Fluorescent probe for near-infrared frequency up-conversion, benzopyrylium salt compound, preparation method and application thereof

Abstract

The present invention belongs to the field of molecular fluorescent probes, and particularly relates to a fluorescent probe for near-infrared frequency up-conversion, a benzopyrylium salt compound, and a preparation method and application thereof. In the present invention, a benzopyrylium salt compound derived from natural materials such as anthocyanins is used as a parent body, and an electron-donating group is introduced as a donor. Then, an organic small molecule with a D-π-A structure is synthesized through a Knoevenagel condensation reaction. This molecule not only has the effect of localizing mitochondria by down-conversion excitation at 650 nm, but also has the characteristic of up-conversion fluorescence targeting mitochondria by excitation at 760 nm, so it can be used as a fluorescent probe in cell imaging. This material has stronger penetration power in biological tissues during fluorescence imaging, so it has a better fluorescence imaging effect. The solution of the present invention also provides a related process for stably synthesizing the fluorescent probe and its core raw material, the benzopyrylium salt compound, so it has higher practical value.

Description

Technical Field

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

[0002] Mitochondria are very important organelles within cells and play a crucial role in maintaining cell homeostasis and completing cell life activities. Mitochondria are responsible for most of the energy supply within cells and are also involved in a series of synthetic reactions and product metabolisms. Currently, traditional Stokes luminescence (i.e., down-conversion fluorescence) is mostly used to identify mitochondria, that is, a material that emits long-wavelength fluorescence after being excited by short-wavelength light is used to target mitochondria. However, such materials still have problems such as relatively large self-fluorescence interference and easy damage to biological tissues when excited by ultraviolet or visible light. To address this issue, technical personnel have further developed anti-Stokes frequency up-conversion fluorescence (FUCL) dyes. FUCL dyes have high absorption rates and adjustable excitation and emission wavelengths, are easy to modify in structure, can avoid photobleaching of the dyes, reduce autofluorescence, deepen the penetration depth, and have good photostability.

[0003] Near-infrared fluorescent dyes have become a research hotspot in recent years due to their unique properties and are applied in various fields. Near-infrared dyes absorb energy in the near-infrared spectral range and emit fluorescence in the visible light range. Therefore, they have a high tissue penetration depth and low autofluorescence. Benzopyrylium salts are a class of compounds present in natural products such as anthocyanins. Due to their good photophysical properties and unique chemical structures, they are considered by technical personnel 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 develop safer fluorescent dyes that can be commercially used from them is becoming a technical problem that needs to be urgently solved by technical personnel in this field. Summary of the Invention

[0004] To solve the problem in the prior art of lacking 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 thereof.

[0005] The present invention is achieved by the following technical solutions:

[0006] A near-infrared frequency up-conversion fluorescent probe has the following molecular structure expression:

[0007] .

[0008] The fluorescence probe for near-infrared frequency up-conversion provided by the present invention has the characteristics of down-conversion fluorescence and frequency up-conversion fluorescence; the ultraviolet absorption spectrum includes 420-750 nm.

[0009] Typically, under the light excitation condition of 650 nm, the fluorescence spectrum generated by the fluorescence probe is 660 nm - 900 nm; under the light excitation condition of 760 nm, the fluorescence spectrum generated is 620 nm - 750 nm.

[0010] The present invention also includes a benzopyrylium salt compound, which is used for synthesizing the aforementioned fluorescence probe for near-infrared frequency up-conversion; the chemical formula of the benzopyrylium salt compound is shown as Formula II:

[0011] 。

[0012] The preparation method of the benzopyrylium salt compound provided by the present invention includes:

[0013] (1) Condensing 8-hydroxyjulolidine with bis(2,4,6-trichlorophenyl) malonate to obtain Intermediate 1 with the chemical formula shown as Formula IV;

[0014] 。

[0015] (2) After subjecting Intermediate 1 to an acidification reaction, Intermediate 2 with the chemical formula shown as Formula V is obtained:

[0016] 。

[0017] (3) Condensing Intermediate 2 with acetophenone to obtain the benzopyrylium salt compound.

