A near-infrared fluorescent probe DCM-MBS for detecting ONOO - and a preparation method and application thereof

Abstract

The present invention discloses a method for detecting ONOO ‑ The near-infrared fluorescent probe DCM-MBS and its preparation method and application belong to the field of peroxynitrite ion detection technology. The probe DCM-MBS provided by the present invention is a water-soluble probe with a simple detection method and responds to ONOO ‑ It is fast, selective, specific and sensitive, and can effectively avoid interference from other biological molecules, active substances and their own background, ensuring the repeatability, accuracy and reliability of the test results, thereby meeting the rapid changes of ONOO in the body. ‑ The demand for real-time horizontal monitoring; the color change is significant and easy to observe, and ONOO can be achieved with the naked eye ‑ Qualitative in situ real-time detection; it has very good biocompatibility and can achieve excellent fluorescence imaging in a variety of cells and living mice, and no obvious toxic side effects are observed; the synthesis method is simple and efficient, and the raw materials are low-cost and readily available, which is conducive to large-scale industrial production and has broad market application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of peroxynitrite ion detection, in particular to a method for detecting ONOO - Near-infrared fluorescent probe DCM-MBS and its preparation method and application. Background Art

[0002] Drug-induced liver injury (DILI) is a common liver disease in clinical practice and a serious threat to human health. According to incomplete statistics, the number of patients hospitalized due to drug-induced liver injury is increasing year by year worldwide, and some patients may even develop liver failure, which is life-threatening. The pathogenesis of DILI is complex, among which the excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) plays a key role in its pathological occurrence and development. Peroxynitrite (ONOO - ) is a highly oxidizing active nitrogen species, which is composed of nitric oxide (NO) and superoxide anion (O2 - ) Rapid reaction generation. In drug-induced liver injury, due to the disorder of drug metabolism, NO and O2 - The imbalance of ONOO - Excessive ONOO - It will attack biological macromolecules such as proteins, lipids and nucleic acids in cells, triggering oxidative stress and nitrative stress, leading to liver cell damage, apoptosis and even necrosis, further aggravating the liver's inflammatory response and dysfunction.

[0003] Therefore, accurate detection of ONOO in drug-induced liver injury is necessary. - The level of ONOO is of great significance for the in-depth understanding of the pathogenesis of DILI, early diagnosis and the development of effective treatment strategies. Traditional detection methods such as chemiluminescence and electron spin resonance can achieve ONOO to a certain extent. - However, it has the disadvantages of complex operation, low sensitivity, poor selectivity and inability to achieve in situ real-time detection. In contrast, fluorescent probe technology has become a popular method for detecting ONOO due to its unique advantages such as high sensitivity, high selectivity, good biocompatibility and ability to perform in situ, real-time and visual detection of targets. - Therefore, a method to specifically detect ONOO - The water-soluble fluorescent probe has important practical significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting ONOO - The near-infrared fluorescent probe DCM-MBS and its preparation method and application are used to solve the problem of ONOO in the prior art. -The detection has problems such as complex operation, low sensitivity, poor selectivity and inability to achieve in-situ real-time detection.

[0005] To achieve the above object, the present invention provides a method for detecting ONOO - The near-infrared fluorescent probe DCM-MBS has the structural formula:

[0006]

[0007] A method for detecting ONOO as described above - The preparation method of the near-infrared fluorescent probe DCM-MBS comprises the following steps:

[0008] S1. Synthesizing Compound 1: Dissolve isophorone and malononitrile in anhydrous ethanol, add piperidine and reflux for reaction. After the reaction, evaporate to dryness to obtain a crude product. Purify the crude product to obtain Compound 1;

[0009] S2, synthesizing compound 2, dissolving compound 1 prepared in S1 and p-acetaminobenzaldehyde in anhydrous acetonitrile, adding piperidine under a protective atmosphere and reflux reacting, and washing the precipitate after the reaction to obtain compound 2;

[0010] S3, synthesizing compound 3, adding compound 2 prepared in S2 to a mixture of concentrated hydrochloric acid and anhydrous ethanol, stirring, neutralizing, extracting, removing water and solvent, and purifying to obtain compound 3;

[0011] S4, synthesizing the probe DCM-MBS, dissolving the compound 3 obtained in S3 in dichloromethane, adding triethanolamine dropwise, stirring, and then adding p-toluenesulfonyl chloride dropwise. Stirring under a protective atmosphere overnight, purifying the organic layer to obtain the probe DCM-MBS;

[0012] The reaction formula is as follows:

[0013]

[0014] Preferably, the equivalent ratio of isophorone:malononitrile:anhydrous ethanol:piperidine in S1 is 1:2:10:0.01; and the reflux reaction conditions are 80-85° C. for 10-15 hours.

