阅读说明：本技术 一种检测cyp 1a1酶的近红外荧光探针 (Near-infrared fluorescent probe for detecting CYP 1A1 enzyme ) 是由 祁争健 代艳鹏 薛珂 赵鑫鑫 于 2021-05-28 设计创作，主要内容包括：本发明公开了一种检测CYP 1A1酶的近红外荧光探针,该荧光探针通过对分子结构进行修饰,使分子结构能够实现对CYP 1A1酶的特异性检测,从而具有高选择性；同时荧光探针中的荧光基团发出的荧光波长在近红外区域,因此在进行荧光检测时能够避免受生物体发绿光的干扰；其次,BrDXMM荧光性能很弱,而在相同条件下HDXMM具有良好的荧光发射光谱特性(590-800nm),从而能很好的进行区分,因此本发明荧光探针在体内外均具有高的信噪比；并且本发明荧光探针还具有良好的生物相容性,用含有BrDXMM(100μM)的培养基孵育细胞24小时,细胞活度仍大于85％；本发明荧光探针可用于溶液中重组单酶、活细胞、离体组织、活斑马鱼及荷瘤裸鼠体内CYP 1A1酶活性的测定,应用范围广。(The invention discloses a near-infrared fluorescent probe for detecting CYP 1A1 enzyme, which modifies a molecular structure to enable the molecular structure to realize specific detection of CYP 1A1 enzyme, thereby having high selectivity; meanwhile, the fluorescent wavelength emitted by the fluorescent group in the fluorescent probe is in a near infrared region, so that the interference of green light emitted by an organism can be avoided during fluorescence detection; secondly, the fluorescence property of BrDXMM is very weak, and HDXMM has good fluorescence emission spectral characteristics (590-800nm) under the same condition, so that the HDXMM can be well distinguished, and therefore, the fluorescent probe has high signal-to-noise ratio both inside and outside the body; the fluorescent probe also has good biocompatibility, and the cell activity is still more than 85 percent after the cell is incubated for 24 hours by using a culture medium containing BrDXMM (100 mu M); the fluorescent probe can be used for measuring the activity of the CYP 1A1 enzyme in recombinant single enzyme, living cells, isolated tissues, living zebra fish and tumor-bearing nude mice in a solution, and has wide application range.)

1. A near-infrared fluorescent probe for detecting CYP 1A1 enzyme, which is characterized in that: the structural formula of the fluorescent probe is as follows:

2. the near-infrared fluorescent probe for detecting a CYP 1a1 enzyme according to claim 1, wherein: the detection process of the fluorescent probe for CYP 1A1 enzyme in vitro cells is as follows: dissolving a fluorescent probe in a PBS (phosphate buffer solution) aiming at in-vitro cells, wherein the pH value of the PBS buffer solution is 7.4-7.5, the temperature of the solution is 37 ℃, and the concentration of the fluorescent probe in the PBS buffer solution is 10 mu M; the cells HepG2 or LO2 live cells were incubated with a medium containing a fluorescent probe, and the amount of a hydrolysate produced by the reaction of the fluorescent probe with CYP 1A1 enzyme in the cells after 60 minutes was measured as an index for evaluating the activity of CYP 1A1 enzyme.

3. The near-infrared fluorescent probe for detecting a CYP 1a1 enzyme according to claim 1, wherein: the detection process of the fluorescent probe for the CYP 1A1 enzyme in the zebra fish body comprises the following steps: and (2) incubating the zebra fish with a culture solution containing a fluorescent probe at room temperature, wherein the concentration of the fluorescent probe in the culture solution is 10 mu M, and measuring the generation amount of a hydrolysate generated by the reaction of the fluorescent probe in the zebra fish and the CYP 1A1 enzyme after 60 minutes as an index for evaluating the activity of the CYP 1A1 enzyme in the zebra fish.

4. The near-infrared fluorescent probe for detecting a CYP 1a1 enzyme according to claim 1, wherein: the detection process of the fluorescent probe to the CYP 1A1 enzyme in the cells in the tumor-bearing nude mouse body is as follows: preparing normal saline containing a fluorescent probe, injecting the normal saline into a tumor in a mouse body, wherein the concentration of the fluorescent probe in the normal saline is 20 mu M, and measuring the generation amount of a hydrolysate generated by the reaction of the fluorescent probe and CYP 1A1 enzyme in a cell after 60 minutes as an evaluation index of the CYP 1A1 enzyme activity.

