A dual-ratio fluorescent / photoacoustic probe, and a preparation method and application thereof

Abstract

The application discloses a dual-ratio fluorescent / photoacoustic probe, a preparation method and application thereof, and the probe structure is shown in the following formula (II). The dual-ratio fluorescent probe provided by the application is a new type of single molecule, can realize precise quantification of the oxygen level of a tumor site during photothermal therapy, and has wide application prospects in precise tumor therapy as a therapy and prognosis evaluation integrated probe. The fluorescent probe has simple synthesis steps, convenient purification and high yield.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a dual-ratio fluorescent / photoacoustic probe and a preparation method and application thereof. BACKGROUND

[0002] With the development of imaging technology, the judgment of early cancer treatment effect monitoring and treatment evaluation by imaging methods is increasingly valued. However, at present, imaging detection often lacks molecular level information, which affects the accurate monitoring of treatment effect. It is urgent to develop new tumor imaging technology through molecular function visualization to realize early tumor treatment monitoring and prognosis evaluation. It is particularly important to design an effective method to accurately detect the hypoxic level before and after tumor treatment.

[0003] In recent years, although there are reports that magnetic resonance imaging nanometer hypoxic probes are used for tumor treatment monitoring, this method cannot evaluate early treatment response or reflect tumor treatment effect at the cellular level. Compared with the uncontrollability of nanometer hypoxic probes in the preparation process, the complexity of the mechanism in the body and the long-term toxicity, small molecule fluorescent hypoxic probes have become a research hotspot due to their simple and controllable synthesis, clear mechanism in the body and easy metabolism.

[0004] At present, tumor treatment and efficacy evaluation are carried out separately. However, due to the difference in probe and drug distribution, separate implementation of imaging and treatment procedures may cause reporting delay. So far, although some fluorescent probes for detecting hypoxia have been developed for real-time monitoring of hypoxic conditions in cells or living bodies; however, these hypoxic fluorescent probes do not have therapeutic function.

[0005] Therefore, it is of great significance for precise tumor treatment to rationally design a high-performance dual-ratio near-infrared fluorescent probe for tumor photothermal treatment and prognosis evaluation to realize accurate detection of the hypoxic level in tumor treatment and provide good prognosis evaluation. SUMMARY

[0006] The purpose of the present application is to provide a dual-ratio fluorescent / photoacoustic probe and a preparation method and application thereof, which can realize accurate quantification of the hypoxic level in tumor sites during photothermal treatment and is an integrated probe for tumor treatment and prognosis evaluation.

[0007] The technical solution for achieving the purpose of the present application is as follows:

[0008] The dual-ratio fluorescent / photoacoustic probe of the present application has the following structure shown in formula (II):

[0009]

[0010] In formula (II), R1 is one of the following S1-S5 structural formulas,

[0011]

[0012] R2 is one of C1-C20 alkyl, substituted alkyl;

[0013] The C1-C20 alkyl is a C1-C20 alkyl group including methyl, ethyl, propyl, butyl, pentyl, isopropyl, and tert-butyl; and the substituted alkyl is a C1-C20 alkyl derivative group including amino, carboxyl, azide, and hydrogen on the terminal carbon of substituted methyl, ethyl, propyl, butyl, pentyl, isopropyl, and tert-butyl.

[0014] The synthesis route of the dual-ratio fluorescent / photoacoustic probe is as follows:

[0015]

[0016]

[0017] The preparation method of the dual-ratio fluorescent / photoacoustic probe comprises the following steps:

[0018] (1) The cyanine dye containing R1 and R2 groups and 3-hydroxythiophenol are respectively dissolved in N,N-dimethylformamide, and after dissolution, the two are mixed uniformly, sodium hydride is added to the mixed solution, and stirring is performed under the conditions of nitrogen protection and 40 DEG C for 6-10 hours to obtain a crude product;

