Near-infrared fluorescent probe for detecting ATP (adenosine triphosphate) and ONOO <-> as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared fluorescent probe for detecting ATP (adenosine triphosphate) and ONOO-as well as a preparation method and application of the near-infrared fluorescent probe, and relates to the technical field of fluorescent probes. The fluorescent probe has a structural formula as shown in a formula (I) or a formula (II): # imgabs0 # triamine / N-diisopropylethylamine and urea are used as recognition groups, rhodamine and methylene blue are used as parent fluorescent dyes, multiple targets can be detected at the same time, and ATP and ONOO-dual-channel near-infrared fluorescence imaging is realized; the rapid and real-time detection of ATP and ONOO <-> of cells and living bodies can be realized; the application range is wide, normal and cancer cells can be selectively distinguished, the cobalt chloride hexahydrate-induced HK-cell pyroptosis process can be monitored, the uric acid protection effect on the cellular level can be evaluated, and acute kidney injury can be detected; the preparation method is simple and low in cost.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and particularly relates to a near-infrared fluorescent probe for detecting ATP and ONOO - , and a preparation method and application thereof. Background Art

[0002] Adenosine-5'-triphosphate (ATP) is mainly synthesized through cellular respiration and is an important energy source for organisms. The disruption of ATP balance is closely related to oxidative stress, which is the result of the production of reactive oxygen species. The occurrence and development of many diseases such as cancer, Parkinson's disease, and ischemic diseases are associated with abnormal ATP metabolism. Peroxynitrite anion (ONOO - ) is an important reactive oxygen species that can inactivate mitochondrial ATPase by oxidation and inhibit the production of ATP. ONOO - is closely related to cancer, neurodegenerative diseases, and inflammatory diseases. Nasopharyngeal carcinoma is a common tumor in otolaryngology, and its incidence in endemic areas is still very high. Although CT, MRI, and PET-CT examinations can accurately image tumors, clinical diagnosis still relies on tissue biopsy. Acute kidney injury is a stress-induced inflammatory disease. Renal ischemia can damage endothelial cells by producing inflammatory mediators (such as IL-6, IL-18, TNF-α, etc.) through tubular cells, and the stress-induced inflammatory response will lead to further damage to renal tissue. When a stress-induced inflammatory response occurs in the body, more ONOO - will be generated. At the same time, ONOO - will exacerbate the inflammatory response and further damage cells and tissues. In summary, the level changes of ATP and ONOO - are closely related to the progression of nasopharyngeal carcinoma and acute kidney injury. Therefore, the development of new methods for accurately detecting the concentrations of ATP and ONOO - is of great significance for deeply understanding the related physiological functions and diseases.

[0003] The accurate detection of the concentrations of ATP and ONOO - in complex biological samples is often restricted by various factors, resulting in unsatisfactory detection results. Traditional methods for detecting the concentrations of ATP and ONOO - include mass spectrometry, ultraviolet spectroscopy, etc. However, these methods are limited by long detection time, low sensitivity, and inability to detect in situ in organisms. Fluorescent probe analysis has the advantages of simplicity, rapidity, high sensitivity, good selectivity, and low cost, which makes it the goal of scientific researchers to achieve the accurate detection of ATP and ONOO - using fluorescent probes. However, the developed fluorescent probes for ATP and ONOO - still have the following deficiencies: First, most of them are single-target detection probes and cannot detect two substances simultaneously. When studying ATP and ONOO- It is restricted when studying the interaction relationship between them; second, the selectivity in complex systems is relatively poor; third, the sensitivity is not high.

[0004] In view of this, it is very important to develop a technology that can detect ATP and ONOO - concentrations with high selectivity, high sensitivity, convenience and rapidity. SUMMARY OF THE INVENTION

[0005] Aiming at the problems existing in the above-mentioned background technology, the present invention provides a dual-channel near-infrared fluorescent probe using diethylenetriamine / N-diisopropylethylamine and urea as recognition groups, and rhodamine and methylene blue as parent fluorescent dyes, which can be used to detect ATP and ONOO - and its preparation method and application.

