect 3.1 Biological toxicity of BZPN This experiment first used the MTT method to test the biological toxicity of BZPN provided in this experiment to living cells. The specific experimental process is as follows: First, BZPN experimental solutions with concentrations of 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, and 5 μM were prepared respectively, and then BZPN solutions of various concentrations were added to multiple Hela cells. The absorbance was measured after incubation for 24 hours, and the cytotoxicity of the fluorescent probe BZPN was calculated based on the absorbance.

[0059] According to the above experimental results, the Figure 6 The bar graphs show the survival rates of Hela cells under the action of different concentrations of BZPN. Figure 6 The experimental data in the experiment show that compared with the cell activity without adding the fluorescent probe BZPN, the cell activity after adding 1-5 μM fluorescent probe incubation has basically no significant change. This shows that the fluorescent probe BZPN provided by the present invention does not cause toxic damage to cells and can be used for cell imaging.

[0060] 2.2 Imaging Effect of BZPN In order to verify the fluorescence imaging effect of BZPN provided by the present invention in cells, a co-localization experimental method was used in this experiment. In the experiment, a mitochondrial commercial dye (Mito-Tracker Green, excitation light of 490nm, emission light collection range of 500nm-550nm) with significantly different excitation light from BZPN of the present invention was selected as a control material to achieve performance calibration.

[0061] During the experiment, Hela cells were first incubated with 0.5 μM fluorescent probe BZPN for 10 min, and then incubated with 0.5 μM mitochondrial commercial dye reagent for 20 min. Then, laser confocal microscopy was used for imaging. The wavelength of the excitation light of the commercial dye was 490 nm; the down-conversion excitation light of BZPN was 650 nm, and the up-conversion excitation light was 760 nm.

[0062] During the experiment, Figure 7 (a) Down-conversion fluorescence imaging of the fluorescent probe BZPN in cells. Figure 7 (b) Upconversion fluorescence imaging of the fluorescent probe BZPN in cells. Figure 7 (c) Cell imaging using commercial mitochondrial dyes.

[0063] Further, Figure 7 (a) with Figure 7 (c) Superposition is performed to obtain Figure 7 (d), Figure 7 (a) with Figure 7 The Pearson correlation coefficient of the data in (c) is 0.92. Figure 7 (b) with Figure 7 (c) Superposition is performed to obtain Figure 7 (e), Figure 7 (b) with Figure 7 The Pearson correlation coefficient for the data in (c) is 0.90.

[0064] Combination Figure 7 From the above experimental results, it can be found that the fluorescent probe BZPN provided by the present invention highly overlaps with the labeling results of the mitochondrial commercial dye cell map, whether it is down-converted at 650 nm excitation or up-converted at 760 nm excitation. This shows that the BAPN of the present invention can achieve a targeting effect comparable to that of traditional commercial mitochondrial dyes, and has outstanding practical value.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A near-infrared frequency up-conversion fluorescent probe, characterized in that: It has the following molecular structure expression: 。 2. The near-infrared frequency up-conversion fluorescent probe according to claim 1, characterized in that: It has down-conversion fluorescence and frequency up-conversion fluorescence characteristics, and the ultraviolet absorption spectrum includes 420-750nm; Moreover, under the condition of 650nm light excitation, the fluorescence spectrum generated by the fluorescent probe is 660nm~900nm; under the condition of 760nm light excitation, the fluorescence spectrum generated is 620nm~750nm.

3. A benzopyrylium salt compound, characterized in that: It is used to synthesize the near-infrared frequency up-conversion fluorescent probe as claimed in claim 1 or 2; the chemical formula of the benzopyrylium salt compound is shown in Formula II: 。 4. A method for preparing a benzopyrylium salt compound as claimed in claim 3, characterized in that: It includes: (1) condensing 8-hydroxyjulolide with bis(2,4,6-trichlorophenyl)malonate to obtain an intermediate 1 represented by formula IV; ； (2) The intermediate 1 is subjected to an acidification reaction to obtain the intermediate 2 represented by the chemical formula V: ； (3) The intermediate 2 is subjected to a condensation reaction with acetophenone to obtain the benzopyrylium salt compound.

5. The method for preparing a benzopyrylium salt compound according to claim 4, wherein: In the condensation reaction of step (1), toluene is used as the reaction solvent, the reaction temperature is 105-115° C., and the reaction time is 3-5 h.

6. The method for preparing a benzopyrylium salt compound according to claim 4, wherein: In the acidification reaction of step (2), 30% sulfuric acid is used as the acidifying agent, the reaction temperature is 120-130° C., and the reaction time is 4-6 h.

7. The method for preparing a benzopyrylium salt compound according to claim 4, wherein: In the condensation reaction of step (3), glacial acetic acid is used as the reaction solvent, and a perchloric acid solution is added to the reaction solvent; during the reaction, the reaction is first carried out at a temperature of 55 to 65° C. for 5 to 7 hours; and then at a temperature of 105 to 115° C. for 11 to 13 hours.

8. A method for preparing a fluorescent probe, characterized in that: It is used to prepare the near-infrared frequency up-conversion fluorescent probe as claimed in claim 1 or 2; the preparation method comprises: Adding the benzopyrylium salt compound as claimed in claim 3 and p-dimethylaminobenzaldehyde as shown in formula III in proportion to an organic solvent, and performing a Knoevenagel condensation reaction in a protective atmosphere to obtain a target product as shown in formula I; 。 9. The method for preparing a fluorescent probe according to claim 8, characterized in that: The organic solvent used in the Knoevenagel condensation reaction is n-butanol and toluene in a volume ratio of 13:7; nitrogen is used as the protective atmosphere; the reaction temperature is 115° C. to 125° C.; and the condensation reaction time under stirring conditions is 9 to 11 hours.

10. A use of the near-infrared frequency up-conversion fluorescent probe as claimed in claim 1 or 2 in cell imaging, characterized in that: The cells are co-incubated with the fluorescent probe to achieve targeting of the mitochondria in the cells.

Images