[0019] (2) The obtained crude product is eluted with an eluent and then purified by a silica gel chromatographic column to obtain a fluorescent dye containing R1 and R2 groups and having the structure shown in formula (I);

[0020] (3) The fluorescent dye having the structure shown in formula (I) obtained in step (2) is dissolved in N,N-dimethylformamide (DMF), and then 2-bromomethyl-5-nitro furan and potassium carbonate are added, and stirring is performed under the conditions of nitrogen protection and 40 DEG C for 12-18 hours;

[0021] (4) After the stirring reaction is completed, the reaction solution is poured into ice water and mixed, extracted with ethyl acetate, dried with anhydrous sodium sulfate, concentrated by a rotary evaporator, eluted with an eluent, and then purified by a silica gel chromatographic column to obtain a dual-ratio fluorescent / photoacoustic probe having the structure shown in formula (II).

[0022] Further, in the preparation method, the mass ratio of the cyanine dye containing R1 and R2 groups to 3-hydroxythiophenol in step (1) is 1:1, and the mass ratio to sodium hydride is 1:2.

[0023] Further, in the preparation method, the crude product in step (2) is eluted with dichloromethane and methanol in a volume ratio of 50:1 as the eluent, and purified by a 200-300 mesh silica gel chromatographic column.

[0024] Further, in the preparation method, the molar ratio of the fluorescent dye to 2-bromomethyl-5-nitrofuran in step (3) is 1:1, and the molar ratio of the fluorescent dye to potassium carbonate is 1:1.

[0025] Further, in the preparation method, the eluent in step (4) is eluted with dichloromethane and methanol in a volume ratio of 50:1 as the eluent, and purified by a 200-300 mesh silica gel chromatographic column.

[0026] In addition, the present application also claims to protect the dual-ratio fluorescent / photacoustic probe with the structure shown in formula (II) for detecting the change of the intracellular hypoxia level for non-disease diagnosis and treatment purposes, specifically for real-time dynamic quantitative detection of hypoxia in the process of photothermal treatment of living cells and tumors in vivo, to realize accurate detection of the hypoxia level, and tumor photothermal treatment and prognosis evaluation.

[0027] The application method of the dual-ratio fluorescent / photacoustic probe of the present application is as follows:

[0028] (1) Dissolve the probe with the structure shown in formula (II) in dimethyl sulfoxide (DMSO) to prepare a probe mother liquor;

[0029] (2) Add the probe mother liquor to the test liquid and biological sample;

[0030] (3) After adding nitroreductase (NTR) and reduced nicotinamide adenine dinucleotide disodium (NADH), observe the change of the fluorescence spectrum of the test liquid containing the probe dual-ratio fluorescent / photacoustic treatment and prognosis evaluation probe by using an ultraviolet and fluorescence spectrometer, culture the cells in an incubator with different oxygen contents (21%-1%) for 6 hours, incubate the cells with the probe for 30 minutes after the culture, and then take fluorescence images under a fluorescence microscope, so as to obtain fluorescence images of different oxygen contents in the cells;

[0031] (4) Incubate the cells in advance under anaerobic and aerobic conditions, incubate the cells for 30 minutes after adding the probe, incubate the cells for 15 minutes after adding green calcein and red propidium iodide, irradiate the cells with a laser, and obtain pictures by using an inverted fluorescence microscope, so as to obtain anaerobic and aerobic dead and live cell staining pictures;

[0032] (5) Use the probe for photothermal treatment of a 4T1 tumor mouse model, and use near-infrared ratio fluorescence imaging and ratio photoacoustic imaging to quantitatively detect tumor hypoxia, so as to obtain near-infrared ratio fluorescence imaging and ratio photoacoustic imaging pictures on the in vivo level.

[0033] The change of the fluorescence spectrum refers to the change of the fluorescence intensity at 648nm in the fluorescence spectrum. The fluorescence spectrum is observed by using a fluorescence spectrometer, and the excitation wavelength of the fluorescence spectrometer is 670nm. The obtained fluorescence imaging diagram refers to that the probe can perform good ratio imaging and tracking on different contents in cells.

