Two-channel fluorescent probe for simultaneously detecting ONOO and viscosity and preparation method of two-channel fluorescent probe

Abstract

The invention is applicable to the technical field of fluorescence imaging, and provides a dual-channel fluorescent probe for simultaneously detecting ONOO <-> and viscosity and a preparation method thereof, and the preparation method specifically comprises the following steps: dissolving QX-OH in a solvent, then adding triethylamine, stirring at room temperature, then adding diphenylphosphinyl chloride, reacting, drying the solvent by distillation after the reaction is completed, and obtaining the dual-channel fluorescent probe for simultaneously detecting ONOO <-> and viscosity. And purifying the residue to obtain a black solid QX-DP. The fluorescent probe can simultaneously respond to two biomarkers of ONOO <-> and viscosity and respectively generate obvious dual-channel fluorescent signals at 668nm and 752nm, the fluorescent probe not only can show application value in an epilepsy mouse model, but also can be successfully applied in a non-alcoholic fatty liver (NAFL) disease model and a tumor ferroptosis model, and the fluorescent probe has a good application prospect. More comprehensive information is provided for disease diagnosis and image-guided surgery; the prepared QX-DP cells are low in toxicity and good in biocompatibility, and the preparation method has the advantages of being simple in synthesis route, high in yield, easy to operate and the like.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescence imaging technology and particularly relates to a method for simultaneously detecting ONOO - Dual-channel fluorescent probe for detecting viscosity and its preparation method. Background Art

[0002] Peroxynitrite ion (ONOO - ) as an important endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS), through superoxide anion (O2 ·- ) reacts with nitric oxide (NO) in biological systems rapidly, with a reaction rate of about 10 10 M -1 s -1 , which has strong oxidation and nitrosation capabilities and is considered to be an important physiologically active substance and signaling molecule that helps maintain the redox homeostasis of organisms. However, excessive ONOO - It can cause irreversible oxidative damage to organelles, proteins and nucleic acids, leading to the occurrence of various diseases, such as inflammation, neurodegenerative diseases, cardiovascular diseases, and even cancer. Viscosity is a key indicator in the intracellular microenvironment of biological systems. It plays an important role in physiological / pathological processes including the transport and signal transmission of intracellular substances and the interaction between biological molecules. Abnormal changes in viscosity are considered to be an important factor leading to liver damage, neurodegenerative diseases and cancer. In summary, ONOO in cells - and viscosity are closely related to a variety of physiological / pathological processes.

[0003] Fluorescence imaging technology based on fluorescent probes has become a powerful tool for detecting bioactive molecules at the cell / tissue / living animal level due to its excellent sensitivity and selectivity, non-invasiveness, real-time in situ response, high spatiotemporal resolution, and simple operation. It is known that near-infrared (NIR) emission can effectively avoid background autofluorescence in the visible region and can penetrate thick tissue samples to improve the imaging signal-to-noise ratio. Many methods have been reported for detecting ONOO in biological systems. - However, most of the probes focus on the detection and imaging of a single analyte, but the pathological processes of many diseases are often accompanied by the simultaneous fluctuations of multiple markers. In particular, abnormalities in intracellular viscosity are always accompanied by fluctuations in pH, polarity, reactive oxygen species, and other reactive molecular levels. Compared with the detection of a single biomarker, simultaneous monitoring of dual biomarkers can provide a more sensitive and reliable strategy for the diagnosis and image-guided surgery of oxidative stress and viscosity-related diseases.