[0006] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0007] In the first aspect, the present invention discloses a near-infrared fluorescent probe for detecting ATP and ONOO - with the structural formula shown in the following formula (I) or formula (II):

[0008]

[0009] In the second aspect, the present invention discloses a preparation method of the near-infrared fluorescent probe for detecting ATP and ONOO as described in the first aspect, including the following steps: - (1) Under the protection of an inert atmosphere, add N,N-diisopropylethylamine (DIPEA) to an organic solvent of rhodamine B and an amino-substituted compound, reflux and react, and remove the solvent under reduced pressure to obtain a crude intermediate product; the amino-substituted compound is diethylenetriamine or 1-(2-aminoethyl)piperazine;

[0010] (2) Purify the crude intermediate product to obtain a high-quality intermediate product;

[0011] (3) Add sodium carbonate to an organic solvent containing the high-quality intermediate product and MB-Cl, stir and react at room temperature, and remove the solvent under reduced pressure to obtain a crude near-infrared fluorescent probe;

[0012] (4) Purify the crude near-infrared fluorescent probe to obtain a high-quality near-infrared fluorescent probe.

[0013] (4) Purify the crude near-infrared fluorescent probe to obtain a high-quality near-infrared fluorescent probe.

[0014] In a preferred embodiment of the present invention, in step (1), the molar ratio between rhodamine B and the amino-substituted compound is 1:1 - 1.5.

[0015] In a preferred embodiment of the present invention, in step (2), the method for purifying the crude intermediate is as follows: The crude intermediate is diluted with dichloromethane and washed with distilled water. The combined organic layers are dried over anhydrous sodium sulfate and then concentrated under vacuum, and the crude product is purified on a silica gel column using an elution solvent.

[0016] In a preferred embodiment of the present invention, in step (3), the molar ratio of sodium carbonate, the refined intermediate, and MB-Cl is 2 - 5:1:1.

[0017] In a preferred embodiment of the present invention, in step (4), the method for purifying the crude near-infrared fluorescent probe is as follows: The crude near-infrared fluorescent probe is purified on a silica gel column using an elution solvent to obtain the crude product.

[0018] Further, the elution solvent is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 30:1.

[0019] In a preferred embodiment of the present invention, the organic solvent in step (1) is acetonitrile, and the organic solvent in step (3) is dichloromethane.

[0020] In a third aspect, the present invention discloses an application of the near-infrared fluorescent probe as described in the first aspect in detecting ATP and / or ONOO - -.

[0021] Preferably, the near-infrared fluorescent probe is used for selectively distinguishing normal cells and cancer cells, monitoring the pyroptosis process of HK-2 cells induced by cobalt chloride hexahydrate, evaluating the protective effect of uric acid at the cellular level, and detecting acute kidney injury by detecting ATP and / or ONOO - -.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The fluorescent probe of the present invention uses diethylenetriamine / N-diisopropylethylamine and urea as recognition groups, and rhodamine and methylene blue as parent fluorescent dyes, which can detect multiple targets simultaneously, realizing dual-channel near-infrared fluorescence imaging of ATP and ONOO - -.

[0024] 2. Fast response, the fluorescent probe can achieve rapid and real-time detection of ATP and ONOO - in cells and in vivo.

[0025] 3. Wide application range, it can not only selectively distinguish normal and cancer cells, monitor the pyroptosis process of HK- cells induced by cobalt chloride hexahydrate, evaluate the protective effect of uric acid at the cellular level, but also detect acute kidney injury;

[0026] 4. The method for synthesizing and preparing the fluorescent probe of the present invention is simple and low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Fluorescence titration diagram of the dual-channel near-infrared fluorescent probe P2 for detecting ATP and ONOO provided by the present invention - ;

[0028] Figure 2 Response time diagram of the dual-channel near-infrared fluorescent probe P2 for ATP and ONOO provided by the present invention - ;

[0029] Figure 3 Selectivity experiment diagram of the dual-channel near-infrared fluorescent probe P2 for ATP and ONOO provided by the present invention - ;

[0030] Figure 4 Diagram of the dual-channel near-infrared fluorescent probe P2 provided by the present invention for selectively distinguishing cancer cells and normal cells

[0031] Figure 5 Imaging diagram of the dual-channel near-infrared fluorescent probe P2 provided by the present invention for ATP and ONOO in HK-2 cells - ;

[0032] Figure 6 Diagram of the dual-channel near-infrared fluorescent probe P2 provided by the present invention for monitoring the process of pyroptosis of HK-2 cells induced by cobalt chloride hexahydrate

[0033] Figure 7 Diagram of the evaluation result of the protective effect of uric acid at the cellular level by the dual-channel near-infrared fluorescent probe P2 provided by the present invention

[0034] Figure 8 Diagram of the result of detecting acute kidney injury by the dual-channel near-infrared fluorescent probe P2 provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] The raw materials and experimental instruments used in the preferred embodiments of the present invention are all commercially available.

