A dual-channel fluorescent probe for simultaneous detection of ONOO − and viscosity and preparation method thereof

Abstract

The application is suitable for the field of fluorescence imaging technology, and provides a dual-channel fluorescent probe for simultaneously detecting ONOO ‑ and viscosity, and a preparation method thereof. The preparation method specifically comprises the following steps: dissolving QX-OH in a solvent, then adding triethylamine, stirring at room temperature, adding diphenyl phosphine chloride, carrying out reaction, evaporating the solvent after the reaction is completed, and purifying the residue to obtain black solid QX-DP. The application can simultaneously respond to two biomarkers of ONOO ‑ and viscosity, and generate significant dual-channel fluorescence signals at 668nm and 752nm respectively. The fluorescence probe not only can show its 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 provide more comprehensive information for disease diagnosis and image-guided surgery. The prepared QX-DP has low cytotoxicity and good biocompatibility, and the preparation method has the characteristics of simple synthetic route, high yield and simple operation.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence imaging technology, and particularly relates to a method for simultaneously detecting ONOO. - Dual-channel fluorescent probes with varying viscosity and their preparation methods. Background Technology

[0002] peroxynitrite ions (ONOO) - As an important endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS), it is generated by superoxide anion (O2). ·- The reaction between nitric oxide (NO) and nitrogen oxide (NO) in biological systems occurs rapidly, with a reaction rate of approximately 10. 10 M -1 s -1 It possesses strong oxidizing and nitrifying capabilities and is considered an important physiologically active substance and signaling molecule, helping to maintain redox homeostasis in organisms. However, excessive ONOO... - It can cause irreversible oxidative damage to organelles, proteins, and nucleic acids, leading to various diseases such as inflammation, neurodegenerative diseases, cardiovascular diseases, and even cancer. Viscosity, as a key indicator of the intracellular microenvironment of biological systems, plays a crucial role in physiological and pathological processes, including intracellular substance transport, signal transduction, and interactions between biomolecules. Abnormal changes in viscosity are considered important factors leading to liver damage, neurodegenerative diseases, and cancer. In summary, intracellular ONOO... - Viscosity is closely related to a variety of physiological / pathological processes.

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

[0004] Most fluorescent probes can only detect and image a single analyte; for example, Feng's team reported a probe capable of simultaneously detecting ONOO. -A dual-channel fluorescent probe (CQ) with good viscosity, but its resistance to ONOO - The response mechanism is based on the oxidative cleavage of ethylene bonds, which disrupts the probe's conjugated structure and limits fluorescence emission in the visible region; although mechanisms capable of extending ONOO have been developed. - ONOO launched at 645nm - And viscosity / polarity dual-channel probes, but these probes are in response to ONOO - Further improvements are needed to achieve stable ONOO. - Real-time detection; in bioimaging, highly sensitive and selective probes are needed to accurately detect biomarkers, but existing probes may fall short in these aspects; due to the lack of suitable multifunctional fluorescent probes, many diseases... - Simultaneous visualization of viscosity has not yet been achieved, for example in epilepsy and tumor ferroptosis models. Therefore, it is necessary to develop a method that can accurately detect viscosity and ONOO simultaneously. - This method is of great significance for the early diagnosis of diseases and the elucidation of their pathological mechanisms. Summary of the Invention

[0005] The purpose of this invention is to provide a method for simultaneously detecting ONOO. - A dual-channel fluorescent probe with high viscosity is intended to address the problems mentioned in the background art.

[0006] The present invention is implemented as follows: a method for simultaneously detecting ONOO. - A dual-channel fluorescent probe QX-DP with high viscosity, the chemical formula of which is shown in Formula 1:

[0007]

[0008] Another objective of this invention is to provide a method for simultaneously detecting ONOO. - The preparation method of the dual-channel fluorescent probe with high viscosity is shown in Formula 2:

[0009]

[0010] Specifically, the following steps are included:

[0011] QX-OH was dissolved in a solvent, then triethylamine was added, and after stirring at room temperature, diphenylphosphonic chloride was added to carry out the reaction. 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℃ and the time is 1-24h.

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

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

[0017] Preferably, the solvent used for purification is dichloromethane and methanol in a volume ratio of 10:1 to 60:1.

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

[0019] This invention provides a method for simultaneously detecting ONOO. - A dual-channel fluorescent probe with high viscosity can simultaneously respond to ONOO - The probe, containing two biomarkers (acid and viscosity), produced significant dual-channel fluorescence signals at 668 nm and 752 nm, respectively. This fluorescent probe not only demonstrated its application value in a mouse model of epilepsy but also proved successful in a non-alcoholic fatty liver disease (NAFL) model and a tumor ferroptosis model, providing more comprehensive information for disease diagnosis and image-guided surgery. The prepared QX-DP exhibited good sensitivity and selectivity, and could simultaneously and rapidly respond to ONOO. - It exhibits low viscosity and low cytotoxicity, good biocompatibility, and its preparation method features a simple synthetic route, high yield, and easy operation. Attached Figure Description

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

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

[0022] Figure 3 The high-resolution mass spectrometry (HR-MS) of QX-DP provided in Embodiment 2 of the present invention;

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

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

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

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

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

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

[0029] Figure 10 The fluorescence lifetime spectra of QX-DP (10 μM) provided in Example 4 of the present invention at different viscosities show that the fluorescence lifetime (τ) increased 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] Figure 11 The effect of pH on the fluorescence intensity of QX-DP (10 μM) at 668 nm in 0% and 90% glycerol, as provided in Example 4 of this invention, is λ. ex =574 / 668nm;

[0031] Figure 12 For the MTT assay results of QX-DP on HeLa cells provided in Example 5 of this invention, control group, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 8 μM, and 10 μM were set up respectively. The data are expressed as the mean ± standard error of three independent experiments, and each experiment was repeated three times.