[0018] 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.

[0019] 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.

[0020] 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 perchloric acid solution is added to the reaction solvent; during the reaction process, first react at a temperature of 55 - 65 °C for 5 - 7 h: then react at a temperature of 105 - 115 °C for 11 - 13 h.

[0021] The present invention also includes a preparation method of a fluorescence probe, which is used for preparing the aforementioned fluorescence probe for near-infrared frequency up-conversion. The preparation method includes:

[0022] The benzopyrylium salt compound as described above and p-dimethylaminobenzaldehyde of formula Ⅲ are added to an organic solvent in a certain proportion, and the Knoevenagel condensation reaction is carried out under a protective atmosphere to obtain the target product of formula Ⅰ;

[0023] 。

[0024] As a further improvement of the present invention, the organic solvent used in the Knoevenagel condensation reaction is n-butanol and toluene with a volume ratio of 13:7; the protective atmosphere is nitrogen; the reaction temperature is 115 °C to 125 °C; the condensation reaction time under stirring conditions is 9 to 11 h.

[0025] The present invention also includes the application of a near-infrared frequency up-conversion fluorescent probe as described above in cell imaging, specifically including: co-incubating cells with the fluorescent probe to achieve targeting of mitochondria in cells.

[0026] The technical solution provided by the present invention has the following beneficial effects:

[0027] The present invention provides a new material as a near-infrared frequency up-conversion fluorescent probe. This material not only has the effect of recognizing mitochondria by down-conversion excitation at 650 nm, but also has the property of up-conversion fluorescence targeting mitochondria by excitation at 760 nm. The fluorescent probe provided by the present invention has stronger penetration in biological tissues during fluorescence imaging, so it has a better fluorescence imaging effect.

[0028] The present invention also provides a method for stably synthesizing the material and its precursor as a fluorescent probe, which makes the synthesis of this material no longer rely on the extraction of natural materials, can greatly improve the product yield and reduce the production cost, thus enhancing the practical value and commercial prospect of this material. Description of the Drawings

[0029] Figure 1 It is the 1H NMR spectrum of the BZPN material prepared in the test experiment of the present invention.

[0030] Figure 2 It is the UV absorption spectrum of the BZPN material provided by the present invention in the test experiment.

[0031] Figure 3 It is the down-conversion emission spectrum of the BZPN material provided by the present invention under 650 nm laser excitation in the test experiment.

[0032] Figure 4 It is the up-conversion emission spectrum of the BZPN material provided by the present invention under 760 nm laser excitation in the test experiment.

[0033] Figure 5The curve of the up-conversion luminescence intensity of BZPN provided by the present invention varying with the excitation power.

[0034] Figure 6 The bar graph of the survival rate of Hela cells in BZPN with different concentrations.

[0035] Figure 7 The comparison graph of the imaging effects of the down-conversion fluorescence imaging and up-conversion fluorescence imaging of BZPN provided by the present invention with the imaging effects of traditional commercial mitochondrial dyes. Detailed implementation manners

[0036] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to 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.

[0037] Embodiment 1

[0038] 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 the parent body (A) and introducing an electron-donating group as the donor (D). This small molecule material has both down-conversion fluorescence and frequency up-conversion fluorescence characteristics. In practical applications, the excitation lights with high absorption rates for down-conversion fluorescence and frequency up-conversion fluorescence are preferably 650 nm and 760 nm respectively. For example, under the excitation condition of 650 nm light, the fluorescence spectrum generated by this fluorescent probe is 660 nm to 900 nm. And under the excitation condition of 760 nm light, the fluorescence spectrum generated is 620 nm to 750 nm.

[0039] Specifically, such a fluorescent probe provided by this embodiment has the following molecular structure expression formula:

[0040] .