5. The method for preparing a near-infrared fluorescent probe for detecting a CYP 1A1 enzyme according to claim 1, wherein said method comprises: placing 2- ((6-hydroxy-2, 3-dihydro-1H-flavone-4-yl) methylene) malononitrile (HDXMM), potassium carbonate and 4- (2-bromoethoxy) benzyl bromide in a reaction bottle, adding N, N-dimethylformamide into the reaction bottle, heating the mixed material to be not less than 90 ℃ under the protection of nitrogen, cooling to room temperature after reaction, adding the reaction liquid into cold water, violently stirring, precipitating a large amount of solid, filtering, and separating a filter cake by adopting a column chromatography.

6. The method for preparing a near-infrared fluorescent probe for detecting a CYP 1A1 enzyme according to claim 5, wherein: the mixing molar ratio of the 2- ((6-hydroxy-2, 3-dihydro-1H-flavone-4-yl) methylene) malononitrile (HDXMM), the potassium carbonate and the 4- (2-bromoethoxy) benzyl bromide is 1: 2: 1.2.

7. the method for preparing a near-infrared fluorescent probe for detecting a CYP 1A1 enzyme according to claim 5, wherein: the reaction time is at least 1 hour.

Technical Field

The invention relates to a near-infrared fluorescent probe for detecting CYP 1A1 enzyme.

Background

Cytochrome P450 enzymes (CYP450) are a ferroheme-containing superfamily of monooxygenases, the most important metabolic enzymes in the body. CYP450 enzymes are involved in biotransformation of many endogenous compounds, including carcinogens, chemicals, drugs, and play an important role in maintaining normal physiological functions of the organism. CYP450 enzymes catalyze the phase reaction of the I-phase, which is a key step in the human body's metabolism of exogenous and endogenous compounds, and is generally the rate-limiting step in the clearance of drugs from the body, and is critical to the half-life, clearance, etc. of the compounds. However, there is a great individual variability in the distribution of CYP 1a1, and the expression of CYP 1a1 enzyme is closely related to factors such as genetics, sex, age, disease, environment, and co-drug administration. To understand the expression of the CYP 1a1 enzyme in different biological samples, fluorescence imaging technology is one of the most powerful tools. The existing fluorescent probes capable of detecting the CYP 1A1 enzyme basically emit green light, and because organisms emit green light, the interference problem can occur during fluorescence imaging, and meanwhile, the fluorescent probes can simultaneously detect a plurality of subtypes of cytochrome P450 enzymes, such as CYP 1A1 and CYP 1A2, so that the selectivity is not high, and the specific selection of the CYP 1A1 enzyme cannot be realized.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems of organism interference and low selectivity in green light emission detection of the CYP 1A1 enzyme fluorescent probe in the prior art, the invention provides the near-infrared fluorescent probe for detecting the CYP 1A1 enzyme, wherein the fluorescent probe modifies the structure, so that the molecular structure can realize the specific detection of the CYP 1A1 enzyme, and the fluorescent wavelength emitted by the fluorescent group in the fluorescent probe is in a near-infrared region, thereby avoiding the interference of green light emission of organisms.

The technical scheme is as follows: the invention relates to a near-infrared fluorescent probe for detecting CYP 1A1 enzyme, which has a structural formula as follows:

the specific process for detecting the CYP 1A1 enzyme activity by the fluorescent probe comprises the following steps: the BrDXMM hydrolysis reaction catalyzed by CYP 1A1 enzyme is selected as a probe reaction, and the actual activity of the CYP 1A1 enzyme in living cells, living bodies and isolated tissues is evaluated by detecting the generation amount of a hydrolysis product 2- ((6-hydroxy-2, 3-dihydro-1H-flavone-4-yl) methylene) malononitrile (HDXMM) in a specific time. The fluorescent properties of the enzymatic reaction probe substrate BrDXMM and the hydrolysate HDXMM are obviously different under the same condition, so that the BrDXMM can evaluate the activity and distribution of CYP 1A1 enzyme in various biological systems.

Wherein the detection process of the fluorescent probe for CYP 1A1 enzyme in vitro cells is as follows: dissolving a fluorescent probe in a PBS (phosphate buffer solution) aiming at in-vitro cells, wherein the pH value of the PBS buffer solution is 7.4-7.5, the temperature of the solution is 37 ℃, and the concentration of the fluorescent probe in the PBS buffer solution is 10 mu M; the cells HepG2 or LO2 live cells were incubated with a medium containing a fluorescent probe, and the amount of a hydrolysate produced by the reaction of the fluorescent probe with CYP 1A1 enzyme in the cells after 60 minutes was measured as an index for evaluating the activity of CYP 1A1 enzyme.