[0037] Example 1: Synthesis of P1

[0038]

[0039] The specific synthesis steps are as follows: Under nitrogen, N,N-diisopropylethylamine (3.3 ml) was added to a solution of rhodamine B (4.4 g, 10 mmol) and diethylenetriamine (1.30 ml, 12 mmol) in acetonitrile (100 ml), and the mixture was heated to reflux for 10 h. After the reaction stopped, the solvent was removed under reduced pressure. The residue was diluted with dichloromethane and washed with distilled water. The combined organic layers were dried over anhydrous sodium sulfate and then concentrated in vacuo. The crude product was purified on a silica gel column (200 - 300 mesh) using dichloromethane / methanol (volume ratio 30 / 1) as the eluent to obtain the desired compound Rh-NH2 as a red solid (3.27 g, 62%).

[0040] Sodium carbonate (318 mg, 3.0 mmol) was added to a dichloromethane solution (20 ml) containing Rh-NH2 (527 mg, 1.0 mmol) and MB-Cl (347 mg, 1.0 mmol). The mixture was stirred at room temperature overnight. After the reaction stopped, the solvent was removed under reduced pressure. The crude product was purified on a silica gel column (200 - 300 mesh) using dichloromethane / methanol (volume ratio 30 / 1) as the eluent to obtain the desired compound P2 as a purple solid (293 mg, 35%). NMR characterization data: 1 1H NMR (400 MHz, CDCl3): 7.89 - 7.87 (m, 1H), 7.45 - 7.43 (m, 2H), 7.31 (d, J = 8.6 Hz, 2H), 7.10 - 7.08 (m, 1H), 6.64 (d, J = 2.7 Hz, 2H), 6.57 (dd, J = 8.8, 2.8 Hz, 2H), 6.41 (d, J = 8.8 Hz, 2H), 6.37 (d, J = 2.6 Hz, 2H), 6.23 (dd, J = 8.9, 2.6 Hz, 2H), 5.43 (s, 1H), 3.31 (q, J = 7.1 Hz, 8H), 3.23 - 3.11 (m, 4H), 2.89 (s, 12H), 2.43 - 2.34 (m, 4H), 2.03 - 1.98 (m, 1H), 1.14 (d, J = 7.0 Hz, 12H); 13 13C NMR (400 MHz, CDCl3): 168.63, 156.02, 153.51, 153.26, 148.82, 148.78, 132.92, 132.46, 131.01, 128.68, 128.49, 128.03, 127.20, 123.83, 122.73, 111.39, 110.84, 108.04, 105.21, 97.60, 65.12, 48.20, 47.60, 44.31, 40.74, 40.18, 12.55. Mass spectrometry characterization data: HRMS m / z: C 49H 58 N8O3S[M] + The theoretically calculated value is 838.4353, and the actual detection is 838.4391.

[0041] Example 2: Synthesis of P2

[0042]

[0043] The specific synthesis steps are as follows: Under nitrogen, N,N-diisopropylethylamine (3.3 ml) was added to a solution of rhodamine B (4.4 g, 10 mmol) and 1-(2-aminoethyl)piperazine (1.57 ml, 12 mmol) in acetonitrile (100 ml), and the mixture was heated to reflux for 20 h. After the reaction stopped, the solvent was removed under reduced pressure. The residue was diluted with dichloromethane and washed with distilled water. The combined organic layers were dried over anhydrous sodium sulfate and then concentrated in vacuo. The crude product was purified on a silica gel column (200 - 300 mesh) using dichloromethane / methanol (volume ratio 30 / 1) as the eluent to obtain the desired compound Rh-N as a red solid (3.9 g, 70%).