[0041] The preparation method of this specific fluorescent probe provided by this embodiment includes:

[0042] Adding the benzopyrylium salt compound of formula II and p-dimethylaminobenzaldehyde of formula III into an organic solvent in proportion, and carrying out the Knoevenagel condensation reaction in a protective atmosphere to obtain the target product of formula I.

[0043] ,

[0044] .

[0045] Among them, the product of Formula I is formed by the condensation of Formula II and Formula III in a molar ratio of 1:1. To ensure the yield, in the reaction system of the Knoevenagel condensation reaction, according to the molar ratio, the dosages of the two reactants can be set to 1:(1 - 1.5), that is, either increasing the dosage of the benzopyrylium salt compound or increasing the dosage of dimethylaminobenzaldehyde can be chosen. In the preparation method of the fluorescent probe provided in this example, various commonly used organic solvents can be used, such as various alcohol solvents and benzene solvents, etc. In the typical scheme of this example, a mixed solvent of n-butanol and toluene configured in a volume ratio of 13:7 is used. The Knoevenagel condensation reaction of this example needs to be carried out under a protective atmosphere. In practical applications, the type of gas used for the protective atmosphere is not limited, and various protective gases well-known to those skilled in the art can be used, such as nitrogen and inert gases, etc. The reaction temperature of this example is set to 115°C - 125°C; the condensation reaction time under stirring conditions is 9 - 11 h. The final product purified after the reaction is a dark blue solid.

[0046] A typical application scenario of the fluorescent probe provided in this example is for cell imaging. This fluorescent probe can specifically bind to mitochondria in cells. For example, after co-incubating cells with the fluorescent probe, it is possible to target the mitochondria in the cells. In this state, after the fluorescent probe generates fluorescence based on the excitation of a specific light field, it is possible to perform fluorescence imaging on the cells and localize the mitochondria in the cells.

[0047] Example 2

[0048] The fluorescent probe provided in Example 1 is mainly synthesized by the condensation reaction of two substances, namely benzopyrylium salt compounds and p-dimethylaminobenzaldehyde. Among them, p-dimethylaminobenzaldehyde belongs to a commercially available raw material that can be stably supplied, while benzopyrylium salt compounds belong to a special new material. Such materials can be separated from natural substances such as anthocyanins, but the purity and yield of the benzopyrylium salt compounds obtained by this separation and purification method are both insufficient, and the separation cost is high, making it difficult to meet market demand. In view of this situation, this example further provides a preparation method of benzopyrylium salt compounds. This method generates benzopyrylium salt compounds through chemical synthesis, thus effectively ensuring the purity and output rate of the products and reducing the manufacturing cost.

[0049] Specifically, the preparation method of the benzopyrylium salt compounds provided in this example includes:

[0050] (1) Condense 8-hydroxyjulolidine with bis(2,4,6-trichlorophenyl) malonate to obtain Intermediate 1 with the chemical formula shown in Formula IV;

[0051] .

[0052] Specifically, in the condensation reaction of this step, toluene can be selected as the reaction solvent, and 8-hydroxyjulolidine and bis(2,4,6-trichlorophenyl) malonate are added to the reaction system in a molar ratio of 1: (1-1.5). The reaction temperature is 105-115 °C, and the reaction time is 3-5 h. After the reaction, the solid substance therein is purified to obtain Intermediate 1.

[0053] (2) After subjecting Intermediate 1 to an acidification reaction, Intermediate 2 having the chemical formula shown in Formula V is obtained:

[0054] .

[0055] Specifically, 30% sulfuric acid can be selected as the acidifying agent in this step of the acidification reaction. The reaction temperature is 120-130 °C, and the reaction time is 4-6 h.

[0056] (3) Condensation reaction of Intermediate 2 with acetophenone gives benzopyrylium salt compounds.

[0057] Specifically, glacial acetic acid is used as the reaction solvent in this step of the condensation reaction, and perchloric acid solution is added to the reaction solvent; during the reaction, it is first reacted at a temperature of 55-65 °C for 5-7 h: then reacted at a temperature of 105-115 °C for 11-13 h. After the reaction, the deep red solid substance purified is the required benzopyrylium salt compound.