[0015] Preferably, the evaporation to dryness in S1 is performed by evaporating to dryness under reduced pressure using a rotary evaporator or a freeze dryer; and the crude product is purified by silica gel column chromatography using petroleum ether as the eluent.

[0016] Preferably, in S2, the equivalent ratio of compound 1: p-acetaminobenzaldehyde: anhydrous acetonitrile: piperidine is 1:1:10:0.3; and washing is performed with acetonitrile.

[0017] Preferably, the equivalent ratio of compound 2: concentrated HCl: anhydrous ethanol in S3 is 0.1:1:2; sodium hydroxide solution is used for neutralization; and extraction is carried out using a mixture of saturated brine and ethyl acetate, with a volume ratio of saturated brine: ethyl acetate of 1:1.

[0018] Preferably, in S3, drying is performed using anhydrous sodium sulfate; desolvation is performed using a reduced pressure desolvation method with a reduced pressure condition of 0.01 MPa; purification is performed using a silica gel column, and the eluent is a mixture of petroleum ether and dichloromethane, with a volume ratio of petroleum ether:dichloromethane = 1:9.

[0019] Preferably, the equivalent ratio of compound 3 in S4: dichloromethane: triethanolamine: toluenesulfonyl chloride is 1:5:1.2:2; triethanolamine is added dropwise and stirred for 15-30 minutes; the organic layer is extracted with ethyl acetate; and purification is performed by silica gel column purification, and the eluent is a mixture of petroleum ether and dichloromethane, and the volume ratio of petroleum ether: dichloromethane = 3:7.

[0020] A method for detecting ONOO using the above-mentioned near-infrared fluorescent probe DCM-MBS - The method is to mix the probe DCM-MBS, the test solution and PBS and incubate them, and then use a fluorescence detection instrument to detect the change of fluorescence signal to achieve ONOO - qualitative and quantitative analysis.

[0021] A method for detecting ONOO as described above - The near-infrared fluorescent probe DCM-MBS was used to prepare ONOO - Application in detection reagents.

[0022] Therefore, the present invention provides a method for detecting ONOO - The near-infrared fluorescent probe DCM-MBS and its preparation method and application have the following specific technical effects:

[0023] (1) The probe DCM-MBS provided by the present invention exhibits fluorescence characteristics at 562 nm in the initial state; when combined with ONOO - After the reaction occurs, its fluorescence spectrum red-shifts to 648nm and produces strong red fluorescence. The color change of the reaction system can be observed by the naked eye, and ONOO can be realized by the naked eye. - Qualitative in situ real-time detection;

[0024] (2) The probe DCM-MBS provided by the present invention responds to ONOO - Fast and highly sensitive, it can reach the highest fluorescence intensity in 180 seconds, and the minimum detection limit can reach 197nM, which can meet the rapid changes of ONOO in the body. - The need for real-time monitoring of levels;

[0025] (3) The probe DCM-MBS provided by the present invention has good selectivity and strong specificity and can accurately identify ONOO in complex biological systems. - , which can effectively avoid interference from other biomolecules, active substances and its own background, ensuring the repeatability, accuracy and reliability of the test results;

[0026] (4) The probe DCM-MBS provided by the present invention is a water-soluble probe with a simple detection method. It can react in a system without adding any organic reagents. It has very excellent biocompatibility and can achieve excellent fluorescence imaging in various cells and living mice without observing obvious toxic side effects.

[0027] (5) The preparation method provided by the present invention can be used to successfully synthesize the probe DCM-MBS. The synthesis method is simple and efficient, and the raw materials are low-cost and readily available, which is conducive to large-scale industrial production and has broad market application prospects.