[0004] Most fluorescent probes can only detect and image a single analyte. For example, Feng's team reported a method that can simultaneously detect ONOO -and viscosity dual-channel fluorescent probe (CQ), but its sensitivity to ONOO - The response mechanism of ONOO is based on oxidative cleavage of the ethylene bond, which destroys the conjugated structure of the probe and limits the fluorescence emission to the visible light region; although - ONOO emitting at 645nm - and viscosity / polarity dual-channel probes, but these probes respond to ONOO - There is still room for improvement to achieve unstable ONOO - Real-time detection of ONOO in many diseases; In biological imaging, highly sensitive and selective probes are required to accurately detect biomarkers, and existing probes may be insufficient in these aspects; Due to the lack of suitable multifunctional fluorescent probes, ONOO - Simultaneous visualization of viscosity and ONOO has not been achieved, for example in epilepsy and tumor ferroptosis models. Therefore, it is necessary to develop a method that can accurately and simultaneously detect viscosity and ONOO - This method is of great significance for the early diagnosis of the disease and the revelation of its pathological mechanism. Summary of the invention

[0005] The purpose of the embodiment of the present invention is to provide a method for simultaneously detecting ONOO - The invention discloses a dual-channel fluorescent probe for detecting fluorescence and viscosity, aiming to solve the problems raised in the above-mentioned background technology.

[0006] The embodiment of the present invention is implemented as follows: a method for simultaneously detecting ONOO - The chemical formula of QX-DP is shown in Formula 1:

[0007]

[0008] Another object of the embodiment of the present invention is to provide a method for simultaneously detecting ONOO - The preparation method of the dual-channel fluorescent probe with a viscosity of 200 μm and 100 μm is shown in Formula 2:

[0009]

[0010] The specific steps include:

[0011] QX-OH was dissolved in a solvent, and then triethylamine was added. After stirring at room temperature, diphenylphosphonyl chloride was added to react. After the reaction was completed, the solvent was evaporated and the residue was purified to obtain a black solid QX-DP.

[0012] Preferably, the solvent is one of acetonitrile, tetrahydrofuran, N,N-dimethylformamide and dichloromethane.

[0013] Preferably, the solvent is dichloromethane.

[0014] Preferably, the reaction temperature is 0-80°C and the reaction time is 1-24h.

[0015] Preferably, the molar ratio of QX-OH to diphenylphosphine chloride is 1:1-1:20.

[0016] Preferably, the purification is performed by silica gel chromatography, and the solvent used is one or more of ethyl acetate, dichloromethane, methanol, acetone and petroleum ether.

[0017] Preferably, the solvents used for the purification are dichloromethane and methanol in a volume ratio of 10:1-60:1.

[0018] Another object of an embodiment of the present invention is to provide an application of a fluorescent probe QX-DP in an epilepsy mouse model, a non-alcoholic fatty liver disease model, or a tumor ferroptosis model.

[0019] A method for simultaneously detecting ONOO provided by an embodiment of the present invention - A dual-channel fluorescent probe that can simultaneously respond to ONOO - The two biomarkers of iodine and viscosity were detected by the fluorescence probe, which produced significant dual-channel fluorescence signals at 668nm and 752nm, respectively. The fluorescent probe not only showed its application value in epilepsy mouse models, but also was successfully applied in non-alcoholic fatty liver (NAFL) disease models and tumor ferroptosis models, providing more comprehensive information for disease diagnosis and image-guided surgery. The prepared QX-DP has good sensitivity and selectivity, and can quickly respond to ONOO - and viscosity changes, and has low cytotoxicity and good biocompatibility. The preparation method has the characteristics of simple synthetic pathway, high yield and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The QX-DP provided in Example 1 of the present invention 1 H NMR spectrum (in DMSO-d6);

[0021] Figure 2 The QX-DP provided in Example 1 of the present invention 13 C NMR spectrum (in MeOD);

[0022] Figure 3 A high resolution mass spectrometer (HR-MS) of QX-DP provided in Example 2 of the present invention;

[0023] Figure 4 QX-DP and ONOO provided in Example 2 of the present invention - Post-reaction process;

[0024] Figure 5 QX-DP (10 μM) and ONOO provided in Example 3 of the present invention - (50μM) fluorescence kinetic response, λ ex / λ em =720 / 752nm;

[0025] Figure 6 The fluorescence intensity (F) of QX-DP (10 μM) provided in Example 3 of the present invention in the presence of different biological species (100 μM) is shown in FIG. 752nm ), λ ex / λ em =720 / 752nm;