The detection process of the fluorescent probe for the CYP 1A1 enzyme in the zebra fish body comprises the following steps: and (2) incubating the zebra fish with a culture solution containing a fluorescent probe at room temperature, wherein the concentration of the fluorescent probe in the culture solution is 10 mu M, and measuring the generation amount of a hydrolysate generated by the reaction of the fluorescent probe in the zebra fish and the CYP 1A1 enzyme after 60 minutes as an index for evaluating the activity of the CYP 1A1 enzyme in the zebra fish.

Wherein the detection process of the fluorescent probe for CYP 1A1 enzyme in cells in a tumor-bearing nude mouse body is as follows: preparing normal saline containing a fluorescent probe, injecting the normal saline into a tumor in a mouse body, wherein the concentration of the fluorescent probe in the normal saline is 20 mu M, and measuring the generation amount of a hydrolysate generated by the reaction of the fluorescent probe and CYP 1A1 enzyme in a cell after 60 minutes as an evaluation index of the CYP 1A1 enzyme activity.

The preparation method of the near-infrared fluorescent probe for detecting CYP 1A1 enzyme comprises the steps of placing 2- ((6-hydroxy-2, 3-dihydro-1H-flavone-4-yl) methylene) malononitrile (HDXMM), potassium carbonate (serving as hydrogen bromide generated by alkali neutralization reaction) and 4- (2-bromoethoxy) benzyl bromide into a reaction bottle, adding N, N-dimethylformamide (serving as a solvent in the reaction system) into the reaction bottle, heating the mixed material to be not less than 90 ℃ under the protection of nitrogen, cooling to room temperature after reaction, adding the reaction liquid into cold water, violently stirring, precipitating a large amount of solids, filtering, and separating a filter cake by adopting a column chromatography method.

Wherein the mixing molar ratio of the 2- ((6-hydroxy-2, 3-dihydro-1H-flavone-4-yl) methylene) malononitrile (HDXMM), the potassium carbonate and the 4- (2-bromoethoxy) benzyl bromide is 1: 2: 1.2.

wherein the reaction time is at least 1 hour.

The specific molecular structure of the fluorescent probe 2- ((6- ((4- (2-bromoethoxy) benzyl) oxy) -2, 3-dihydro-1H-xanthine-4-yl) methylene) malononitrile (BrDXMM) only can enter the enzyme cavity of CYP 1A1 and can perform enzyme-catalyzed oxidative hydrolysis reaction with the enzyme cavity, so that the specific detection of CYP 1A1 enzyme can be realized, the carbon-oxygen bond of 2-bromoethoxy in the 2- ((6- ((4- (2-bromoethoxy) benzyl) oxy) -2, 3-dihydro-1H-xanthine-4-yl) methylene) malononitrile can be hydrolyzed by the CYP 1A1 enzyme, and then a hydrolysis product 2- ((6-hydroxy-2 is generated through intramolecular 1, 6-elimination reaction, 3-dihydro-1H-flavone-4-yl) methylene) malononitrile (HDXMM), which upon photoexcitation produces an intense fluorescence emission; the activity of the CYP 1a1 enzyme was measured in different biological samples by measuring the amount of HDXMM produced over time.

The reaction formula is as follows:

has the advantages that: 1. the fluorescent probe disclosed by the invention has the advantages that the molecular structure is modified, so that the molecular structure can realize the specific detection of CYP 1A1 enzyme, and the fluorescent probe has high selectivity; 2. the fluorescent wavelength emitted by the fluorescent group in the fluorescent probe is in the near infrared region, so that the interference of green light emitted by organisms can be avoided during fluorescence detection; 3. the fluorescence property of BrDXMM is very weak, and HDXMM has good fluorescence emission spectral characteristics (590-800nm) under the same condition, so that the HDXMMM can be well distinguished, and therefore, the fluorescent probe has high signal-to-noise ratio both inside and outside a body; 4. the fluorescent probe has good biocompatibility, and the cell activity is still more than 85 percent after the cell is incubated for 24 hours by using a culture medium containing BrDXMM (100 mu M); 5. the fluorescent probe can be used for measuring the activity of the CYP 1A1 enzyme in recombinant single enzyme, living cells, isolated tissues, living zebra fish and tumor-bearing nude mice in a solution, and has wide application range.