[0028] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0030] Figure 1 is the H NMR spectrum of the probe DCM-MBS prepared in Example 1 of the present invention;

[0031] Figure 2 This is the carbon NMR spectrum of the probe DCM-MBS prepared in Example 1 of the present invention;

[0032] Figure 3 This is a high-resolution mass spectrum of the probe DCM-MBS prepared in Example 1 of the present invention;

[0033] Figure 4 The probe DCM-MBS in Example 2 of the present invention is added with ONOO in different co-solvents and different buffers. - wherein (A) is the fluorescence spectrum in different organic solvents, and (B) is the fluorescence spectrum in different buffer solutions;

[0034] Figure 5 The probe DCM-MBS with ONOO in Example 2 of the present invention - Fluorescence emission spectra, fluorescence intensity and fluorescence intensity ratio (I 655 / I 572 ) and ONOO - The linear relationship of concentration; (A) is the probe DCM-MBS with ONOO under naked eye observation - Color changes with increasing concentration; (B) probe DCM-MBS and different concentrations of ONOO - Titration fluorescence emission spectrum and fluorescence intensity; (C) is the fluorescence intensity ratio (I 655 / I 572 ) and ONOO - Linear relationship graph of concentration;

[0035] Figure 6 The color photographs of the probe DCM-MBS after reacting with different ions in Example 2 of the present invention; (A) is a photo of the color change under ultraviolet light; (B) is a photo of the color change under fluorescent light; 1 in the figure is Blank; 2 is ONOO - ; 3 is SO3 2- ; 4 is Cl - 5 is 1 - ; 6 is SO4 2- ; 7 is IO4 - ; 8 is Fe 2+ ; 9 is ClO - ; 10 is Cu 2+ ; 11 is Co 2+ ; 12 is HSO4 - ; 13 is S2O5 2- ; 14 is CN - ; 15 is Fe 3+ ; 16 is HSO3 - ; 17 is ClO4 - ; 18 is S 2- ; 19 is AcO - ; 20 is K + ; 21 is S2O3 2- ; 22 is O2 1 ; 23 is Ca 2+ ; 24 is Mg 2+ ; 25 is Al 3+ ; 26 is Br - ; 27 is Pb 2+ ; 28 is Sn 2+ ; 29 is Cu + ;30 is Ba 2+ ;

[0036] Figure 7 The probe DCM-MBS in Example 2 of the present invention detects ONOO - Selectivity and competitiveness statistics of ONOO -; 2 is Black; 3 is SO3 2- ; 4 is Cl - 5 is 1 - ; 6 is SO4 2- ; 7 is IO4 - ; 8 is Fe 2+ ; 9 is ClO - ; 10 is Cu 2 + ; 11 is Co 2+ ; 12 is HSO4 - ; 13 is S2O5 2- ; 14 is CN - ; 15 is Fe 3+ ; 16 is HSO3 - ; 17 is ClO4 - ; 18 is S 2- ; 19 is AcO - ; 20 is K + ; 21 is S2O3 2- ; 22 is O2 1 ; 23 is Ca 2+ ; 24 is Mg 2+ ; 25 is Al 3+ ; 26 is Br - ; 27 is Pb 2+ ; 28 is Sn 2 + ; 29 is Cu + ;30 is Ba 2+ ;

[0037] Figure 8 The results of the investigation on the effect of time on the probe DCM-MBS in Example 2 of the present invention are as follows; wherein (A) is the relationship between the probe DCM-MBS and ONOO - Fluorescence emission spectra before and after response; (B) is the fluorescence spectrum of probe DCM-MBS and ONOO - UV absorption spectra before and after response; (C) is the curve of fluorescence intensity changing with time;

[0038] Figure 9 This is the result of investigating the effect of pH on the probe DCM-MBS in Example 2 of the present invention;

[0039] Figure 10 These are the experimental results of the cytotoxicity of different concentrations of the probe DCM-MBS to HCT116 cells in Example 2 of the present invention;

[0040] Figure 11 The different concentrations of ONOO in Example 3 of the present invention - Confocal imaging in A549 cells;

[0041] Figure 12 The different concentrations of ONOO in Example 3 of the present invention - Confocal imaging in HCT116 cells;

[0042] Figure 13 The different concentrations of ONOO in Example 3 - Confocal imaging in Hk2 cells;

[0043] Figure 14 These are the in vivo imaging images of the probe DCM-MBS in Example 4 of the present invention in mice with drug-induced liver injury and normal mice, and the liver tissue section images after dissection; wherein (A) is the in vivo fluorescence imaging image of normal mice and liver injury model mice after injection of the probe DCM-MBS over time; (B) is the liver tissue section image of normal mice and liver injury model mice 1 hour after injection of the probe DCM-MBS; (C) is the in vivo fluorescence imaging image of the kidney, lung, pancreas, liver, and heart of normal mice and liver injury model mice after injection of the probe DCM-MBS; (D) is a photo of the kidney, lung, pancreas, liver, and heart of normal mice and liver injury model mice after injection of the probe DCM-MBS in daylight. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0045] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0046] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources; the method steps not described in detail are conventional techniques in the art.