[0026] Figure 7 QX-DP (10 μM) provided in Example 3 of the present invention in the presence or absence of ONOO - (50 μM), the fluorescence intensity (F 752nm ), λ ex / λ em =720 / 752nm;

[0027] Figure 8 A is the QX-DP (10 μM) in DMSO / PBS (4 / 6, v / v, pH 7.4) provided in Example 3 of the present invention in the presence of ONOO - (100 μM) and the absorption spectra of QX-OH (10 μM), the color of the solution changes from pink-purple to blue-green; Figure 8 B is in DMSO / PBS (4 / 6, v / v, pH 7.4), ONOO - Fluorescence spectra of QX-DP (10 μM) in the presence of (0–50 μM); Figure 8 C is in the range of 0 to 20 μM, I 752nm With ONOO - Linear response between concentrations, λ ex =720nm; Figure 8 D is the absorption spectrum of QX-DP (10 μM) provided in Example 4 of the present invention in a water-glycerol mixture with a viscosity ranging from 0.89 to 945 cP; Figure 8 E is the fluorescence spectrum; Figure 8 F is the linear response between logI and logη, λ ex =574nm;

[0028] Fig. 9 A is the absorption spectrum A of QX-DP (10 μM) in different solvents provided in Example 4 of the present invention, Fig. 9 B is the fluorescence spectrum, with an excitation wavelength of 574 nm;

[0029] Fig.10 The fluorescence lifetime spectra of QX-DP (10 μM) provided in Example 4 of the present invention at different viscosities, the fluorescence lifetime (τ) increases from 1.120 ns (0.89 cP), 1.141 ns (1.54 cP), 1.181 ns (3.18 cP), 1.270 ns (8.82 cP), 1.404 ns (45.86 cP) to 1.657 ns (945 cP);

[0030] Fig.11 The effect of pH on the fluorescence intensity of QX-DP (10 μM) at 668 nm in 0% glycerol and 90% glycerol provided in Example 4 of the present invention, λ ex =574 / 668nm;

[0031] Fig.12 The results of MTT detection of QX-DP on HeLa cells provided in Example 5 of the present invention are respectively provided with a control group, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 8 μM, and 10 μM. The data are expressed as the mean ± standard error of three independent experiments, and each experiment was repeated three times;

[0032] Fig.13 A is the cell imaging experiment result (viscosity) provided in Example 5 of the present invention. The fluorescence image of HeLa cells incubated with QX-DP (5 μM) is used as a control, and pretreated with Mon (monensin) (10 μM) or Nys (nystatin) (10 μM) and then stained with QX-DP (5 μM). Viscosity channel: λ ex =561nm,λ em = 600-690 nm, scale bar: 20 μm; Fig.13 B is the mean fluorescence intensity of the viscosity channel image in A (±SD, n = 3);

[0033] Fig.14 A is the cell imaging experiment result provided in Example 5 of the present invention (ONOO - ), the fluorescence image of HeLa cells incubated with QX-DP (5 μM) was used as the control; the other four groups were pretreated with SIN-1 (3-morpholino norepinephrine) (100 μM) and then stained with QX-DP (5 μM); pretreated with SIN-1 (100 μM) and minocycline (100 μM) and then stained with QX-DP (5 μM); pretreated with LPS (lipopolysaccharide) (100 μg / mL), IFN-γ (interferon-γ) (50 ng / mL) and PMA (phorbol ester) (10 nM) and then stained with QX-DP (5 μM); pretreated with AG (semicarbazide, a kind of uric acid that reduces intracellular ONOO- The cells were pretreated with 5 μM nitrite oxidase inhibitor (5 mM), LPS (100 μg / mL), IFN-γ (50 ng / mL), and PMA (10 nM), and then stained with QX-DP (5 μM). - Channel: Lambda ex =633nm,λ em =700-754 nm, scale bar: 20 μm; Fig.14 B is the mean fluorescence intensity of the image shown in A (±SD, n = 3);