[0034] Compared with the prior art, the advantages and beneficial effects of the present application are:

[0035] (1) The dual-ratio type fluorescence / photoacoustic probe provided by the present application is a new type of single molecule, which can realize precise quantification of the oxygen level of the tumor site during photothermal therapy, and has wide application prospects in precise tumor therapy as a therapy and prognosis evaluation integrated probe.

[0036] (2) The synthesis steps of the probe are simple, the purification is convenient, and the yield is high. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is the change of the titration ultraviolet spectrum of TDRSEP 1 prepared in Example 1 to NTR and its linear relationship fitting diagram; 1 H NMR spectrum;

[0038] Figure 2 is the change of the titration fluorescence spectrum of TDRSEP 1 prepared in Example 1 to NTR and its linear relationship fitting diagram; 13 C NMR spectrum;

[0039] Figure 3 is the change of the titration ultraviolet spectrum of TDRSEP 1 prepared in Example 1 to NTR and its linear relationship fitting diagram;

[0040] Figure 4 is the change of the titration fluorescence spectrum of TDRSEP 1 prepared in Example 1 to NTR and its linear relationship fitting diagram;

[0041] Figure 5 is the ratio fluorescence imaging diagram of TDRSEP 1 prepared in Example 1 to cells under different oxygen conditions;

[0042] Figure 6 is the dead and live cell staining imaging diagram of TDRSEP 1 prepared in Example 1 under normoxia and anaerobic laser irradiation;

[0043] Figure 7 is the dark toxicity and phototoxicity test result diagram of TDRSEP 1 prepared in Example 1 to normoxic and anaerobic cells;

[0044] Figure 8 is the near-infrared ratio fluorescence imaging prognosis evaluation test result diagram of TDRSEP 1 prepared in Example 1 in the treatment process of different groups of 4T1 tumor mouse models;

[0045] Figure 9Figure of the ratio photoacoustic imaging prognosis evaluation test results of TDRSEP 1 prepared in Example 1 in different groups of treatment process in 4T1 tumor mouse model;

[0046] Figure 10 Figure of the change of the titration UV spectrum of TDRSEP 2 prepared in Example 2 to NTR and its linear relationship fitting figure;

[0047] Figure 11 Figure of the change of the titration fluorescence spectrum of TDRSEP 2 prepared in Example 2 to NTR and its linear relationship fitting figure;

[0048] Figure 12 Figure of the change of the titration UV spectrum of TDRSEP 3 prepared in Example 3 to NTR and its linear relationship fitting figure;

[0049] Figure 13 Figure of the change of the titration fluorescence spectrum of TDRSEP 3 prepared in Example 3 to NTR and its linear relationship fitting figure;

[0050] Figure 14 Figure of the change of the titration UV spectrum of TDRSEP 4 prepared in Example 4 to NTR and its linear relationship fitting figure;

[0051] Figure 15 Figure of the change of the titration fluorescence spectrum of TDRSEP 4 prepared in Example 4 to NTR and its linear relationship fitting figure. DETAILED DESCRIPTION

[0052] The present application will be further described below in conjunction with the examples and the accompanying drawings, but the present application is not limited by the following examples.

[0053] Example 1

[0054] Preparation of the dual-ratio fluorescence / photoacoustic probe: R1 is selected as R2 is ethyl, the fluorescence probe is prepared, and is named as TDRSEP 1;

[0055] Preparation of TDRSEP 1:

[0056] (1) The cyanine dye containing R1 as R2 is ethyl (93 mg, 0.1 mmol) and 3-hydroxythiophenol (25 mg, 0.1 mmol) are respectively dissolved in N,N-dimethylformamide, after dissolution, the two are mixed uniformly, sodium hydride (4.8 mg, 0.2 mmol) is added in the mixed solution, stirring is carried out under the conditions of nitrogen protection and 40°C for 8 hours, and the obtained crude product is obtained;