Drawings

FIG. 1 is a fluorescent selective response of BrDXMM to a single enzyme;

FIG. 2 is a graph showing the comparison of fluorescence intensity of BrDXMM reacted with different enzymes;

FIG. 3 is a graph showing inhibition of CYP 1A1 enzyme activity by quinophthalol;

FIG. 4 shows cytotoxicity of BrDXMM on HepG2 and LO 2;

FIG. 5 is fluorescence imaging of BrDXMM in HepG2 cells;

FIG. 6 is fluorescence imaging of BrDXMM in LO2 cells;

FIG. 7 is time-dependent fluorescence imaging of BrDXMM in HepG2 cells;

FIG. 8 is time-dependent fluorescence imaging of BrDXMM in LO2 cells;

FIG. 9 is fluorescence imaging of BrDXMM in zebrafish;

FIG. 10 is time-dependent fluorescence imaging of BrDXMM in live tumor-bearing nude mice;

fig. 11 is fluorescence imaging of BrDXMM in ex vivo tissues.

Detailed Description

The technical solution of the present invention is further described with reference to the following specific embodiments.

Example 1

The fluorescent probe 2- ((6- ((4- (2-bromoethoxy) benzyl) oxy) -2, 3-dihydro-1H-xanthine-4-yl) methylene) malononitrile (BrDXMM) is prepared by the following method:

1mmol of HDXMM (the HDXMM of the invention is prepared by the synthetic method disclosed in the following document: Sensors&The activators B.chemical 273(2018)167-Adding 8mL of N, N-dimethylformamide into the mouth bottle, heating the reaction materials to 90 ℃ under the protection of nitrogen, and reacting and stirring for 1 hour; after the reaction, the reaction solution was cooled to room temperature, and the reaction solution was added to cold water, vigorously stirred to precipitate a large amount of dark red solid, filtered, and the filter cake was separated by column chromatography (developing solvent: ethyl acetate: petroleum ether: 5:1, v: v) to obtain dark red solid (yield 65.2%).¹H NMR(600MHz,DMSO)δ8.17(s,1H),7.44(d,J＝8.6Hz,1H),7.41(d,J＝8.7Hz,2H),7.36–7.34(m,2H),7.02–6.98(m,2H),6.93(dd,J＝8.6,2.4Hz,1H),5.11(s,2H),4.28–4.25(m,2H),3.96–3.94(m,2H),2.74(t,J＝6.0Hz,2H),2.63–2.59(m,2H),1.75(dt,J＝12.2,6.2Hz,2H).¹³C NMR(150MHz,DMSO)δ161.14,159.19,157.95,153.66,149.83,132.27,129.81,128.63,128.43,125.75,117.71,116.22,114.75,114.60,113.79,109.24,101.42,69.70,67.95,43.08,28.04,24.24,19.99.

Example 2: determination of the selectivity of the fluorescent Probe BrDXMM for each of the CYP450 subtypes

Preparation of Phosphate Buffer Solution (PBS): sodium dihydrogen phosphate dihydrate (0.7410g), disodium hydrogen phosphate dodecahydrate (7.2495g) and magnesium chloride hexahydrate (0.2030g) are weighed by an analytical balance and placed in a 100mL beaker, secondary distilled water (200 mL in total) is added into the beaker for 4 times to be fully dissolved, the beaker is placed still and transferred into a 250mL volumetric flask, the secondary distilled water is added to the volumetric flask for constant volume, the flask is fully and uniformly shaken, and then a pH meter is used for checking the pH value of the buffer solution. PBS buffer (0.1M, pH 7.4, c (MgCl)₂) 4.0mM), stored at 4 ℃.

Preparation of reaction substrate solution: BrDXMM was weighed and dissolved in dimethyl sulfoxide to prepare a mother liquor with a concentration of 5 mM. mu.L of the stock solution was added to 4.96mL of PBS buffer solution to prepare a 40. mu.M reaction substrate solution.