[0034] Fig.15 A is a dual-channel fluorescence image of normal tissue (as a control) and epileptic brain tissue incubated with QX-DP (20 μM) provided in Example 6 of the present invention, viscosity channel: λ ex =561nm,λ em =600-690nm, ONOO - Channel: Lambda ex =633nm,λ em =700-754 nm, scale bar: 100 μm; Fig.15 B is the mean fluorescence intensity of the image shown in A (±SD, n = 3);

[0035] Fig.16 A is a dual-channel fluorescence image of the liver of normal mice (as a control), non-alcoholic fatty liver (NAFL) and NAFL+NAC (N-acetyl-L-cysteine) mice provided in Example 7 of the present invention 30 minutes after intravenous injection of QX-DP (100 μM, 200 μL), viscosity channel: λ ex =580nm,λ em =670nm,ONOO - Channel: Lambda ex =680nm,λ em =790nm; Fig.16 B is the mean fluorescence intensity of the image shown in A (±SD, n=3)

[0036] Fig.17 A is a dual-channel fluorescence image of a control group, non-alcoholic fatty liver (NAFL) and NAFL+NAC tissue sections stained with QX-DP (20 μM, 30 minutes) provided in Example 7 of the present invention; Fig.17 B is the average fluorescence intensity of each image in A (±SD, n=3), viscosity channel: λ ex =561nm,λ em =600-690nm, ONOO - Channel: Lambda ex =633nm,λ em=700-754nm, scale bar: 100μm.

[0037] Fig.18 A is the result of the cancer ferroptosis model provided in Example 8 of the present invention, with HeLa cells incubated with QX-DP (5 μM) as the control group; pre-treated with erastin (a ferroptosis inducer) (50 μM), followed by the addition of QX-DP (5 μM); and pre-treated with Fer-1 (Ferrostatin-1, a ferroptosis inhibitor) (50 μM) and erastin (50 μM), followed by the addition of QX-DP (5 μM), respectively, dual-channel fluorescence images; Fig.18 B is the mean fluorescence intensity of the image in A (±SD, n=3), viscosity channel: λ ex =561nm,λ em =600-690nm. ONOO - Channel: Lambda ex =633nm,λ em =700-754nmScale bar: 20μm;

[0038] Fig.19 A is the dual-channel fluorescence imaging result of tumor-bearing mice in vivo provided in Example 8 of the present invention, with injection of normal saline as the blank control, injection of QX-DP (100 μM, 30 min) as the control group, injection of erastin (2 mM, 24 h) followed by injection of QX-DP (100 μM, 30 min), injection of erastin (2 mM, 24 h) and Fer-1 (2 mM, 4 h) followed by injection of QX-DP (100 μM, 30 min), viscosity channel: λ ex =580nm,λ em =670nm. ONOO - Channel: Lambda ex =680nm,λ em =790nm; Fig.19 B is the mean fluorescence intensity of the image shown in A (±SD, n=3). DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0040] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0041] Example 1: A method for simultaneously detecting ONOO -The reaction formula of the dual-channel fluorescent probe QX-DP for the preparation of the viscosity is shown in Formula 3:

[0042]

[0043] The specific steps include:

[0044] Firstly, the intermediate QX-OH was synthesized according to a reported method, specifically, 2-methylquinoline and 2-hydroxy-4-methoxybenzaldehyde were used as starting materials to react with iodoethane / 2-bromocyclohex-1-ene-1-carboxaldehyde respectively, and the two intermediate products thus obtained were subjected to Knoevenagel condensation reaction and further demethylated to prepare QX-OH;

[0045] QX-OH (51 mg, 0.1 mmol) was dissolved in dichloromethane (2 mL), and then triethylamine (42 μL, 0.3 mmol) was added. After stirring at room temperature for 30 min, diphenylphosphonyl chloride (40 μL, 0.2 mmol) was added. The reaction was carried out at 50°C for 2 h. After the reaction was completed, it was monitored and confirmed by thin layer chromatography (TLC), and the solvent was evaporated. The residue was then purified by column chromatography to obtain a black solid QX-DP (30 mg, 42.3% yield).

