阅读说明：本技术 基于苯并吲哚的ntr-1响应型荧光探针、制备方法及用途 (NTR-1 response type fluorescent probe based on benzindole, preparation method and application ) 是由 蔡进 吉民 杨敏 黄铭琪 王雨红 陈茜茜 于 2020-12-15 设计创作，主要内容包括：本发明公开了基于苯并吲哚的NTR-1响应型荧光探针、制备方法及用途,结构如下。先将对羟基苯甲醛与对硝基溴化苄反应合成以醚键连接的稳定中间体NFP-1-M,然后再将1-乙基-2-甲基苯并[cd]吲哚-1-氯化物与中间体NFP-1-M缩合,生成探针NFP-1。该探针可以用于衡量细胞缺氧程度,有助于肿瘤早期诊断等。(The invention discloses a benzoindole-based NTR-1 response type fluorescent probe, a preparation method and application thereof, and the structure is as follows. Firstly, p-hydroxybenzaldehyde and p-nitrobenzyl bromide react to synthesize a stable intermediate NFP-1-M connected by ether bond, and then 1-ethyl-2-methylbenzo [ cd]Condensing indole-1-chloride with intermediate NFP-1-M to generate probe NFP-1. The probe can be used for measuring the degree of cell hypoxia, and is beneficial to early diagnosis of tumors and the like.)

1. An NTR-1 response type fluorescent probe based on benzindole has the following structure:

2. the benzindole-based NTR-1-responsive fluorescent probe according to claim 1, characterized in that: the detection limit is 17 ng/mL.

3. The benzindole-based NTR-1-responsive fluorescent probe according to claim 1, characterized in that: the probe NTR-1 has different fluorescence intensities in different pH ranges.

4. The method for preparing a benzindole-based NTR-1 responsive fluorescent probe according to claim 1, wherein the method comprises the following steps: firstly, p-hydroxybenzaldehyde and p-nitrobenzyl bromide react to synthesize a stable intermediate NFP-1-M connected by ether bond, then 1-ethyl-2-methylbenzo [ cd ] indole-1-chloride and the intermediate NFP-1-M are condensed to generate a probe NFP-1, and the process comprises the following steps:

5. use of the benzindole-based NTR-1 responsive fluorescent probe of claim 1 in the preparation of NTR-1 detection reagents.

6. Use according to claim 5, characterized in that: the NTR-1 detection reagent is used for NTR detection in living cells.

7. Use according to claim 6, characterized in that: the living cells are tumor cells.

8. Use of the benzindole-based NTR-1 responsive fluorescent probe of claim 1 in cellular imaging.

9. Use of the benzindole-based NTR-1 responsive fluorescent probe according to claim 1 in the preparation of a tumor diagnostic reagent.

Technical Field

The invention relates to a fluorescent probe, a preparation method and application, in particular to an NTR-1 response type fluorescent probe based on benzindole, a preparation method and application.

Background

Nitroreductases (NTRs) belong to the flavoenzymes and usually contain either a flavin mononucleotide unit or a flavin adenine dinucleotide unit. In the presence of NADPH or NADH, the nitroreductase can reduce the nitro groups on the nitro-substituted heterocyclic and aromatic rings to produce the corresponding nitrite, hydroxylamine or amino derivatives. It has been demonstrated that NTR activity is related to intracellular oxygen content. Over-expression of NTR can be observed in hypoxic cells or tissues. Hypoxia is an important pathological process for rapid proliferation of tumor cells, and has become a main characteristic of solid tumors. Hypoxic microenvironments play an important role in the process of tumorigenesis and metastasis, and can promote malignant biological behaviors including tumor cell proliferation, invasion, metastasis and apoptosis. Thus, the amount of NTR may reflect O in the tumor₂And (4) horizontal. In addition, it has been reported that NTR has good application prospect in the fields of drug screening, clinical diagnosis and the like. Therefore, the detection of nitroreductase activity in tumor cells and tissues has important significance in the research of biological functions and clinical diagnosis and treatment.