[0058] In order to verify the properties and performance of the materials provided in this embodiment, technicians verified the solutions of Embodiments 1 and 2 in subsequent test examples and experiments, synthesized relevant samples, analyzed the physical and chemical properties of the synthesized substances in subsequent experiments, and tested various performances of this material used as a fluorescent probe.

[0059] Test Example 1

[0060] (1) Preparation of Intermediate 1

[0061] In proportion, 5.68 g, 0.03 mol of 8-hydroxyjulolidine, 13.89 g, 0.03 mol of bis(2,4,6-trichlorophenyl) malonate and 70 mL of toluene are added to a 250 mL flask to form a reactant system; the above reactant system is heated under reflux at 110 °C for 12 h. After the reaction, it is cooled to room temperature, filtered by suction, washed with petroleum ether and anhydrous ether, and dried to obtain Intermediate 1.

[0062] (2) Preparation of Intermediate 2

[0063] Proportionally, add 2.2 g (0.008 mol) of Intermediate 1 to a 100 mL flask, and then add 34 mL of 30% sulfuric acid solution to the flask to form a reactant system. Heat the above reactant system under reflux at 125 °C for 5 h, and after the reaction is completed, cool it to room temperature. Next, adjust the solution to neutral with NH₃·H₂O under ice bath conditions, transfer the product system to a separatory funnel, and extract it with dichloromethane and deionized water for three times. After the extraction is completed, dry the obtained organic phase with anhydrous sodium sulfate and then filter it by suction. Rotate the filtrate under reduced pressure to remove the solvent to obtain a crude product; perform column chromatography separation on the crude product using a mixture of petroleum ether and ethyl acetate (the volume ratio of petroleum ether to ethyl acetate is 50:1) as the eluent, and rotate the obtained eluate under reduced pressure to remove the eluent to obtain Intermediate 2.

[0064] (3)Preparation of benzopyrylium salt compounds

[0065] Proportionally, dissolve 1.08 g (0.005 mol) of Intermediate 2 and 2.5 ml of acetophenone in 15 mL of glacial acetic acid, then stir the reactant system at room temperature and dropwise add 8.4 mL of 70% perchloric acid solution. Heat it under reflux at 60 °C for 6 h, and then heat it under reflux at 110 °C for 12 h. After the reaction is completed, cool it to room temperature, add 100 mL of deionized water. At this time, a dark red precipitate is produced. Filter it by suction under reduced pressure and wash it with ethyl acetate, and then dry it in a vacuum drying oven. The obtained dark red solid is the benzopyrylium salt compound.

[0066] (4)Synthesis of fluorescent probe materials

[0067] Proportionally, add 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 to a round-bottom flask, heat it to 120 °C under nitrogen protection, and react for 10 h under stirring conditions. After the reaction is completed, cool it to room temperature, filter it by suction under reduced pressure and wash the filter cake with petroleum ether and anhydrous ether, and then dry it in a vacuum drying oven to obtain a dark blue solid product.

[0068] It should be noted that: although only one scheme for reaction conditions such as raw material ratio, reaction solvent, reaction time, and reaction temperature is provided in the above test examples, those skilled in the art know that based on the same reaction principle, fine-tuning of the above parameters can still ensure the occurrence of the reaction. The difference is only that there are differences in the product yield and purity, etc. And these differences can be compensated by purifying the product.