[0047] Example 1

[0048] Preparation of fluorescent probe DCM-MBS, the synthesis route is as follows:

[0049]

[0050] The specific steps are as follows:

[0051] (1) Synthesis of compound 1.

[0052] Accurately weigh isophorone (5.0 g, 36.2 mmol, 1.0 equiv.) and malononitrile (5.0 g, 75.7 mmol, 2.0 equiv.) in a 250.0 mL round-bottom flask, add 150.0 mL of anhydrous ethanol (EtOH, 10 equiv.) to fully dissolve, then add piperidine (112.0 mg, 0.362 mmol, 0.01 equiv.) and reflux at 82°C for 12 h. After the reaction is complete, evaporate to dryness under reduced pressure on a rotary evaporator (reduced pressure condition is 0.01 MPa), and the resulting product is purified by silica gel column chromatography (eluent is petroleum ether, PE) to obtain a white solid, which is compound 1, totaling 4.5 g (yield is 66.0%, R f =0.39, and the developing solvent was petroleum ether:ethyl acetate (PE:EtOAc) with a volume ratio of 10:1).

[0053] (2) Synthesis of compound 2.

[0054] In a 25.0 mL round-bottom flask, p-acetaminobenzaldehyde (163.2 mg, 1.0 mmol, 1.0 equiv.) and the prepared compound 1 (186.3 mg, 1.0 mmol, 1.0 equiv.) were added and dissolved with 5.0 mL of anhydrous acetonitrile (10.0 equiv.). 60 μL of piperidine (0.6 mmol, 0.3 equiv.) was added at room temperature under nitrogen protection, and the mixture was refluxed at 82°C for 1 hour. After the reaction was completed, the precipitate was washed with acetonitrile to obtain an orange solid, which was compound 2, totaling 246.2 mg (yield 74.3%, R f =0.55, developing solvent was PE:EtOAc volume ratio =6:1).

[0055] (3) Synthesis of compound 3.

[0056] The prepared compound 2 (125.0 mg, 0.38 mmol, 0.1 equiv.) was added to a mixture of concentrated HCl (10 mL, 1.0 equiv.) and EtOH (20 mL, 2.0 equiv.), and the mixture was stirred at 80°C for 5.5 h. After the reaction was completed, the pH was adjusted to 7.0 with 0.1 M sodium hydroxide solution. Then, the aqueous solution was extracted with a mixture of saturated brine and ethyl acetate (volume ratio of 1:1), and the obtained product was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure (reduced pressure condition of 0.01 MPa) to obtain a crude compound 3. The crude compound 3 was purified by silica gel column (eluent: PE:DCM volume ratio = 1:9), and the red solid compound obtained was compound 3, a total of 90.2 mg (yield 82.0%, R f =0.65, developing solvent: PE:EtOAc=6:1).

[0057] (4) Synthesis of probe DCM-MBS.

[0058] Accurately weighed compound 3 (28.0 mg, 0.1 mmol, 1.0 equiv.) prepared above was placed in a 25 mL reaction flask and dissolved in 5 mL dichloromethane (DCM, 5.0 equiv.). Then triethanolamine (TEA, 18 μL, 0.12 mmol, 1.2 equiv.) was added dropwise under ice-bath condition and stirred for 20 min. p-Toluenesulfonyl chloride (38.6 mg, 0.2 mmol, 2.0 equiv.) was added dropwise into the flask and stirred overnight under nitrogen condition. The resulting product was extracted with ethyl acetate and the organic layer was collected and purified by silica gel column (eluent: PE:DCM = 3:7 by volume) to obtain the final yellow solid compound, probe DCM-MBS, 20 mg (yield: 42%, Rf= 0.55, eluent: PE:EtOAc = 4:1 by volume). f

[0059] The 1H NMR spectrum of probe DCM-MBS is shown in Figure 1 The 13C NMR spectrum of probe DCM-MBS is shown in Figure 2 The high resolution mass spectrum of probe DCM-MBS is shown in Figure 3

[0060] The 1H NMR data of probe DCM-MBS are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 7.67 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 8.2 Hz, 2H), 7.23 (s, 1H), 7.18 (s, 1H), 7.10 (d, J = 8.7 Hz, 2H), 6.81 (s, 1H), 2.60 (s, 3H), 2.32 (s, 4H), 0.99 (s, 6H).