[0046] The prepared QX-DP was tested and the 1 H NMR spectrum Figure 1 As shown, 1 H NMR (400MHz, DMSO-d6) δ8.83(d,J=8.8Hz,1H),8.61(d,J=9.2Hz,1H),8.55(d,J=14.8Hz,1H),8.43(d,J=9.2Hz,1H),8.30(d,J=7.2Hz, 1 H),8.10(t,J=8.0Hz,2H),7.97–7.90(m,3H),7.86(t,J=7.5Hz,1H),7.76–7 .39(m,1H),7.69–7.63(m,2H),7.60–7.56(m,4H),7.47(s,1H),7.32(d,J=8 .4Hz,1H),7.09(d,J=8.3Hz,1H),6.96(s,1H),6.86(d,J=15.2Hz,1H),4.98 –4.97(m,2H),2.60–2.61(m,4H),1.80–1.74(m,4H),1.54(t,J=7.1Hz,3H).

[0047] get 13 C NMR spectrum Figure 2 As shown, 13C NMR(100MHz,MeOD)δ156.3,156.1,154.3,153.2,143.3,143.0,139.6,138.3 ,136.9,135.6,134.2,132.7,132.6,132.1,132.0,131.8,131.2,130.6,130. 0,129.9,129.1,128.9,128.8,128.6,127.6,121.4,120.2,118.8,117.7,114 .0,112.4,109.4,46.9,30.1,25.2,21.3,13.7.HR-MS(ESI-TOF):calcd.forC 38 H 33 NO3P + [M+H] + 582.2193; found 582.2236.

[0048] Example 2: QX-DP for ONOO - Verification of the response mechanism:

[0049] In order to verify the effect of QX-DP on ONOO - The response mechanism of QX-DP was analyzed by high-resolution mass spectrometry, and the results were as follows Figure 3 As shown, with ONOO - After the reaction, the mass spectrum peak of QX-DP at m / z = 582.2236 ([QX-DP] + calcd:582.2193) disappeared, and a new peak appeared at m / z=382.1823 that was similar to QX-OH ([QX-OH] + calcd:382.1802,found:382.1828), which indicates that the peroxynitrite anion (ONOO - ) leads to the cleavage of the diphenyl phosphate moiety in QX-DP, forming a phenolic hydroxyl intermediate, which then undergoes a rapid elimination reaction to release the methylene quinone and give the highly fluorescent QX-OH, thus leading to the ONOO-dependent - The "on" fluorescence properties of Figure 4 shown.

[0050] Example 3: QX-DP for ONOO - Spectral response:

[0051] The effects of QX-DP on ONOO were studied in PBS buffer (pH 7.4, 10 mM, containing 40% DMSO). - The spectral response of Figure 8As shown in A, QX-DP itself is at 546nm (ε 546nm =2.07×10 4 Lmol -1 cm -1 ) shows the maximum absorption, and ONOO - After the reaction, the wavelength was red-shifted to 720nm (ε 720nm =3.61×10 4 Lmol -1 cm -1 ), and QX-OH(ε 720nm =4.52×10 4 L mol -1 cm -1 ) is close, and at the same time, the color of the solution changes from pink purple to blue green; Figure 8 As shown in B, QX-DP responds to different concentrations of ONOO at an excitation wavelength of 720 nm. - QX-DP showed very weak fluorescence at 752 nm. However, when ONOO - When the concentration of increases from 0 μM to 50 μM, the fluorescence increases by about 3.5 times; Figure 8 C, in the range of 0 to 20 μM (R 2 =0.9959), QX-DP fluorescence intensity (I 752nm ) and ONOO - There is an excellent linear relationship between the concentrations, and the detection limit (3 / slope) is 127.4nM;

[0052] In addition, the embodiment of the present invention also tracks the QX-DP to ONOO - The time-dependent fluorescence response of Figure 5 As shown in ONOO - In the presence of , the fluorescence intensity of QX-DP gradually increased and reached a plateau within 60 s, indicating that the probe can effectively monitor ONOO - Rapid fluctuations in biological systems;

[0053] The present invention also demonstrates that QX-DP can effectively inhibit ONOO in complex biological environments. - Highly selective, such as Figure 6 As shown; and under physiological conditions, the pH value is 7.4 and shows obvious fluorescence enhancement, as shown Figure 7 These results suggest that QX-DP monitors endogenous ONOO under physiological / pathological conditions. - There is huge potential in terms of volatility.