The conventional NTR detection method includes: electron Paramagnetic Resonance (EPR), Nuclear Magnetic Resonance (NMR), clarke electrode, and the like. However, most of these methods require complex and precise instruments, are complicated in sample processing, cannot be monitored in real time, and have low resolution, which greatly limits the application of the conventional detection method. The advent of fluorescence imaging technology has enabled the detection of NTR to find new directions. Although a series of organic small-molecule fluorescent probes for detecting NTR have been developed in recent years, these probes still have the disadvantages of poor stability, low sensitivity, low fluorescence quantum yield, large influence of cell self conditions, and the like, and therefore, it is still of great significance to develop novel organic small-molecule fluorescent probes with novel structures and better performance.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a fluorescent probe capable of detecting NTR rapidly and with high sensitivity. The probe takes EMC as a probe framework, and introduces aromatic nitro as an NTR specific recognition group. When Nicotinamide Adenine Dinucleotide (NADH) is taken as an electron donor, the probe NFP-1 can react with NTR specifically, and shows remarkable fluorescence enhancement characteristics. The probe has the advantages of good stability, high selectivity, small cytotoxicity, response time of 25min and detection limit as low as 17 ng/mL. Cell imaging experiments prove that the probe NFP-1 can be used as an important index for measuring the hypoxia degree of cells for living cells based on NTR, and is favorable for early diagnosis of tumors.

The invention also aims to provide a preparation method and application of the probe.

The technical scheme is as follows: the structure of the NTR-1 response type fluorescent probe based on the benzindole is as follows:

further, the benzindole-based NTR-1 responsive fluorescent probe according to claim 1, characterized in that: the detection limit is 17 ng/mL.

Further, the probe NTR-1 has different fluorescence intensities in different pH ranges.

The preparation method of the NTR-1 response type fluorescent probe based on the benzindole comprises the steps of firstly reacting parahydroxyben-zaldehyde with p-nitrobenzyl bromide to synthesize a stable intermediate NFP-1-M connected by ether bonds, and then condensing 1-ethyl-2-methylbenzo [ cd ] indole-1-chloride with the intermediate NFP-1-M to generate the probe NFP-1, wherein the process comprises the following steps:

specifically, the method comprises the following steps:

(1) synthesis of 1-ethyl-2-methylbenzo [ cd ] indole-1-chloride (EMC):

1, 8-naphthalene imide is dissolved in DMF, and sodium hydride is added. After the system was warmed to room temperature, ethyl iodide was added dropwise and stirred at room temperature. The reaction mixture was extracted with ethyl acetate, the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate, and after filtration, the filtrate was concentrated under reduced pressure, and the resulting solid was separated and purified by silica gel column chromatography to give 1-ethylbenzo [ cd ] indol-2 (1H) -one.

1-ethylbenzo [ cd ] indol-2 (1H) -one was dissolved in anhydrous THF and methyl magnesium chloride was added dropwise. The mixed solution was heated to reflux under nitrogen and monitored by TLC for completion. Cooling the reaction liquid to room temperature, slowly dropwise adding a small amount of water for quenching, then adding a small amount of NaOH solution to adjust the pH of the reaction liquid to be alkaline, extracting with ethyl acetate for three times, adding a small amount of HCl solution to the combined organic phase to adjust the pH to be acidic, extracting with water, adding a large amount of acetone to the combined water phase, stirring, and separating out a large amount of solids. And (4) carrying out suction filtration, washing a filter cake with acetone, and drying to obtain EMC.

(2) Synthesis of Probe NFP-1:

p-hydroxybenzaldehyde and K₂CO₃Dissolving in dry DMF, heating and stirring, adding p-nitrobenzyl bromide and potassium iodide, and continuously stirring for complete reaction. Cooling the system to room temperature, adding ethyl acetate for extraction, combining organic phases, drying by anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, dissolving the obtained solid in a small amount of CH₂Cl₂And adding petroleum ether, stirring, separating out a solid, and drying the solid after suction filtration to obtain an intermediate NFP-1-M.

NFP-1-M and compound EMC were dissolved in a mixed solution of ethanol/acetonitrile, stirred overnight at room temperature in the dark until the reaction was complete. Filtering the mixed solution, slowly adding a proper amount of methyl tert-butyl ether into the filtrate to precipitate a solid, collecting the solid after filtering, adding THF, pulping to remove impurities, filtering again, washing a filter cake with a small amount of THF, and drying to obtain the product NFP-1 (mauve solid).

The application of the NTR-1 response type fluorescent probe based on the benzindole in preparing an NTR-1 detection reagent.

Further, the NTR-1 detection reagent is used for NTR detection in living cells.