Incubation system NADPH reduction system configuration: β -nicotinamide adenine dinucleotide phosphate (1.0 mM); glucose-6-phosphate (10.0 mM); glucose-6-phosphate dehydrogenase (1.0 unit/mL); all three of the above accessory enzymes were 0.1M PBS buffer (0.1M, pH 7.4, c (MgCl)₂) 4.0mM) was dissolved. CYP450 monoase (CYP 1A1, CYP 1A2, CYP 2A6, CYP 2C9, CYP 2C19, CYP 2D6, CYP 2E1, CYP 2J2, CYP 3A4 orCYP 3A5)。

100. mu.L of the above-mentioned glucose-6-phosphate solution, 50. mu.L of the above-mentioned β -nicotinamide adenine dinucleotide phosphate solution, 5. mu.L of the glucose-6-phosphate dehydrogenase solution, and 5. mu.L of the CYP monoose (final concentration: 50nM) and 10. mu.L of the reaction substrate solution (final concentration: 10. mu.M in the incubation system for DXMM) were added to the incubation system having a final volume of 400. mu.L, respectively, and finally 30. mu.L of the PBS buffer was added. The mixture was then mixed and incubated for 60 minutes at 37 ℃ in a constant temperature water bath. At the end of the incubation, dimethyl sulfoxide (200. mu.L) was added to the reaction system to terminate the reaction and mixed well. The mixture was centrifuged at low temperature (13,300 Xg, 4 ℃) for 20 minutes, and the supernatant was used for fluorescence analysis, and it can be seen from FIG. 1 that the fluorescent probe BrDXMM of the present invention has high selectivity for CYP 1A 1. As can be seen from FIG. 2, the fluorescent probe BrDXMM of the present invention has higher contrast in response to CYP 1A1 than other subtypes.

Example 3: chemical inhibition experiment of fluorescent probe BrDXMM

100 mu L of the glucose-6-phosphate solution, 50 mu L of the beta-nicotinamide adenine dinucleotide phosphate solution, 5 mu L of the glucose-6-phosphate dehydrogenase solution, 5 mu L of CYP 1A1 (final concentration is 50nM) and resveratrol (final concentration is 25 mu M and CYP 1A1 specific inhibitor) are respectively added into an incubation system with the final volume of 400 mu L, 10 mu L and 40 mu M of BrDXMM (final concentration is 10 mu M) are added after mixing, and finally PBS buffer solution is used for diluting to the responding concentration. The mixture was then mixed and incubated for 60 minutes at 37 ℃ in a constant temperature water bath. At the end of the incubation, dimethyl sulfoxide (200. mu.L) was added to the reaction system to terminate the reaction and mixed well. The mixture was centrifuged at low temperature (13,300 Xg, 4 ℃) for 20 minutes, and the supernatant was used for fluorescence analysis, and it can be seen from FIG. 3 that the fluorescent probe BrDXMM of the present invention indeed produces a fluorescent response due to the oxidative hydrolysis of CYP 1A 1.

Example 4: cytotoxicity detection of fluorescent probe BrDXMM

Cell activity was determined by the MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) method. HepG2 cells (human hepatoma cells) and LO2 cells (human normal hepatocytes) were first seeded into 96-well culture plates, respectively, and cultured in the presence of 10% Fetal Bovine Serum (FBS)Base (100. mu.L) and at 5% CO₂Incubated at 37 ℃ for 24 hours. The medium for HepG2 and LO2 cells was changed to medium (100. mu.L) containing various concentrations of BrDXMM (0,5,10,20,30,40,80, 100. mu.M) and incubation was continued for 24 hours. The medium was aspirated and the cells were washed with PBS (100. mu.L), and 100. mu.L of medium containing 0.05% MTT was added to each well and incubation was continued for 4 hours. After incubation, the medium was removed and 150 μ L of dimethyl sulfoxide was added to each well and the formazan formed in the wells was dissolved by shaking at low speed for 10 minutes. Finally, the absorbance of each well at 490nm was measured with a microplate reader and compared with the absorbance of the control group (100% survival rate) to obtain the relative survival rate of the cells at different concentrations of the fluorescent probe, as can be seen from FIG. 4, the fluorescent probe of the present invention has good biocompatibility.