[0057] (2) The crude product was eluted with dichloromethane and methanol in a volume ratio of 50:1, and purified by silica gel chromatography using a 200-300 mesh column to obtain a fluorescent dye containing R1 and R2 groups (molecular formula: C). 42 H 36 FN2O4S + );

[0058] (3) Take 40 mg (0.058 mmol) of the fluorescent dye from step (2) (molecular formula: C 42 H 36 FN2O4S + Place the solution in a round-bottom flask, add 8 mL of N,N-dimethylformamide to dissolve it completely, then add 11.8 mg (0.058 mmol) of 2-bromomethyl-5-nitrofuran (molecular formula: C5H4BrNO3) and 8 mg (0.058 mmol) of potassium carbonate, and stir at 40 °C for 15 hours under nitrogen protection.

[0059] (4) After the reaction was completed, the reaction solution was poured into ice water and mixed. It was extracted with ethyl acetate, dried with anhydrous sodium sulfate, concentrated by rotary evaporator, eluted with dichloromethane and methanol in a volume ratio of 50:1, and purified by silica gel chromatography column of 200-300 mesh to obtain 9.4 mg of blue-green solid.

[0060] The structural formula of the probe TDRSEP 1 prepared in Example 1 is as follows:

[0061]

[0062] Reference Figure 1 , 1 H NMR (600MHz, DMSO-d6) 1 H NMR(600MHz,DMSO-d6)δ8.52(d,J=8.8Hz,1H),8.44(d,J=14.6Hz,1H),8.37( d,J=8.9Hz,1H),8.16(d,J=8.9Hz,1H),8.10(d,J=8.9Hz,1H),8.00(dd,J=8.6 ,5.5Hz,2H),7.80(d,J=3.7Hz,1H),7.69(d,J=8.7Hz,1H),7.57(d,J=2.5Hz, 1H),7.46–7.42(m,3H),7.32(d,J=11.1Hz,1H),7.24(d,J=11.2Hz,1H),7.14.

[0063] Reference Figure 2 , 13C NMR (150 MHz, DMSO-d6) δ 175.04, 153.70, 147.60, 144.32, 139.26, 137.14, 133.51, 130.13, 129.52, 129.46, 126.57, 124.81, 123.90, 123.82, 116.59, 116.45, 114.89, 114.10, 113.57, 110.34, 62.35, 34.11, 31.76, 29.47, 24.95, 22.56, 14.43. HRMS (ESI) m / z calcd for C 42 H 36 FN2O4S + ([M] + ): 683.2374; found 683.2371.

[0064] Spectral experiment of the probe TDRSEP 1 prepared in Example 1 for NTR response:

[0065] The DMSO stock solution of the fluorescent probe prepared in Example 1 was configured with a concentration of 1 mM; then 1970 μL of liquid (PBS:DMSO = 9:1) and 30 μL of probe stock solution were added in the ultraviolet and fluorescence dish respectively, NTR and NADH were added in the dish until the ultraviolet and absorption of the compound no longer changed. When 5 μg / mL of NTR was added, the ultraviolet spectrum of the compound was found to be red-shifted by 80 nm, and the fluorescence spectrum was red-shifted by 46 nm, which indicated that the probe could be used as a ratiometric fluorescent / photacoustic probe for nitroreductase, and the results are shown in Figure 3 、 Figure 4 .

[0066] Ratiometric fluorescence imaging experiment of the probe TDRSEP 1 prepared in Example 1 on cells under different oxygen content:

[0067] The DMSO stock solution of the fluorescent probe prepared in Example 1 was configured with a concentration of 1 mM. 1 mL of cell culture solution was added in a confocal dish treated in advance with different oxygen content (21%, 15%, 10%, 5%, 1%), 5 μL of probe stock solution was added and incubated in a cell incubator for 30 min, and then washed with PBS for 2-3 times; then the fluorescence imaging of the cells was performed by a two-photon laser microscope, and the excitation wavelength of the two channels was 638 nm, the collection range of the green channel was 648-730 nm, and the collection range of the red channel was 750-800 nm, and the results are shown in Figure 5 .