Further, the living cell is a tumor cell.

The application of the NTR-1 response type fluorescent probe based on the benzindole in cell imaging.

The NTR-1 response type fluorescent probe based on the benzindole is used for preparing a tumor diagnosis reagent.

The research result shows that the concentration of NTR is 0-2 mug/mL, and the probe NFP-1 has I_490nmHas good linear relation with NTR concentration (R)²0.9915). The detection limit of NFP-1 is 17ng/mL, the sensitivity is high, and the low-concentration NTR can be accurately and quantitatively detected. The stability of the probe NFP-1 is better, and the probe I is added with NTR_490nmThe increase of the response time is enhanced, and the reaction with NTR can be completely carried out within 25 min. In addition, the probe NFP-1 is suitable for the detection of NTR under physiological pH conditions. Moreover, other analytes do not cause a significant change in the fluorescence of NFP-1, and only in response to NTR does probe NFP-1 exhibit a significant fluorescence-enhanced response. The probe NFP-1 has higher selectivity to NTR.

The cytotoxicity experiment result shows that the probe NFP-1 has low cytotoxicity. Cell fluorescence imaging experimental results show that almost no fluorescence signal is found in cells cultured under the normoxic condition, and a stronger fluorescence signal is observed in a green fluorescence channel in cells cultured under the anoxic condition. Fluorescence imaging results prove that the probe NFP-1 can image endogenous NTR in living cells, and has good application potential in the aspect of tumor diagnosis.

Has the advantages that:

1. the invention relates to a fluorescence enhancement type small molecule probe NFP-1. The probe is capable of specifically recognizing NTR in the presence of NADH as an electron donor, exhibiting significant fluorescence enhancement.

2. The stability of the probe NFP-1 is good, the detection limit is 17ng/mL, and the rapid and high-sensitivity detection of NTR can be realized within 25 min.

3. Cell experiments prove that the probe NFP-1 has low cytotoxicity and can successfully detect NTR generated by hypoxia induction in living cells.

Drawings

FIG. 1 probes NFP-1 (10. mu.M) and NTR (5. mu.M)g/mL) and/or the ultraviolet-visible absorption spectrum and the fluorescence spectrum b after reaction of NADH (500. mu.M), the fluorescence spectrum after response of probe NFP-1 (10. mu.M) and NTR with different concentrations when NADH (500. mu.M) exists, the fluorescence intensity I of dNTP-1_490nmWith the change of NTR concentration (0-5. mu.g/mL), the insets: i is_490nmLinear dependence of NTR (0-2. mu.g/mL), λ_ex＝420nm；

FIG. 2 fluorescence intensity (I) at 490nm after different time (0-70 min) for probe NFP-1 (10. mu.M) and/or NTR (5. mu.g/mL) response_490nm)，λ_ex＝420nm；

FIG. 3 fluorescence intensity at 490nm after response of probe NFP-1 (10. mu.M) and/or NTR (5. mu.g/mL) at different pH, lambda_ex＝420nm；

FIG. 4 shows fluorescence spectra of probe NFP-1 after reaction with different analytes, FIG. 4a shows fluorescence spectra of probe NFP-1 after reaction with different analytes, and FIG. 4b shows fluorescence spectra of probe NFP-1 after reaction with different analytes_{At 490nm}Fluorescence intensity of NFP-1 after reaction with different analytes, (1) blank; (2) k⁺(1mM)；(3)Na⁺(1mM)；(4)Mg²⁺(1mM)； (5)·O^tBu(500μM)；(6)TBHP(500μM)；(7)ClO^-(500μM)；(8)O₂ ^·-(500μM)；(9)H₂O₂ (500μM)；(10)Pro(1mM)；(11)Gly(1mM)；(12)Lys(1mM)；(13)Leu(1mM)；(14)Ser(1 mM)；(15)GSH(1mM)；(16)Cys(1mM)；(17)NTR(5μg/mL).λ_ex＝420nm；

FIG. 5 MTT assay results of probe NFP-1 (10. mu.M) on HeLa cells;

FIG. 6 confocal images of HeLa cells cultured under different oxygen conditions after coculture with NFP-1 (10. mu.M), a, b, c: normal oxygen (20% O)₂) (ii) a d. e, f: hypoxia (1% O)₂)，Green Channel：492nm～577nm，λ_ex＝420nm.Scale bar：10μm。