[0061] The 13C NMR data of probe DCM-MBS are as follows: 13 C NMR (101 MHz, DMSO-d6) δ 170.39, 156.07, 143.60, 139.14, 137.08, 136.60, 131.50, 129.87, 129.05, 128.58, 126.81, 122.47, 119.39, 76.02, 42.36, 38.20, 31.76, 27.49, 21.05.

[0062] Example 2

[0063] The fluorescent probe DCM-MBS prepared in Example 1 was tested for performance, as follows: ​​

[0064] (1) Fluorescence spectrum test.

[0065] 1) Determine the best co-solvent. - Ionic solution (0.1mmol / L) was added to a colorimetric tube, followed by the addition of 1.0mL of different organic reagents (DMSO, ACN, DMF, MeOH, THF, DXA, with deionized water as a control) and 1.0mL of phosphate buffer solution (PBS). The reaction system was then fixed to 5.0mL with deionized water. The fluorescence spectrum and UV absorption spectrum of each reaction system were scanned. The excitation and emission slit widths of the fluorescence spectrum were set to 10nm and 10nm, respectively, and the voltage was set to 700V. The results are shown in Figure 2. Figure 4 As shown in (A), the experiment shows that in the system of deionized water and PBS buffer, the fluorescence intensity of the fluorescent probe is optimal and the Stokes shift is maximum. Therefore, no organic co-solvent is added in subsequent experiments.

[0066] 2) Determine the optimal buffer. Add 100.0 μL of probe DCM-MBS (0.1 mmol / L), 100.0 μL of ONOO - Ionic solution (0.1mmol / L) was added to a colorimetric tube, followed by 1.0mL of deionized water and 1.0mL of different buffer solutions (PBS, HEPES, Tris). The reaction system was then fixed to 5.0mL with deionized water. The fluorescence spectrum and UV absorption spectrum of each reaction system were scanned. The excitation and emission slit widths of the fluorescence spectrum were set to 10nm and 10nm, respectively, and the voltage was set to 700V. The results are shown in Figure 2. Figure 4 As shown in (B), the probe response performance is best in PBS, and the probe response effect is best in the deionized water and PBS system, that is, in the system without adding any organic reagents.

[0067] 3) Determine the minimum detection limit.

[0068] 100.0 μL of probe DCM-MBS (0.1 mmol / L) and 100.0 μL of different concentrations of ONOO - Ion solution (4.0-16.0 μM) was added to a colorimetric tube, and then 1.0 mL of deionized water and 1.0 mL of PBS buffer were added, and then the reaction system was fixed to 5.0 mL with deionized water. The fluorescence spectrum and UV absorption spectrum of each reaction system were scanned. The excitation and emission slit widths of the fluorescence spectrum were set to 10 nm and 10 nm, respectively, and the voltage was set to 700 V. The results are shown in Figure 2. Figure 5 As shown, the probe DCM-MBS reacts with ONOO - (4.0~10.0μM) showed good linear response, and the correlation coefficient R2 to 0.9901, and the lowest detection limit was 197.0 nM, indicating that the probe DCM-MBS can be used to monitor the concentration changes of ONOO - .

[0069] 4) The selectivity and anti-interference ability (competitive) of the probe DCM-MBS were investigated. 100.0 μL of the probe DCM-MBS (0.1 mmol / L), 100.0 μL of 0.1 M ONOO - ion solution or some common interfering ions (such as Fe 3+ , K + , Ca 2+ , Mg 2+ , etc.) were added into a colorimetric tube, then 1.0 mL of deionized water and 1.0 mL of PBS buffer were added, respectively, and then the reaction system was fixed to 5.0 mL with deionized water. The fluorescence spectrum and the ultraviolet absorption spectrum of each reaction system were scanned, the excitation and emission slit widths of the fluorescence spectrum were set to 10 nm and 10 nm, respectively, and the voltage was set to 700 V. The photos of the probe DCM-MBS after reaction with different ions are shown in Figure 6 , and the statistical results are shown in Figure 7 . After the addition of ONOO - , the fluorescence signal of the probe DCM-MBS at 655 nm was significantly enhanced, while the addition of other analytes hardly produced fluorescence shift.