[0068] Fluorescence imaging experiment of the probe TDRSEP 1 prepared in Example 1 and calcein (green) and propidium iodide (red) on cells:

[0069] The fluorescent probe prepared in Example 1 was formulated in DMSO with a concentration of 1 mM. 1 mL of cell culture solution was added to a confocal dish which was pre-treated with normoxia and anoxia, and 10 μL of the probe mother liquor and 0.5 μL of calcein (green) and propidium iodide (red) (1 mM) were added. After incubation in a cell incubator for 30 min, the cells were washed with PBS for 2-3 times. Subsequently, the cells were imaged under an inverted microscope after laser irradiation for 5 min, and the results are shown in Figure 6 , which show that the probe can not only be used as a ratiometric fluorescent probe to quantitatively detect the nitroreductase content in tumor cells under different hypoxic conditions, but also has good potential for photothermal treatment of cancer under hypoxic conditions.

[0070] Cell dark toxicity and phototoxicity test experiment of the probe TDRSEP 1 prepared in Example 1 under normoxic and anoxic conditions:

[0071] The digested HeLa (human cervical cancer) cell suspension was inoculated in a 96-well culture plate at a density of 1 x 10 5 cells per well in 180 μL -1 , and was placed in a cell incubator for 24 hours. When the cell density was observed to be 80-90% under a microscope, 20 μL of TDRSEP 1 (0, 5, 10, 15, 20 and 25 μM) was added to each well, and the cells were cultured under normoxic and anoxic conditions for 6 hours, respectively. After the culture, laser irradiation groups and non-laser irradiation groups were set up, and then MTT reagent (10 μL, 5 mg / mL MTT) was uniformly added and incubated for 4-6 hours. The cell supernatant was removed, 100 mL of DMSO was added to each well, and the plate was placed on a shaker for low-speed shaking for 10-15 min to fully dissolve the purple formazan crystals. The absorbance of formazan at 570 nm was tested by an enzyme-labeled instrument, and the cell survival rate was calculated. The cell survival rate was expressed as the percentage of the average value of the experimental group to the average value of the blank group. The results are shown in Figure 7 , which show that the probe has good cell dark toxicity under normoxic conditions without light irradiation, and good phototoxicity under hypoxic conditions with light irradiation, and can specifically kill cells.

[0072] Prognosis evaluation test experiment of near-infrared ratiometric fluorescence imaging of the probe TDRSEP 1 prepared in Example 1 in different groups of 4T1 tumor mouse models during treatment:

[0073] Mouse breast cancer cells (4T1 cells) were injected into mice to form subcutaneous breast tumors. The mice were randomly divided into 4 groups (5 mice in each group) to receive different treatments. The probe TDRSEP 1 and normal saline were injected in situ into the tumor, and the mice after treatment were irradiated with a laser for 5 min at 1 h after injection, and then imaged by a small animal live fluorescence imager, and the results are shown in Figure 8As shown, the results show that the probe has a very strong inhibitory effect on tumors under light conditions, and the change in two-channel fluorescence intensity is not much different from that of normal tissues.

[0074] Prognosis evaluation test experiment of ratio photoacoustic imaging of the probe TDRSEP 1 prepared in Example 1 in the treatment process of different groups of 4T1 tumor mouse models:

[0075] Mouse breast cancer cells (4T1 cells) were injected into mice to form subcutaneous breast tumors. The mice were randomly divided into 4 groups (5 mice in each group) to receive different treatments. The probe TDRSEP 1 and normal saline were injected in situ into the tumor, and the mice after treatment were irradiated with laser for 5 minutes 1 hour after injection, and then imaged with a multispectral photoacoustic tomography instrument, and the results are shown in Figure 9 As shown, the photoacoustic imaging results are consistent with the fluorescence imaging results.