Detailed Description

Example 1 Synthesis of Probe NFP-1:

synthesis of 1-ethyl-2-methylbenzo [ cd ] indole-1-chloride (EMC)

1, 8-Naphthaleneimide (8.03g, 47.4mmol) was dissolved in 100mL DMF and sodium hydride (3.528g, 150mmol) was added slowly at 0 ℃. After warming the system to room temperature, iodoethane (7.403g, 50mmol) was added dropwise and stirred at room temperature for 1 h. The reaction mixture was extracted with ethyl acetate (150mL × 2), the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate, the filtrate was concentrated under reduced pressure after filtration, and the resulting solid was isolated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 10: 1) to give 1-ethylbenzo [ cd ] indol-2 (1H) -one 3(7.762g of yellow solid, yield 83%) which was used directly in the next reaction.

3(1.972g, 10mmol) was dissolved in 40mL dry THF (40mL) and methyl magnesium chloride (15mL, 45mmol) was added dropwise. The mixed solution was heated to reflux at 60 ℃ under nitrogen protection, and the reaction was monitored by TLC for 2h to completion. Cooling the reaction liquid to room temperature, slowly dropwise adding a small amount of water for quenching, then adding a small amount of NaOH solution (1M) to adjust the pH of the reaction liquid to be alkaline, extracting the reaction liquid for three times by using ethyl acetate, adding a small amount of HCl solution (1M) to the combined organic phase to adjust the pH to be acidic, extracting the reaction liquid for three times by using a small amount of water, adding a large amount of acetone to the combined water phase, stirring the mixture, and separating out a large amount of solids. Suction filtration, filter cake washed with acetone and dried in 40 ℃ oven to get 1.753g EMC (green solid) with 76% yield. m.p.159-160 ℃;¹H NMR(400MHz，MeOD)δ8.93(d，J＝ 7.3Hz，1H)，8.79(d，J＝8.1Hz，1H)，8.47(d，J＝6.3Hz，1H)，8.46(d，J＝7.1Hz，1H)，8.23 -8.16(m，1H)，8.07-8.02(m，1H)，4.83-4.79(m，2H)，3.36-3.34(m，3H)，1.72(t，J＝7.4 Hz，3H).¹³C NMR(100MHz，D₂O)：δ170.68，138.33，137.25，134.31，130.84，130.48， 129.40，128.08，127.73，121.41，120.40，42.29，14.51，12.24.MS[ES-API]：calcd for C₁₄H₁₄N⁺，196.1，found：196.1[M]⁺。

2. synthesis of intermediate NFP-1-M:

p-hydroxybenzaldehyde (0.244g, 2mmol) and K₂CO₃(0.277g, 2mmol) was dissolved in 15ml dry DMF and heated to 50 deg.C and stirred for 10min, then p-nitrobenzyl bromide (0.518g, 2.4mmol) and potassium iodide (0.332g, 2mmol) were added and stirring was continued for 4h to complete the reaction. After cooling the system to room temperature, 20ml of ethyl acetate were addedExtracting for 2 times, mixing organic phases, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and dissolving the obtained solid in small amount of CH₂Cl₂Adding petroleum ether, stirring, separating out solid, and drying the solid in an oven at 40 ℃ after suction filtration to obtain 0.375g of intermediate crude product NFP-1-M (light yellow solid) which is directly used for the next reaction.

Synthesis of NFP-1:

crude NFP-1-M (0.154g, 0.6mmol) and compound EMC (0.116g, 0.5mmol) were dissolved in 10mL of a mixed solution of ethanol/acetonitrile (9: 1) and stirred overnight at room temperature in the dark until the reaction was complete. Filtering the mixed solution, slowly adding a proper amount of methyl tert-butyl ether into the filtrate to precipitate a solid, collecting the solid after filtering, adding 1mL of THF, pulping to remove impurities, filtering again, washing a filter cake with a small amount of THF, and drying in an oven at 40 ℃ to obtain 0.198 g of product NFP-1 (mauve solid) with the yield of 52%.¹H NMR(400MHz，DMSO)：δ9.37(s，1H)，8.85 (d，J＝14.1Hz，1H)，8.72(s，1H)，8.39(s，2H)，8.31(d，J＝6.4Hz，4H)，8.16(s，1H)，7.95(d， J＝13.2Hz，2H)，7.79(d，J＝7.6Hz，2H)，7.31(d，J＝6.4Hz，2H)，5.48(s，2H)，4.88(s，2H)， 1.52(s，3H).¹³C NMR(101 MHz，MeOD)：δ163.08，162.27，153.82，147.70，143.99，138.87， 136.71，134.53，132.61，130.76，129.88，129.59，128.66，127.83，123.70，123.31，118.34， 115.73，111.71，68.71，41.27，14.29.MS[ES-API]：calcd for C₂₈H₂₃N₂O₃ ⁺，435.2，found： 435.2[M]⁺。