[0070] In the competitive experiment, 100.0 μL of the probe DCM-MBS (0.1 mmol / L), 100.0 μL of ONOO - (0.1 mmol / L), 1.0 mL of deionized water and 1.0 mL of PBS buffer were added, and then 100.0 μL of each interfering ion solution (0.1 mmol / L) was added, respectively. Then the reaction system was fixed to 5.0 mL with deionized water. The fluorescence spectrum and the ultraviolet absorption spectrum of each reaction system were scanned, the excitation and emission slit widths of the fluorescence spectrum were set to 10 nm and 10 nm, respectively, and the voltage was set to 700 V. The photos of the probe DCM-MBS after reaction with different ions are shown in Figure 6 , and the statistical results are shown in Figure 7 . It was found that other interference factors did not affect the detection of ONOO - by the probe DCM-MBS. The above results show that the probe DCM-MBS has excellent selectivity and strong anti-interference ability for ONOO - .

[0071] 5) The stability and pH tolerance of the probe DCM-MBS were investigated.

[0072] Firstly, the probe DCM-MBS (10.0 μM) and ONOO- (4.0~10.0μM) reaction kinetics. Figure 8 As shown in (A), without adding ONOO - When ONOO - When the probe fluorescence signal was red-shifted, the fluorescence signal at 648 nm was significantly enhanced and reached a peak within 3 min.

[0073] Then, the ability of the probe DCM-MBS to detect ONOO at physiological pH was evaluated. - By changing the pH value of the buffer solution in the reaction system (2 to 11), the spectral scanning was performed. The experimental results showed that the probe DCM-MBS had a strong affinity for ONOO in the range of pH = 6 to 10. - Good fluorescence response ( Figure 9 ), indicating that the probe DCM-MBS can detect ONOO under physiological conditions - Changes in concentration.

[0074] (2) Biocompatibility testing.

[0075] The CCK-8 method was used to evaluate the toxicity of the probe DCM-MBS on HCT-116 cells. First, 100.0 μL of cell suspension was added to a 96-well plate, followed by 100 μL of DMEM culture medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 U / mL streptomycin. The 96-well plate was placed in an incubator at 37°C and 5% CO2 for 24 hours. Subsequently, 8 different concentrations of probe DCM-MBS were added to the above system, including 0.0 μM, 0.5 μM, 1.0 μM, 2.5 μM, 5.0 μM, 10.0 μM, 15.0 μM, and 20.0 μM, and the cells were incubated for 24 hours. 0.1 mL of CCK-8 solution was then added and the cells were incubated for another 4 hours. Finally, the absorbance at 450 nm of each well (or reaction system) was recorded, and the cell viability was calculated using the following formula:

[0076] Cell viability (%) = (A 加药 -A 空白 ) / (A 不加药 -A 空白 )×100%

[0077] Among them, A 加药 Refers to the cell group after co-incubation of the probe solution and CCK-8 solution; A 空白 Refers to the blank control, which is a cell group to which neither probe solution nor CCK-8 solution is added; A 不加药 The negative control group is a cell group incubated with only CCK-8 solution.

[0078] The results are as follows Figure 10 As shown, even at 20 μM ONOO - Even at this concentration, the cell survival rate can still reach over 80%.

[0079] Example 3

[0080] The fluorescent probe DCM-MBS prepared in Example 1 was used to detect intracellular peroxynitrite ions, as follows:

[0081] An appropriate amount of HCT-116, A549 and Hk2 cell suspensions were routinely cultured in a 37°C, 5% CO2 incubator until the logarithmic growth phase. After the culture was completed, 100 μL of a 10.0 μM probe DCM-MBS solution was added to each culture system and incubated for 20 minutes to allow the probe to fully interact with the cells. After the incubation was completed, the cells were washed three times with PBS to completely remove the unbound probes. Subsequently, four different concentrations (0.0 μM, 5.0 μM, 10.0 μM, 20.0 μM, respectively) of peroxynitrite ions (ONOO - ) solution and continue incubating for 15 minutes to allow ONOO - After the reaction is complete, wash the sample 3 times with PBS to terminate the reaction and remove the residual ONOO - The treated cell samples were placed under a confocal microscope for fluorescence imaging analysis to obtain information such as the distribution and intensity changes of the intracellular fluorescence signal.