[0076] Example 2

[0077] Preparation of a double-ratio fluorescence / photoacoustic probe: R1 is selected as R2 is ethyl, a fluorescent probe is prepared, named TDRSEP 2, and the preparation method is the same as Example 1 except that the R1 and R2 groups of the cyanine dye are different from Example 1;

[0078] The structural formula of the probe TDRSEP 2 prepared in Example 2 is:

[0079]

[0080] Spectral experiment of the probe TDRSEP 2 prepared in Example 2 in response to NTR, according to the experimental method of Example 1, the results are shown in Figure 10 、 Figure 11 As shown, when 5 μg / mL of NTR is added, the ultraviolet spectrum of the compound is red-shifted by 100 nm, and the fluorescence spectrum is red-shifted by 38 nm, which shows that the probe TDRSEP 2 can also be used as a nitroreductase ratio fluorescence / photoacoustic probe, with similar performance to TDRSEP 1.

[0081] Other experiments of the probe TDRSEP 2 were carried out according to the experimental method of Example 1.

[0082] Example 3

[0083] Preparation of a double-ratio fluorescence / photoacoustic probe: R1 is selected as R2 is ethyl, a fluorescent probe is prepared, named TDRSEP 3, and the preparation method is the same as Example 1 except that the R1 and R2 groups of the cyanine dye are different from Example 1;

[0084] The structural formula of the probe TDRSEP 3 prepared in Example 3 is:

[0085]

[0086] The spectral experiment of the probe TDRSEP 3 prepared in Example 3 to NTR response was carried out according to the experimental method of Example 1, and the results are shown in Figure 12 、 Figure 13 When 5 μg / mL of NTR was added, it was found that the ultraviolet spectrum of the compound was red-shifted by 81 nm, and the fluorescence spectrum was red-shifted by 46 nm, which indicated that the probe TDRSEP 3 could also be used as a ratio fluorescence / photacoustic probe for nitroreductase, and had similar performance to TDRSEP 1.

[0087] Other experiments of the probe TDRSEP 3 were all carried out according to the experimental method of Example 1.

[0088] Example 4

[0089] Preparation of a double-ratio fluorescence / photacoustic probe: R1 is selected as R2 is ethyl, a fluorescence probe is prepared, named as TDRSEP 4, and the preparation method thereof is the same as that of Example 1 except that the groups of the cyanine dye R1 and R2 are different from those of Example 1.

[0090] The structural formula of the probe TDRSEP 4 prepared in Example 4 is:

[0091]

[0092] The spectral experiment of the probe TDRSEP 4 prepared in Example 4 to NTR response was carried out according to the experimental method of Example 1, and the results are shown in Figure 14 、 Figure 15 When 5 μg / mL of NTR was added, it was found that the ultraviolet spectrum of the compound was red-shifted by 100 nm, and the fluorescence spectrum was red-shifted by 50 nm, which indicated that the probe TDRSEP 4 could also be used as a ratio fluorescence / photacoustic for nitroreductase, and had similar performance to TDRSEP 1.

[0093] Other experiments of the probe TDRSEP 3 were all carried out according to the experimental method of Example 1.

[0094] Example 5

[0095] Preparation of a double-ratio fluorescence / photacoustic probe: R1 is selected as R2 is ethyl, a fluorescence probe is prepared, named as TDRSEP 5, and the preparation method thereof is the same as that of Example 1 except that the groups of the cyanine dye R1 and R2 are different from those of Example 1.

[0096] The structural formula of the probe TDRSEP 5 prepared in Example 5 is:

[0097]

[0098] The probe TDRSEP 5 prepared in Example 5 was used in all experiments, and the experimental method was the same as that in Example 1.

[0099] The probes prepared in Examples 1-5 were proved by experiments to have the function of realizing accurate quantification of the oxygen level at the tumor site in photothermal therapy, and are integrated probes for tumor treatment and prognosis evaluation, and have wide application prospects in accurate treatment of tumors.