[0054] Example 4: Spectral response of QX-DP to viscosity:

[0055] In order to determine whether QX-DP responds to viscosity, the absorption and emission spectra were tested in different viscosity (η) systems, such as Figure 8 As shown in D, the maximum absorption wavelength of QX-DP in water is 540nm (ε 540nm =1.61×10 4 M –1 cm –1 ), while in glycerol it red-shifts to 574 nm (ε 574nm =1.70×10 4 M –1 cm –1 );like Figure 8 As shown in Figure E, with the increase of glycerol volume fraction, the fluorescence intensity of QX-DP at 668 nm increased by about 24 times, which may be due to the inhibition of TICT effect in high viscosity system; Figure 8 As shown in F, by fitting Equation (logI 668nm =1.0108logη+5.1194,R 2 =0.9858and logI 668nm =0.2520logη+5.6279,R 2 =0.9904), and when the viscosity was 0.89-5.04 cP and 5.04-945 cP, respectively, there was a good linear relationship between the fluorescence intensity (logI) and the viscosity (logη);

[0056] In addition, the absorption and emission spectra of QX-DP in solvents of different polarities are shown in Figure 2. Fig. 9 The viscosity-dependent fluorescence lifetime characteristics are shown in Fig.10 The effect of pH on fluorescence intensity in 0% glycerol and 90% glycerol is shown in Fig.11 As shown, it is further demonstrated that QX-DP has high selectivity and sensitivity to viscosity;

[0057] In summary, QX-DP has excellent fluorescence properties and a large Stokes shift of 94 nm, and is expected to be widely used in the field of biological imaging.

[0058] Example 5. Fluorescence imaging of QX-DP in living cells:

[0059] Cell culture and cytotoxicity assays:

[0060] Before cell fluorescence imaging, the cytotoxicity of QX-DP was first evaluated using the MTT method. The cytotoxicity of QX-DP on living HeLa cells was determined by a standard MTT assay. Approximately 1 × 10 4Cells / well were seeded with 200 μL of cell culture medium, and then the original culture medium was replaced with culture medium containing different concentrations of QX-DP (0, 0.1, 0.5, 1, 2, 5, 8, 10 μM) for 12 hours respectively; six replicate wells were used for each control and test concentration; after washing the cells three times with fresh culture medium, 20 μL of MTT and 180 μL of culture medium were added to each well and cultured for another 4 hours; the MTT solution was discarded, 200 μL of DMSO was added to each well, and the cells were shaken at a low speed on a shaker for 10 minutes; each well was then analyzed using an ELISA microplate reader, and the absorbance at a wavelength of 570 nm was detected. Cell viability was expressed based on the 100% metabolic activity of the control cells:

[0061] Cell activity (%) = (A withprobe -A blank ) / (A control -A blank )×100%

[0062] The result is as follows Fig.12 As shown, according to Fig.12 It can be seen that after treatment with QX-DP (10 μM), the survival rate of HeLa cells exceeded 89%, indicating that QX-DP has low cytotoxicity and can be used for cell imaging;

[0063] Cell imaging experiments:

[0064] To image ONOO in living cells - HeLa cells were incubated with QX-DP (5 μM) for 30 min as a control. Based on the effect of QX-DP on viscosity and ONOO - The remarkable dual-channel sensing properties of QX-DP were used to monitor viscosity and ONOO in living HeLa cells. - Changes, as intracellular viscosity stimulators monensin (Mon) or nystatin (Nys) can cause mitochondrial dysfunction and increased viscosity levels, the results are as follows Fig.13 As shown, the fluorescence signal emitted by cells treated with QX-DP alone was negligible, while significantly enhanced fluorescence emission was detected in cells pretreated with Mon- or Nys-, indicating that QX-DP has good visualization imaging capabilities for changes in intracellular viscosity.