Example 2 spectroscopic Properties of Probe NFP-1:

all uv-vis absorption spectra and fluorescence spectra of probe NFP-1 were recorded in DMSO-PBS (1: 9 v/v, 10mM, pH 7.4) in solution. At λ_exThe solution was collected at 420nm for fluorescence emission from 430nm to 700nm with excitation and emission slit widths of 5nm/5 nm. NFP-1 was used as a 1mM stock solution in DMSO. NTR was dissolved in ultrapure water to make 100. mu.g/mL of mother liquor for use. Unless otherwise specified, 500. mu.M NADH was added to all the reaction systems.

NADH (500. mu.M) was added to NFP-1 (10. mu.M), incubated with NTR (0-10. mu.g/mL) at various concentrations for 30min at 37 ℃ and UV-visible absorption and fluorescence spectra were recorded. In the selectivity study, NFP-1 (10. mu.M) was incubated with NTR (5. mu.g/mL), active oxygen (500. mu.M), a portion of metal ions (1mM), and amino acids (1mM) at 37 ℃ for 30min, and their fluorescence spectra were recorded. In the kinetic response experiment, fluorescence spectra of NFP-1(10 μ M) and/or at different time points (0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70min) without/with NTR (5 μ g/mL) were recorded. In the pH response study, probes NFP-1 (10. mu.M) and NTR (5. mu.g/mL) were incubated at 37 ℃ for 30min at different pH (1-14), and their fluorescence spectra were recorded.

The ultraviolet absorption spectrum and fluorescence spectrum of the probe NFP-1 before and after the reaction with NTR and NADH are shown in FIG. 1. FIG. 1a shows UV-visible absorption spectrum, FIG. 1b shows fluorescence spectrum, FIG. 1c shows fluorescence spectrum of probe NFP-1(10 μ M) in response to different concentrations of NTR in the presence of NADH (500 μ M), and FIG. 1d shows fluorescence intensity I of NFP-1_490nmLambda is varied with NTR concentration (0-5. mu.g/mL)_ex420 nm. FIG. 1a shows that when only NADH (500. mu.M) or NTR (5. mu.g/mL) is added to the probe, the absorption peak of the reaction system is not obviously changed and is all around 500 nm; when NADH and NTR exist simultaneously, a new maximum absorption peak appears at 440 nm. FIG. 1b shows that the probe itself has very weak fluorescence under 420nm excitation, almost no fluorescence, and the fluorescence intensity of the reaction system is not significantly changed by adding only NADH or NTR, while a significant fluorescence enhancement is observed near 490nm when NTR and NADH exist at the same time. The above analysis shows that when NADH exists, the nitro group on the probe NFP-1 can be reduced by NTR, and the reaction system shows fluorescence enhancement response. Therefore, in the subsequent fluorescence response experiment, 500. mu.M NADH was added to the reaction system as an electron donor.

To evaluate the concentration response of probe NFP-1 to NTR, fluorescence spectra were recorded after reaction of NFP-1 with different concentrations of NTR (0-10. mu.g/mL). As shown in FIG. 1c, the fluorescence intensity of probe NFP-1 at 490nm (I) increases with increasing NTR concentration_490nm) Gradually increase and reach saturation at 5. mu.g/mL. As shown in FIG. 1d, the NTR concentration is in the range of 0-2 μ g/mLIn the periphery, I of the probe_490nmHas good linear relation with NTR concentration (R)²0.9915). The results show that the probe NFP-1 can effectively and quantitatively detect NTR with a certain concentration.