[0082] The results are as follows Figures 11-13 As shown in these figures, it can be clearly observed that as ONOO - With the increase of concentration, the red fluorescence in the cells gradually increased.

[0083] Example 4

[0084] The in vivo imaging effect of the fluorescent probe DCM-MBS prepared in Example 1 in mice with drug-induced liver injury was investigated as follows:

[0085] BALB / C mice weighing 18 to 22 g were used as the experimental model, and the experimental procedures adhered to international ethical guidelines. The mice were randomly divided into two groups: a control group and a drug-induced liver injury model group (referred to as the model group), with 10 mice in each group. The model group received an intraperitoneal injection of 300 mg / kg of acetaminophen (APAP) solution to establish a drug-induced liver injury mouse model. The control group received an intraperitoneal injection of an equal volume of saline. Twelve hours after APAP or saline injection, both groups received a 10 mg / kg injection of a fluorescent probe via the tail vein. At 0.5, 1, 2, 4, and 6 hours after injection of the fluorescent probe, the mice were placed on the stage of a live imaging system. The position of the mice was adjusted, and the distribution and intensity of the fluorescence signal in the mice were recorded at an excitation wavelength of 520 nm.

[0086] The results are as follows Figure 14 As shown in the figure, the in vivo imaging results showed that 0.5 hours after the injection of the fluorescent probe, only a weak fluorescent signal appeared in the liver of the control group mice, and the fluorescence intensity did not change significantly over time. However, 0.5 hours after the injection of the fluorescent probe, the liver of the mice in the drug-induced liver injury model group showed a clear fluorescent signal, and although the fluorescence intensity decreased over time, a strong fluorescent signal could still be observed at 4 hours. Figure 14 A). The control and model group mice were then dissected and their visceral tissues removed. Observed under daylight, the fresh livers of the control mice were darker in color, had clear structures, tight and shiny membranes, and clearly defined liver lobes. In contrast, the livers of the liver injury group mice showed signs of collapse and a lighter color, with unclear lobes, loose capsules, flat, collapsed areas, pale color, and thin edges ( Figure 14 D). Under fluorescence observation, the fluorescence intensity of the liver of mice in the drug-induced liver injury model group was significantly higher than that of the control group ( Figure 14 This clearly shows that the fluorescent probe can be specifically enriched in the liver tissue of mice with drug-induced liver damage and bind to ONOO produced in the liver tissue due to drug damage. - Reactions occur, producing obvious fluorescent signals, thereby achieving the detection of ONOO in drug-induced liver injury. - effective detection.

[0087] In addition, the mouse liver tissue was stained with HE ( Figure 14 B). In the control group, liver cells were arranged in an orderly manner, and no pathological conditions were observed. In contrast, liver tissue treated with APAP showed signs of hepatocellular necrosis, hepatocellular edema, and frequent venous congestion. These liver tissue observations confirmed the successful establishment of the liver injury model. This result further confirms that the fluorescent probe can accurately detect ONOO in drug-induced liver injury liver tissue in vivo.- The generation and distribution of the probe provide a powerful visual tool for in-depth study of the pathological mechanism of drug-induced liver injury.

[0088] Therefore, the probe DCM-MBS provided by the application is water-soluble, the detection method is simple, the response ONOO - The probe has fast speed, good selectivity, strong specificity, high detection sensitivity, can effectively avoid the interference of other biological molecules, active substances and self background, ensures the repeatability, accuracy and reliability of the detection result, and further meets the demand of real-time monitoring of ONOO - in vivo; the color change is obvious and easy to observe, naked eye can realize the qualitative in-situ real-time detection of ONOO - The probe has very excellent biocompatibility, can realize excellent fluorescence imaging in various cells and in vivo of living mice, and no obvious toxic side effects are observed; the synthesis method is simple and efficient, the raw material cost is low and easy to obtain, is conducive to large-scale industrial production, and has wide market application prospect.