[0069] Test experiment

[0070] I. Composition analysis of the product

[0071] 1.1 Benzo[c]chromen-6-ium salts

[0072] In this experiment, the red solid obtained in step (3) of Test Example 1 was first analyzed by nuclear magnetic resonance hydrogen spectrum ( 1 HNMR), and the analysis results are as follows:

[0073] 1 H NMR (600 MHz, DMSO- d 6) δ 8.20 (d, J J = 7.7 Hz, 2H), 7.99 (s, 1H), 7.81 (s, 1H), 7.67 (d, J J = 6.9 Hz, 1H), 7.64 (t, J J = 7.5 Hz, 2H), 3.55 (t, J J = 5.8 Hz, 4H), 3.01 (t, J J = 6.3 Hz, 2H), 2.89 (t, J J = 6.1 Hz, 2H), 2.74 (s, 3H), 2.01 – 1.96 (m, 2H), 1.92 (p, J J = 6.3 Hz, 2H).

[0074] Combined with the above characterization data, it can be seen that the dark red solid synthesized in Test 1 is a benzo[c]chromen-6-ium salt compound, and its chemical structural formula is as follows:

[0075] .

[0076] 1.2 Final product

[0077] In this experiment, the dark blue solid obtained in step (4) of Test Example 1 was further analyzed by nuclear magnetic resonance hydrogen spectrum, and the nuclear magnetic resonance hydrogen spectrum is as shown in Figure 1 the figure, and the analysis results are as follows:

[0078] 1 H NMR (600 MHz, DMSO- d 6) δ 8.30 (d, J J = 15.2 Hz, 1H), 8.19 – 8.15 (m, 2H), 8.12 (s, 1H), 8.06 (d, J 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).

[0079] Combined with the above characterization data, the chemical structural formula of the dark blue solid synthesized in Test 1 is as follows:

[0080] .

[0081] II. Fluorescence property test

[0082] 2.1. UV absorption performance

[0083] In this experiment, the finally synthesized dark blue solid product in Test Example 1 was used as a fluorescent probe (BZPN), and the UV absorption spectrum of this fluorescent probe was tested. Specifically, the experimental process included:

[0084] The fluorescent probe BZPN was dissolved in dimethyl sulfoxide (DMSO) solvent to prepare a stock solution with a concentration of 10 mM. 20 μL of the stock solution was diluted with DMSO to a working concentration of 10 μM. The solution was placed in a 1.0 cm quartz cell to measure the UV absorption performance. And the UV absorption spectrum as shown Figure 2 was plotted according to the experimental results.

[0085] Analysis Figure 2 The experimental data shows that the UV absorption spectrum of the product synthesized in this test example is in the range of 420 nm to 750 nm.

[0086] 2.2. Fluorescence spectrum test

[0087] In this experiment, the fluorescence spectrum of the 10 μM solution prepared in the aforementioned quartz cell was further tested. In the experiment, first, the emission spectrum generated by BZPN under the excitation of a 650 nm laser was tested, and the result is as shown Figure 3 . Then, the emission spectrum generated by BZPN under the excitation of a 760 nm laser was tested, and the result is as shown Figure 4 .

[0088] Pair Figure 3 And Figure 4 After comprehensive analysis, it was found that: under the condition of light excitation at 650 nm, the fluorescence spectrum generated by this fluorescent probe was 660 nm - 900 nm. And under the condition of light excitation at 760 nm, the generated fluorescence spectrum was 620 nm - 750 nm. Among them, Figure 3 The spectrum of Figure 4 Belongs to down-conversion fluorescence,

[0089] 2.3. Laser Power and Frequency Up-Conversion Fluorescence Performance

[0090] Combined with the frequency up-conversion fluorescence characteristics of BZPN in the aforementioned tests, this experiment further studied the influence of the emission power of the laser used for excitation on the emission peak intensity of the fluorescence of BZPN. The experimental process included:

[0091] First, dissolve the fluorescent probe BZPN in dimethyl sulfoxide solvent to prepare a stock solution with a concentration of 10 mM. Take 20 μL of the stock solution and dilute it to a working concentration of 10 μM with DMSO. Place the solution in a 1.0 cm quartz cell for up-conversion emission spectrum testing.