Example 5: fluorescent probe BrDXMM cell fluorescence imaging experiment

HepG2 or LO2 cells were seeded into confocal imaging cell culture dishes, 1mL of the corresponding medium (containing 10% fetal bovine serum) was added, and the mixture was incubated in an incubator (5% CO)₂Incubation at 37 ℃ for 24 hours. The cells were then washed 3 times with PBS (1mL) and incubated with additional BrDXMM (10. mu.M) in medium. After 30 min, cells were washed 3 times with PBS (1 mL). In the inhibitor control group, cells were first incubated with a culture medium containing resveratrol (25. mu.M) inhibitor for 30 minutes, then washed 3 times with PBS (1mL), and finally the cells were incubated with a medium containing BrDXMM (10. mu.M). After 30 min, cells were washed 3 times with PBS (1 mL). And finally, observing the distribution state of fluorescence in the cells under a confocal microscope. The excitation wavelength is 561nm, the fluorescence collection range is 570-670nm, and as can be seen from the graphs in FIGS. 5-6, the fluorescent probe BrDXMM can be used for qualitative detection of CYP 1A1 enzymes in HepG2 and LO2 cells.

Example 6: time-dependent fluorescence imaging experiments of fluorescent probe BrDXMM in living cells

HepG2 or LO2 cells were inoculated into confocal imaging cell culture dishes, respectively, and 1mL of the corresponding medium (containing 10% fetal bovine serum) was added thereto in an incubator (5% CO)₂Incubation at 37 ℃ for 24 hours; then, the cells were washed 3 times with PBS (1mL), and then the cells were incubated with a medium containing BrDXMM (10. mu.M)Co-incubation; after 30 min, cells were washed 3 times with PBS (1 mL); and finally, observing the change of the fluorescence emission intensity in the cells at different time points under a confocal microscope. The excitation wavelength is 561nm, the fluorescence collection range is 570-670nm, and as can be seen from the graphs in FIGS. 7-8, the fluorescent probe BrDXMM can be used for rapid (short fluorescence response time) qualitative detection of CYP 1A1 enzyme in HepG2 and LO2 cells.

Example 7: fluorescence imaging experiment of fluorescent probe BrDXMM in zebra fish

Commercially available zebrafish embryos are placed in a clean petri dish and cultured for 4 days at room temperature with the addition of E3 culture medium. Prior to fluorescence imaging, zebrafish were incubated for 30 minutes in E3 medium containing BrDXMM (10. mu.M), and then washed three times with E3 medium. In the inhibitor control group, zebrafish were first cultured for 30 minutes in a culture solution containing resveratrol (25. mu.M) inhibitor, washed 3 times with E3 culture solution, then cultured for 30 minutes in E3 culture solution containing BrDXMM (10. mu.M), and then washed three times with E3 culture solution. And finally, observing the fluorescence distribution state in the zebra fish body under a confocal microscope. The excitation wavelength is 561nm, the fluorescence collection range is 570-670nm, and as can be seen from FIG. 9, the fluorescent probe BrDXMM can be used for qualitative detection of CYP 1A1 enzyme in zebra fish.

Example 8: time-dependent fluorescence imaging experiment of fluorescent probe BrDXMM in tumor-bearing nude mice

The purchased tumor-bearing nude mice were stopped from feeding for 24 hours, during which time only distilled water was fed. Before live body imaging, sodium chloride injection containing BrDXMM (20 mu M) is directly injected into nude mouse tumor bodies by using a disposable syringe, and live body fluorescence imaging photographing is carried out at different time points immediately after injection. The excitation wavelength is 580nm, the fluorescence collection range is 600-700 nm, as can be seen from figure 10, the fluorescence response time is short, the fluorescent probe can rapidly react with the CYP 1A1 enzyme in vivo, and the fluorescent probe BrDXMM can be used for rapid qualitative detection of the CYP 1A1 enzyme in a tumor-bearing nude mouse.

Example 9: fluorescence imaging experiment in fluorescent probe BrDXMM in vitro tissue

The tumor-bearing nude mice were stopped from feeding for 24 hours, during which time only distilled water was fed. Before dissection, sodium chloride injection containing BrDXMM (20 mu M) is directly injected into a tumor of a nude mouse by a disposable syringe, and various tissues (comprising heart, liver, spleen, lung, kidney and tumor) of the nude mouse with the tumor are dissected after 60 minutes to take a picture by biological imaging. The excitation wavelength is 580nm, the fluorescence collection range is 600-700 nm, and as shown in figure 11, the fluorescent probe BrDXMM can stay in a tumor for a long time and does not diffuse after being used for CYP 1A1 enzyme detection in a tumor-bearing nude mouse, namely, the fluorescent probe can specifically emit light in a response region and cannot diffuse to other internal organs quickly, so that the fluorescent probe can perform fluorescent tracing on the tumor or tissues with high CYP 1A1 enzyme expression in vivo.