[0044] Sodium carbonate (318 mg, 3.0 mmol) was added to a dichloromethane solution (10 ml) containing Rh-N (553 mg, 1.0 mmol) and MB-Cl (347 mg, 1.0 mmol). The mixture was stirred at room temperature overnight. After the reaction stopped, the solvent was removed under reduced pressure. The crude product was purified on a silica gel column (200 - 300 mesh) using dichloromethane / methanol (volume ratio 30 / 1) as the eluent to obtain the desired compound P2 as a purple solid (583 mg, 67%). NMR characterization data: 1 HNMR(400MHz,CDCl3):7.86 - 7.84(m,1H),7.49(d,J = 9.0Hz,2H),7.42 - 7.38(m,2H),7.06 - 7.04(m,1H),6.62(d,J = 2.8Hz,2H),6.55(dd,J = 8.9,2.8Hz,2H),6.40(d,J = 8.8Hz,2H),6.34(d,J = 2.6Hz,2H),6.22(dd,J = 8.8,2.5Hz,2H),3.31(q,J = 7.1Hz,8H),3.19(t,J = 7.3Hz,2H),3.13 - 3.10(m,4H),2.89(s,12H),2.09 - 2.07(m,4H),2.01(t,J = 7.4Hz,2H),1.14(t,J = 7.0Hz,12H); 1313C NMR (400 MHz, CDCl3): 167.91, 158.18, 153.54, 153.21, 148.63, 148.11, 132.21, 131.56, 131.16, 131.09, 128.98, 127.87, 123.90, 123.68, 122.61, 111.65, 110.91, 107.89, 105.56, 97.60, 64.68, 55.41, 52.29, 45.83, 44.33, 40.81, 12.56. Mass spectrometry characterization data: HRMS m / z: C 51 H 60 N8O3S [M+H] + The theoretical calculated value is 865.4587, and the actual detection value is 865.4534.

[0045] Example 3: Fluorescence titration of dual-channel fluorescent probe P2 for detecting ATP and ONOO - The fluorescence titration of

[0046] Taking the dual-channel fluorescent probe P2 as an example, the fluorescence titration curve of P2 was obtained by testing in a PBS / DMSO = 4:1 (volume ratio) solution at 37 °C and pH = 7.4. As Figure 1 A, when different concentrations of ATP (concentration range 0 - 15 mM) were added to the probe P2 (10 μM) solution, a new fluorescence emission peak appeared at 585 nm, and its fluorescence intensity gradually increased with the increase of the ATP concentration. There was a linear relationship between the fluorescence intensity of the probe P2 at 585 nm and the ATP concentration (5 - 15 mM) (the linear equation was F 585nm = 126622.2 × [ATP] mM – 617326.0). Using the calculation formula 3σ / k (where k represents the slope of the linear equation and σ represents the standard deviation), the detection limit of the probe P2 for ATP was calculated to be 11.6 μM. As Figure 1 B, when different concentrations of ONOO - (0 - 25 μM) were added to the P2 (10 μM) solution system, a new fluorescence emission peak appeared at 690 nm, and its fluorescence intensity gradually increased with the increase of the ONOO - concentration. There was a linear relationship between the fluorescence intensity of the probe P2 at 690 nm and the ONOO - concentration (1 - 6 μM) (the linear equation was F 690nm = 8572.9 × [ONOO - ] μM + 15841.5). Using the calculation formula 3σ / k (where k represents the slope of the linear equation and σ represents the standard deviation), the detection limit of the probe P2 for ONOO -The detection limit is 138 nM. This example demonstrates that the dual-channel fluorescent probe P2 can highly sensitively detect ATP and ONOO in a relatively wide concentration range. - .

[0047] Example 4: Response time of the dual-channel fluorescent probe P2 to ATP and ONOO - .

[0048] Taking the dual-channel fluorescent probe P2 as an example, the response time of P2 to ATP and ONOO - was measured in a PBS / DMSO = 4:1 (v / v) solution at 37 °C and pH = 7.4. As Figure 2 shown in Fig. A, ATP (5, 10, 15 mM) was added to the probe P2 (10 μM) solution, and the change in fluorescence intensity at 585 nm over time was recorded. It was found that the fluorescence intensity approached the saturation value at about 20 min. The above results indicate that the probe P2 can rapidly respond to ATP in the mM concentration range, which is consistent with the ATP concentration range in vivo. As Figure 2 shown in Fig. B, ONOO - was added at 600 s, and the fluorescence intensity of the probe P2 at 690 nm increased rapidly and reached the saturation value within 50 s and then remained basically unchanged. This example demonstrates that the dual-channel fluorescent probe P2 can rapidly detect ATP and ONOO in vitro. - .