[0089] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the application but not to limit it, although the application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: the technical solutions of the application can still be modified or replaced by equivalents, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the application.

Claims

1. A method for detecting ONOO - The near-infrared fluorescent probe DCM-MBS is characterized by: The structural formula of the near-infrared fluorescent probe is:

2. A method for detecting ONOO according to claim 1 - The preparation method of the near-infrared fluorescent probe DCM-MBS is characterized in that: The steps include: S1. Synthesizing Compound 1: Dissolve isophorone and malononitrile in anhydrous ethanol, add piperidine and reflux for reaction. After the reaction, evaporate to dryness to obtain a crude product. Purify the crude product to obtain Compound 1; S2, synthesizing compound 2, dissolving compound 1 prepared in S1 and p-acetaminobenzaldehyde in anhydrous acetonitrile, adding piperidine under a protective atmosphere and reflux reacting, and washing the precipitate after the reaction to obtain compound 2; S3, synthesizing compound 3, adding compound 2 prepared in S2 to a mixture of concentrated hydrochloric acid and anhydrous ethanol, stirring, neutralizing, extracting, removing water and solvent, and purifying to obtain compound 3; S4, synthesizing the probe DCM-MBS, dissolving the compound 3 obtained in S3 in dichloromethane, adding triethanolamine dropwise, stirring, and then adding p-toluenesulfonyl chloride dropwise. Stirring under a protective atmosphere overnight, purifying the organic layer to obtain the probe DCM-MBS; The reaction formula is as follows:

3. A method for detecting ONOO according to claim 2. - The preparation method of the near-infrared fluorescent probe DCM-MBS is characterized by: The equivalent ratio of isophorone:malononitrile:anhydrous ethanol:piperidine in S1 is 1:2:10:0.01; and the reflux reaction conditions are 80-85° C. for 10-15 hours.

4. A method for detecting ONOO according to claim 2 - The preparation method of the near-infrared fluorescent probe DCM-MBS is characterized by: S1 was evaporated to dryness using a rotary evaporator or a freeze dryer; the crude product was purified by silica gel column chromatography using petroleum ether as eluent.

5. A method for detecting ONOO according to claim 2. - The preparation method of the near-infrared fluorescent probe DCM-MBS is characterized by: In S2, the equivalent ratio of compound 1: p-acetaminobenzaldehyde: anhydrous acetonitrile: piperidine is 1:1:10:0.3; washing is carried out with acetonitrile.

6. A method for detecting ONOO according to claim 2. - The preparation method of the near-infrared fluorescent probe DCM-MBS is characterized by: In S3, the equivalent ratio of compound 2: concentrated HCl: anhydrous ethanol is 0.1:1:2; sodium hydroxide solution is used for neutralization; and extraction is carried out using a mixture of saturated brine and ethyl acetate, with a volume ratio of saturated brine: ethyl acetate of 1:

1.

7. A method for detecting ONOO according to claim 2. - The preparation method of the near-infrared fluorescent probe DCM-MBS is characterized by: In S3, anhydrous sodium sulfate was used for drying; the solvent was removed by vacuum desolvation at a pressure of 0.01 MPa; and the product was purified by silica gel column purification using a mixture of petroleum ether and dichloromethane as the eluent, with a volume ratio of petroleum ether to dichloromethane of 1:

9.

8. A method for detecting ONOO according to claim 2. - The preparation method of the near-infrared fluorescent probe DCM-MBS is characterized by: The equivalent ratio of compound 3 in S4: dichloromethane: triethanolamine: toluenesulfonyl chloride is 1:5:1.2:2; triethanolamine is added dropwise and stirred for 15-30 minutes; the organic layer is extracted with ethyl acetate; and purification is performed on a silica gel column using a mixture of petroleum ether and dichloromethane as the eluent, with a volume ratio of petroleum ether: dichloromethane = 3:

7.

9. A method for detecting ONOO using the near-infrared fluorescent probe DCM-MBS according to claim 1 - The method is characterized in that: After the probe DCM-MBS, the test solution and PBS are mixed and incubated, the fluorescence signal change is detected by a fluorescence detection instrument to achieve ONOO - qualitative and quantitative analysis.

10. A method for detecting ONOO as claimed in claim 1. - The near-infrared fluorescent probe DCM-MBS was used to prepare ONOO - Application in detection reagents.

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