[0065] To evaluate whether QX-DP can track ONOO in living cells - To further analyze the effect of QX-DP on exogenous / endogenous ONOO under different conditions -The fluorescence response of the cells was imaged by changes in fluorescence response. The treatment processes of the four groups were as follows: pretreatment with SIN-1 (3-morpholino norepinephrine, 100 μM) for 30 min, followed by addition of QX-DP (5 μM) and incubation for another 30 min; pretreatment with minocycline (100 μM) for 30 min, followed by treatment with SIN-1 (100 μM) for 30 min, followed by addition of QX-DP (5 μM) and incubation for 30 min; pretreatment with LPS (lipopolysaccharide, 100 μg / mL) for 4 h, followed by treatment with PMA (phorbol ester, 10 nM) and IFN-γ (interferon-γ, 50 ng / mL) for 30 min, followed by addition of QX-DP (5 μM) and incubation for 30 min; pretreatment with AG (semicarbazide, a kind of uric acid that reduces intracellular ONOO - The cells were pretreated with nitrite oxidase inhibitor (5 mM) for 30 min, followed by LPS (100 μg / mL) for 4 h, PMA (10 nM) and IFN-γ (50 ng / mL) for 30 min, and then QX-DP (5 μM) was added for 30 min. Fluorescence images were taken with ONOO - Channel(λ ex =633nm,λ em =700-754nm) was obtained on a confocal laser scanning microscope; the results are as follows Fig.14 As shown, compared with the weak fluorescence signal of control cells, the fluorescence of cells treated with SIN-1 (3-morpholinoimide hydrochloride, an ONOO - Brighter fluorescence was observed in cells pretreated with minocycline (an ONOO - After HeLa cells were stimulated with scavengers, the fluorescence in the cells disappeared, proving that QX-DP can monitor exogenous ONOO - In addition, HeLa cells were co-incubated with LPS (lipopolysaccharide), IFN-γ (interferon-γ) and PMA (phorbol ester) to verify the effect of QX-DP on endogenous ONOO - As expected, an enhanced fluorescence signal was detected, while AG (aminoguanidine, a nitric oxide synthase inhibitor, can reduce the intracellular ONOO - ) can effectively inhibit fluorescence enhancement. The above results show that QX-DP can image exogenous / endogenous ONOO in living cells. - .

[0066] Example 6. Simultaneous visualization of viscosity and ONOO in an epilepsy model - :

[0067] In the present embodiment, PTZ (pentylenetetrazole, a clinical acute epilepsy drug) was subcutaneously injected to establish an acute epilepsy mouse model. The control group of mice was subcutaneously injected with an equal amount of normal saline, and then the brain tissue was separated and the frozen sections were stained with QX-DP. The results are as follows: Fig.15 As shown, the control brain tissue showed weak fluorescence, while the epileptic brain tissue emitted strong fluorescence signals in both channels, showing the viscosity and ONOO in the brain of acute epileptic mice - The levels of viscosity and ONOO were increased for the first time during PTZ-induced acute seizures. - The obvious upregulation of the level proves that QX-DP has great potential in the visual diagnosis and pathological research of epilepsy.

[0068] Example 7. Simultaneous visualization of viscosity and ONOO in a non-alcoholic fatty liver disease model - :