[0049] Example 5: Selectivity experiment of the dual-channel fluorescent probe P2 to ATP and ONOO - .

[0050] Taking the dual-channel fluorescent probe P2 as an example, the selectivity of P2 to ATP and ONOO - was measured in a PBS / DMSO = 4:1 (v / v) solution at 37 °C and pH = 7.4. Figure 3 Fig. A shows the selectivity test of the probe P2 to ATP. Different bioactive species were added to the probe P2 (10 μM) solution: blank probe, adenosine diphosphate, adenosine monophosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, sodium carbonate, sodium sulfate, sodium nitrate, sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, zinc chloride, glutathione, D-glucose, ATP. This example demonstrates that the probe P2 has high selectivity for ATP. In the experiment, only ATP caused a significant increase in the fluorescence intensity of the probe at 585 nm, while the influence of other bioactive species was very small. Figure 3 Fig. B shows the selectivity of the probe P2 to ONOO -Selectivity test: Different bioactive species were added to the solution of probe P2 (10 μM): blank probe, sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, sodium carbonate, sodium sulfate, sodium nitrate, potassium chloride, magnesium sulfate, calcium chloride, zinc chloride, glutathione, hydrogen peroxide, sodium hypochlorite, hydroxyl radical, singlet oxygen, ONOO - . This example proves that probe P2 has high selectivity for ONOO - . Only ONOO - in the experiment caused a significant increase in the fluorescence intensity of the probe at 690 nm, while other bioactive species had little effect.

[0051] Example 6: Dual-channel fluorescent probe P2 selectively differentiates cancer cells and normal cells.

[0052] As Figure 4 , human nasopharyngeal epithelial cell line NP69, human highly differentiated nasopharyngeal carcinoma cell line CNE-1, human poorly differentiated nasopharyngeal carcinoma cell line CNE-2, and human highly metastatic nasopharyngeal carcinoma cell line 5-8F were selected to verify the ability of probe P2 to selectively differentiate normal and nasopharyngeal carcinoma cell lines. At 37 °C, probe P2 (20 μM) was incubated with NP69, CNE-1, CNE-2, and 5-8F for 20 minutes, and then confocal imaging was performed. In the green light channel, there was no significant difference in fluorescence intensity among several cells, while in the red light channel, the fluorescence signal of normal nasopharyngeal cells (NP69) was significantly lower than that of several cancer cells (CNE1, CNE2, 5-8F). This example proves that probe P2 has the ability to selectively identify normal and cancer cells by imaging endogenous ATP and ONOO - in different cells.

[0053] Example 7: Imaging of endogenous ATP and ONOO - in HK-2 cells by dual-channel fluorescent probe P2.

[0054] As Figure 5 , renal tubular epithelial cell line HK-2 was selected to verify the fluorescence imaging ability of probe P2. At 37 °C, probe P2 (20 μM) was incubated with HK-2 cells for 20 minutes, and then confocal imaging was performed. A weak fluorescence signal was quickly detected in the green light channel, while a weak fluorescence signal was detected in the red light channel after 10 minutes. As the time extended to 30 minutes, the fluorescence signals in both channels were slightly enhanced compared with before. This example proves that probe P2 has the ability to image endogenous ATP and ONOO - in HK-2 cells.

[0055] Example 8: Dual-channel fluorescent probe P2 monitors the pyroptosis process of HK-2 cells induced by cobalt chloride hexahydrate.

[0056] As Figure 6, at 37 °C, HK-2 cells were pretreated with CoCl2·6H2O at different concentrations (0.1 mM, 0.3 mM, 0.6 mM) for 24 hours, and then stained with probe P2 (20 μM) for 20 minutes followed by confocal imaging. As the concentration of CoCl2·6H2O increased, the fluorescence signal in the green channel gradually decreased, while the fluorescence signal in the red channel gradually increased. This example demonstrated that probe P2 has the ability to dynamically monitor the process of CoCl2·6H2O-induced pyroptosis.

[0057] Example 9: Evaluation of the protective effect of uric acid by the dual-channel fluorescent probe P2 at the cellular level.