[0092] Among them, during each up-conversion emission spectrum test, fix the excitation wavelength at 760 nm and gradually increase the laser power of the laser light source. Test and record the up-conversion emission peak values of the fluorescence generated by BZPN under different laser power conditions. Fit the experimental data obtained from the test to obtain the Figure 5 Variation curve of the up-conversion luminescence intensity of BZPN with the excitation power as shown.

[0093] Analysis Figure 5 It can be found from the experimental data in that under the condition of fixing the laser wavelength at 760 nm, the up-conversion emission peak intensity of BZPN is positively correlated with the excitation light power, and there is an obvious linear relationship between the two, with a slope of 0.83. This also proves that the up-conversion emission process of BZPN provided in this experiment is a single-photon absorption process.

[0094] III. Biological Toxicity and Cell Imaging Effect

[0095] 3.1. Biological Toxicity of BZPN

[0096] 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:

[0097] First, prepare BZPN experimental solutions with concentrations of 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, and 5 μM respectively. Then, add BZPN solutions of various concentrations to multiple portions of Hela cells. After incubating for 24 h, measure the absorbance, and calculate the cytotoxicity of the fluorescent probe BZPN based on the absorbance.

[0098] Based on the above experimental results, draw a bar graph of the survival rate of Hela cells under the action of BZPN at different concentrations as shown in Figure 6 . Analyzing the experimental data in Figure 6 , it can be found that: compared with the cell activity without adding the fluorescent probe BZPN, after incubating with 1 - 5 μM of the fluorescent probe, the cell activity shows no obvious change basically. This indicates that the fluorescent probe BZPN provided by the present invention will not cause toxic damage to cells and can be applied to cell imaging.

[0099] 2.2 Imaging effect of BZPN

[0100] To verify the fluorescence imaging effect of BZPN provided by the present invention in cells, a co - localization experiment method was adopted in this experiment. In the experiment, a mitochondrial commercial dye (Mito - Tracker Green, excitation light is 490 nm, emission light collection range is 500 nm - 550 nm) with a significant difference in excitation light from BZPN of the present invention was selected as a control material to achieve performance calibration.

[0101] During the experiment, first, Hela cells were co - incubated with 0.5 μM of the fluorescent probe BZPN for 10 min, and then incubated with 0.5 μM of the mitochondrial commercial dye reagent for 20 min. Then, imaging was performed using a laser confocal microscope. Among them, the wavelength of the excitation light of the commercial dye is 490 nm; the down - conversion excitation light of BZPN is 650 nm, and the up - conversion excitation light is 760 nm.

[0102] During the experiment, Figure 7 (a) is the down - conversion fluorescence imaging of the fluorescent probe BZPN in cells, Figure 7 (b) is the up - conversion fluorescence imaging of the fluorescent probe BZPN in cells, Figure 7 (c) is the cell imaging diagram of the mitochondrial commercial dye.

[0103] Furthermore, overlay Figure 7 (a) with Figure 7 (c) to obtain Figure 7 (d), Figure 7 The Pearson correlation coefficient of the data in Figure 7 (a) and Figure 7 (c) is 0.92. Overlay Figure 7 (b) with Figure 7 (c) to obtain​Figure 7 (b) and Figure 7 The Pearson correlation coefficient with the data in (c) is 0.90.

[0104] Combined with Figure 7 the above experimental results in, it can be found that: whether BZPN provided by the present invention is excited by down-conversion at 650 nm or up-conversion at 760 nm, the fluorescence probe BZPN highly overlaps with the labeling results of the mitochondrial commercial staining cell diagram, which indicates that BAPN of the present invention can achieve a targeting effect equivalent to that of traditional commercial mitochondrial dyes, and has prominent practical value.

[0105] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within 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: Formula Ⅰ.

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 is 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 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 a benzopyrylium salt compound of formula II and p-dimethylaminobenzaldehyde of formula III into an organic solvent in proportion, and performing a Knoevenagel condensation reaction in a protective atmosphere to obtain a target product of formula I; Formula II, Formula III.

4. The method for preparing a fluorescent probe according to claim 3, wherein: 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.

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

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