[0069] The analysis of QX-DP can be performed by simultaneously detecting viscosity and ONOO - To diagnose non-alcoholic fatty liver disease (NAFL), a NAFL mouse model was established by feeding a high-fat diet and intraperitoneal injection of dexamethasone for 9 days. The control group mice were fed a normal diet and intraperitoneally injected with normal saline. Based on the advantages of NAC (N-acetyl-L-cysteine) in effectively reducing ROS and reducing viscosity in the body as a mucolytic agent, the treatment group achieved the treatment of non-alcoholic fatty liver disease (NAFL) by simultaneously administering NAC intragastrically. Subsequently, the three groups of mice were intravenously injected with QX-DP for 30 minutes, and then the livers were isolated and fluorescent imaging was performed. The results are as follows Fig.16 As shown in the figure, the dual-channel fluorescence intensity of the NAFL model group was significantly higher than that of the control group, indicating that the viscosity and ONOO in the liver of NAFL mice - At the same time, as expected, the fluorescence signal of the NAC-treated group was significantly weakened, further indicating that the dual-channel fluorescence enhancement was indeed due to the viscosity and ONOO induced by NAFL. - In addition, similar phenomena were also observed in NAFL liver tissue sections (e.g. Fig.17 As shown), it confirms that the viscosity and ONOO in NAFL - The levels were increased simultaneously, and the sensitivity of QX-DP for early diagnosis or screening of NAFL.

[0070] Example 8: Simultaneous monitoring of viscosity and ONOO during ferroptosis in tumor mice - Changes:

[0071] First, a cancer ferroptosis model was constructed by pretreating HeLa cells with erastin, a typical ferroptosis inducer. Fig.18As shown, HeLa cells treated with erastin showed significant differences in viscosity and ONOO compared with the control group. - The fluorescence emission in the two channels was enhanced, and Fer-1 (Ferrostatin-1, a ferroptosis inhibitor) could effectively reduce the fluorescence intensity of the two channels, confirming that the fluorescence increase was due to the viscosity and ONOO generated during ferroptosis. - Due to the increase in levels;

[0072] To further demonstrate the ability of QX-DP to visualize ferroptosis in vivo, dual-channel fluorescence imaging of ferroptosis within tumors in living mice was analyzed. Fig.19 As shown in A, compared with the fluorescence of blank mice (treated with saline only), a strong fluorescence signal was detected in the control mice (intratumorally injected with QX-DP), which was attributed to the viscosity and ONOO in the tumor. - Level increase (such as Fig.19 B); When the tumor was pretreated with erastin for 24 hours and then injected with QX-DP, the fluorescence intensity of the two channels increased significantly. Next, after the tumor was pretreated with erastin for 24 hours, it was treated with Fer-1 for 4 hours and then injected with QX-DP. The fluorescence intensity of the two channels in the tumor decreased significantly, further proving that intratumoral ferroptosis is accompanied by viscosity and ONOO - Viscosity and ONOO levels were observed non-invasively and directly during ferroptosis in living mice. - These simultaneous changes make QX-DP a highly promising dual-channel imaging tool for tracking ferroptosis-related pathological processes.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneous detection of ONOO - The dual-channel fluorescent probe QX-DP for spectroscopy and viscosity is characterized by: The chemical formula of the QX-DP is shown in Formula 1:

2. A method for simultaneously detecting ONOO as claimed in claim 1. - A method for preparing a dual-channel fluorescent probe with a viscosity of , characterized in that: Its chemical reaction formula is shown in Formula 2: The specific steps include: QX-OH was dissolved in a solvent, and then triethylamine was added. After stirring at room temperature, diphenylphosphonyl chloride was added to react. After the reaction was completed, the solvent was evaporated and the residue was purified to obtain a black solid QX-DP.

3. The preparation method according to claim 2, characterized in that: The solvent is one of acetonitrile, tetrahydrofuran, N,N-dimethylformamide and dichloromethane.

4. The preparation method according to claim 2, characterized in that: The reaction temperature is 0-80°C and the reaction time is 1-24h.

5. The preparation method according to claim 2, characterized in that: The molar ratio of QX-OH to diphenylphosphine chloride is 1:1-1:

20.

6. The preparation method according to claim 2, characterized in that: The purification is carried out by silica gel chromatography, and the solvent used is one or more of ethyl acetate, dichloromethane, methanol, acetone and petroleum ether.

7. Use of the fluorescent probe QX-DP as claimed in claim 1 in an epilepsy mouse model, a non-alcoholic fatty liver disease model or a tumor ferroptosis model.

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