[0058] As Figure 7 , an oxygen-glucose deprivation method was used to simulate an HK-2 ischemia / hypoxia stimulation model. The experimental group of HK-2 cells was changed to a sugar-free and serum-free medium, and cultured in a 95% N2 + 5% CO2 environment for 2 hours and then changed to a complete medium and cultured for another 24 hours. At 2 hours of ischemia / hypoxia, the fluorescence signal in the green channel significantly decreased, and the fluorescence signal in the red channel increased. When uric acid (500 μM) was added simultaneously during ischemia / hypoxia, compared with the 2-hour ischemia / hypoxia group, the fluorescence signal in the green channel increased, and the fluorescence signal in the red channel decreased. This example demonstrated that probe P2 has the ability to evaluate the protective effect of urea on ischemic / hypoxic cells.

[0059] Example 10: Detection of acute kidney injury by the dual-channel fluorescent probe P2.

[0060] As Figure 8 , after deeply anesthetizing C57BL / 6 mice, the right renal pedicle was clamped for 15 minutes and then reperfused, and the left kidney was not treated. At 24 hours, 48 hours, and 72 hours after the operation, P2 (50 μM, 50 μL) was injected in situ into the kidney, and after staining for 30 min, in vivo imaging was performed. The fluorescence signal in the green channel of the left kidney hardly changed, and the fluorescence signal in the green channel of the right kidney showed a trend of first decreasing and then slowly rising compared with the left kidney; there was almost no fluorescence in the red channel of the left kidney, and the fluorescence signal in the red channel of the right kidney increased sharply at 24 hours compared with the left kidney. As the time extended to 72 hours, the enhanced fluorescence signal slowly decreased but still remained at a relatively high level. This example demonstrated that probe P2 has the ability to early diagnose acute kidney injury.

[0061] Those of ordinary skill in the art in this technical field should recognize that the above examples are only used to illustrate the present invention, rather than to limit the present invention. As long as it is within the scope of the essential spirit of the present invention, changes and modifications to the above examples will fall within the scope of the claims of the present invention.

Claims

1. A near-infrared fluorescence probe for detecting ATP and ONOO - , characterized in that It has a structural formula shown in the following formula (I) or formula (II):

2. A preparation method of a near-infrared fluorescence probe for detecting ATP and ONOO - , characterized in that It includes the following steps: (1) Under the protection of an inert atmosphere, add N,N-diisopropylethylamine (DIPEA) to an organic solvent of rhodamine B and an amino-substituted compound, reflux and react, and remove the solvent under reduced pressure to obtain a crude intermediate product; the amino-substituted compound is diethylenetriamine or 1-(2-aminoethyl)piperazine; (2) Purify the crude intermediate product to obtain a refined intermediate product; (3) Add sodium carbonate to an organic solvent containing the refined intermediate product and MB-Cl, stir and react at room temperature, and remove the solvent under reduced pressure to obtain a crude near-infrared fluorescent probe; (4) Purify the crude near-infrared fluorescent probe to obtain a refined near-infrared fluorescent probe.

3. The preparation method according to claim 2, wherein: In step (1), the molar ratio between rhodamine B and the amino-substituted compound is 1:1 - 1.

5.

4. The preparation method according to claim 2, characterized in that: In step (2), the method for purifying the crude intermediate product is: dilute the crude intermediate product with dichloromethane and wash it with distilled water, dry the combined organic layers with anhydrous sodium sulfate and then perform vacuum concentration, and purify the crude product on a silica gel column using an elution solvent.

5. The preparation method according to claim 2, characterized in that: In step (3), the molar ratio between sodium carbonate, the refined intermediate product and MB-Cl is 2 - 5:1:

1.

6. The preparation method according to claim 2, wherein: In step (4), the method for purifying the crude near-infrared fluorescent probe is: purify the crude near-infrared fluorescent probe on a silica gel column using an elution solvent.

7. The preparation method according to claim 4 or 6, characterized in that: The elution solvent is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 30:

1.

8. The preparation method according to claim 2, characterized in that: The organic solvent in step (1) is acetonitrile, and the organic solvent in step (3) is dichloromethane.

9. Use of the near-infrared fluorescent probe according to claim 1 for detecting ATP and / or ONOO - therein.

10. The application according to claim 9, characterized in that, The near-infrared fluorescence probe is used for detecting ATP and / or ONOO - It is used for selectively differentiating normal cells and cancer cells, monitoring the pyroptosis process of CoCl₂·6H₂O-induced HK-2 cells, evaluating the protective effect of uric acid at the cellular level, and detecting acute kidney injury.

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