The fluorescence intensity of probe NFP-1 (10. mu.M) (blank) at 420nm excitation was measured 3 times to obtain the standard deviation of fluorescence intensity at 490nm for the blank. And calculating the detection limit according to a linear regression equation of NFP-1(10 mu M) and NTR concentration (0-2 mu g/mL) in the fluorescence response. The calculation formula is as follows:

DL＝3σ/k

where σ is the standard deviation of blank measurements and k is the slope between the fluorescence intensity at 490nm after reaction of NFP-1(10 μ M) with NTR (0-2 μ g/mL) and different NTR concentrations. The detection limit of the probe NFP-1 is calculated to be 17ng/mL according to a formula, which shows that the sensitivity of the probe NFP-1 is higher, and the accurate quantitative detection can be carried out on the NTR with low concentration.

Example 3 kinetic response of Probe NFP-1:

in order to study the dynamic response of the probe NFP-1 to NTR, the fluorescence intensity I of the probe NFP-1(10 μ M) and the NTR (5 μ g/mL) in different response time (0-70 min) is recorded_490nmA change in (c). As shown in fig. 2. The results show that the stability of the probe NFP-1 is better, and the probe I after adding NTR_490nmIncreases with increasing response time and reaches a plateau at 25 min. The above analysis shows that the probe NFP-1 can completely react with NTR within 25 min. To ensure complete reaction, the reaction time was chosen to be 30min in all response experiments.

Example 4 pH response of probe NFP-1:

pH is an important factor influencing the fluorescent response performance of the probe, so that the fluorescence intensity I of the pH to the probe is evaluated by recording the fluorescence spectrum of NFP-1 reacted with NTR (5 mu g/mL) at different pH values in an experiment_490nmThe influence of (c). The results are shown in FIG. 3. The result shows that the fluorescence intensity of the probe NFP-1 does not change significantly under different pH values, and only shows very weak emission intensity, which indicates that the probe is very stable and has little background interference. After NTR is added, the probe NFP-1 shows extremely strong fluorescence emission at pH 6-10, and the pH value is 7A maximum value is reached in the vicinity. The result shows that the probe NFP-1 is suitable for detecting NTR under the physiological pH condition.

Example 5 Probe NFP-1 Selectivity study:

the fluorescent probe with excellent performance can specifically identify a target substance to be detected and is not interfered by other active substances. Thus, to evaluate the selectivity of probe NFP-1, the fluorescence spectra of NFP-1 after reaction with NTR and other analytes were compared, and the addition of probe NFP-1 to the different analytes (K) was recorded⁺、Na⁺、 Mg²⁺、·O^tBu、TBHP、ClO^-、O₂ ^·-、H₂O₂Pro, Gly, Lys, Leu, Ser, GSH, Cys). The results are shown in FIG. 4, in which FIG. 4a shows the fluorescence spectra of the probe NFP-1 after reaction with different analytes and FIG. 4b shows the fluorescence spectra of FIG. 4a_{At 490nm}Fluorescence intensity of NFP-1 after reaction with different analytes. The results show that other analytes do not cause a significant change in the fluorescence of NFP-1 compared to the control group, and that probe NFP-1 only shows a significant fluorescence-enhanced response when it responds to NTR. Therefore, the probe NFP-1 has higher selectivity to NTR.

Example 6 cytotoxicity experiments:

the cytotoxicity of the probe NFP-1 on HeLa cells was evaluated by MTT method. As shown in FIG. 5, the cell survival rate was still over 90% at 25. mu.M concentration of probe NFP-1, indicating that probe NFP-1 is less cytotoxic.

Example 7 cellular fluorescence imaging experiment:

prior to imaging experiments, experimental groups of HeLa cells were seeded in culture dishes under hypoxic conditions (1% O)₂) Cultured for 12h to promote NTR overexpression in the cells. Control cells were in normoxic conditions (20% O)₂) And culturing for 12 h. Then, two groups of cells were incubated with the probe NFP-1 (10. mu.M) for 30min, and fluorescence images were taken using a laser confocal microscope after three rinses of PBS buffer. The results are shown in FIG. 6. The experimental results showed that almost no fluorescence signal was observed in the cells cultured under normoxic conditions (FIGS. 6 a-c), whereas strong fluorescence signal was observed in the green fluorescence channel in the cells cultured under hypoxic conditions (FIG. 6 a-c)Numbers (FIGS. 6 d-f). Fluorescence imaging results prove that the probe NFP-1 can image endogenous NTR in living cells, and has good application potential in the aspect of tumor diagnosis.