[0100] Finally, it should be noted that the above examples do not limit the present application in any form. For those skilled in the art, some modifications and improvements can be made on the basis of the present application. Therefore, any modification or improvement made without departing from the spirit of the present application shall fall within the scope of the present application.

Claims

1. A dual-ratio type fluorescent / photoacoustic probe, characterized by, The probe has a cation as shown in the structural formula of TDRSEP 1, TDRSEP 2, TDRSEP 3, TDRSEP 4 or TDRSEP 5; The structural formula of TDRSEP 1 is as follows: ； The structural formula of TDRSEP 2 is as follows: ； The structural formula of TDRSEP 3 is as follows: ； The structural formula of TDRSEP 4 is as follows: ； The structural formula of TDRSEP 5 is as follows: 。 2. A method of preparing the dual-ratio type fluorescent / photoacoustic probe according to claim 1, characterized by, The synthesis route of the probe is as follows: ； (Ⅰ) ； (Ⅰ) (Ⅱ) The preparation method comprises the following steps: (1) The cyanine dye containing R1 as , R2 as ethyl group and 3-hydroxythiophenol are dissolved in N,N-dimethylformamide respectively, and then mixed uniformly. Sodium hydride is added into the mixture, and the mixture is stirred under nitrogen protection at 40°C for 6-10 hours to obtain the crude product. (2) The obtained crude product is eluted with an eluent and then purified by a silica gel chromatographic column to obtain a fluorescent dye containing R1 and R2 groups; (3) The fluorescent dye obtained in step (2) is dissolved in N,N-dimethylformamide, and then 2-bromomethyl-5-nitrofuran and potassium carbonate are added, and the reaction is stirred under the protection of nitrogen and at 40°C for 12-18 hours; (4) After the stirring reaction is completed, the reaction liquid is poured into ice water and mixed, extracted with ethyl acetate, dried with anhydrous sodium sulfate, concentrated by a rotary evaporator, eluted with an eluent, and then purified by a silica gel chromatographic column to obtain a dual-ratio fluorescent / photoacoustic probe TDRSEP 1; The preparation method of the TDRSEP 2 is same as that of the TDRSEP 1 except that the flower cyanine dye R1 is , R2 is ethyl; and the rest is same as that of the TDRSEP 1. The preparation method of the TDRSEP 3 is same as that of the TDRSEP 1 except that the flower cyanine dye R1 is , R2 is ethyl, and the rest is same as that of the TDRSEP 1; The preparation method of the TDRSEP4 is same as that of the TDRSEP1 except that the cyanine dye R1 is , R2 is ethyl, and the rest is same as that of the TDRSEP1. The preparation method of the TDRSEP5 is same as that of the TDRSEP1 except that the cyanine dye R1 is , R2 is ethyl.

3. The method of claim 2, wherein: In step (1), the molar ratio of the fluorin dye containing R1 and R2 groups to 3-hydroxythiophenol is 1:1, and the molar ratio to sodium hydride is 1:

2.

4. The method of claim 2, wherein: In step (2), the crude product is eluted with an eluent of dichloromethane and methanol in a volume ratio of 50:1, and purified by a silica gel chromatographic column with a mesh size of 200-300.

5. The method of claim 2, wherein: In step (3), the molar ratio of the fluorescent dye to 2-bromomethyl-5-nitrofuran is 1:1, and the molar ratio to potassium carbonate is 1:

1.

6. The method of claim 2, wherein: In step (4), the eluent is an eluent of dichloromethane and methanol in a volume ratio of 50:1, and the purification is performed by a silica gel chromatographic column with a mesh size of 200-300.

7. Use of the dual-ratio fluorescent / photoacoustic probe of claim 1, characterized in that, The probe is applied to the detection of changes in the degree of intracellular hypoxia for non-disease diagnosis and treatment purposes.

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