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

[0033] Figure 14 A represents the cell imaging experiment results provided in Example 5 of this invention (ONOO) - The fluorescence image of HeLa cells incubated with QX-DP (5 μM) served as a control; the other four groups were pretreated with SIN-1 (3-morpholinoenoimide) (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); and pretreated with AG (aminourea, a drug that reduces intracellular ONOO).- Pretreatment with nitrite oxidase inhibitor (5 mM), LPS (100 μg / mL), IFN-γ (50 ng / mL), and PMA (10 nM) followed by QX-DP (5 μM) staining, ONOO - Channel: λ ex =633nm,λ em =700-754nm, scale bar: 20μm; Figure 14 B represents the average fluorescence intensity (±SD, n=3) of the image shown in A;

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

[0035] Figure 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 min after intravenous injection of QX-DP (100 μM, 200 μL). Viscosity channel: λ ex =580nm, λ em =670nm, ONOO - Channel: λ ex =680nm, λ em =790nm; Figure 16 B represents the average fluorescence intensity (±SD, n=3) of the image shown in A.

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

[0037] Figure 18 A shows the results of the cancer ferroptosis model provided in Example 8 of this invention, with HeLa cells incubated with QX-DP (5μM) as the control group; pretreated with erastin (a ferroptosis inducer) (50μM) and then added with QX-DP (5μM); and dual-channel fluorescence images of cells pretreated with Fer-1 (Ferrostatin-1, a ferroptosis inhibitor) (50μM) and erastin (50μM) and then added with QX-DP (5μM), respectively. Figure 18 B represents the average fluorescence intensity (±SD, n=3) of the image in A, viscosity channel: λ ex =561nm,λ em =600-690nm. ONOO - Channel: λ ex =633nm,λ em =700-754nm scale bar: 20μm;

[0038] Figure 19 A represents the in vivo dual-channel fluorescence imaging results in tumor-bearing mice provided in Example 8 of this invention. Saline was injected as a blank control, and QX-DP (100 μM, 30 min) was injected as a control group. After injection of erastin (2 mM, 24 h), QX-DP (100 μM, 30 min) was injected. Similarly, after injection of erastin (2 mM, 24 h) and Fer-1 (2 mM, 4 h), QX-DP (100 μM, 30 min) was injected. Viscosity channel: λ ex =580nm, λ em =670nm. ONOO - Channel: λ ex =680nm, λ em =790nm; Figure 19 B represents the average fluorescence intensity (±SD, n=3) of the image shown in A. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

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

[0042]

[0043] Specifically, the following steps are included:

[0044] First, the intermediate QX-OH was synthesized according to the reported method. Specifically, 2-methylquinoline and 2-hydroxy-4-methoxybenzaldehyde were reacted with iodoethane / 2-bromocyclohex-1-en-1-carboxaldehyde as starting materials. The two intermediates obtained were then 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, diphenylphosphonic 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 confirmed by thin-layer chromatography (TLC). The solvent was evaporated to dryness, and 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 obtained 1 H NMR spectrum as shown 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 The C NMR spectrum is as follows 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 to ONOO - Verification of the response mechanism:

[0049] To verify QX-DP's compatibility with ONOO - The response mechanism was investigated, and high-resolution mass spectrometry analysis of QX-DP was performed, yielding the following results: Figure 3 As shown, with ONOO - After the reaction, the mass spectrum peak of QX-DP at m / z = 582.2236 ([QX-DP]) was observed. + The peak at calcd:582.2193 disappears, and a new peak appears at m / z = 382.1823, corresponding to QX-OH ([QX-OH]). + The calcd:382.1802, found:382.1828 correspondence indicates that the peroxynitrite anion (ONOO) - The strong nucleophilicity and oxidizing properties of ) lead to the cleavage of the diphenyl phosphate moiety in QX-DP, forming a phenolic hydroxyl intermediate. This intermediate then undergoes a rapid elimination reaction, releasing methylenequinone and yielding the highly fluorescent QX-OH, thus resulting in an ONOO-dependent reaction. - The "on" fluorescence properties, such as Figure 4 As shown.

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

[0051] The effect of QX-DP on ONOO was investigated in PBS buffer (pH 7.4, 10 mM, containing 40% DMSO). - The spectral response, such as Figure 8As shown in Figure A, QX-DP itself is at 546nm (ε 546nm =2.07×10 4 Lmol -1 cm -1 (Displays maximum absorption, with ONOO) - After the reaction, the red shift was to 720 nm (ε 720nm =3.61×10 4 Lmol -1 cm -1 ), with QX-OH(ε 720nm =4.52×10 4 L mol -1 cm -1 As they approach each other, the color of the solution changes from pinkish-purple to blue-green; for example... Figure 8 As shown in Figure B, QX-DP responds to different concentrations of ONOO at a 720 nm excitation wavelength. - The fluorescence emission changes showed that QX-DP exhibited very weak fluorescence at 752 nm; however, when ONOO... - When the concentration was increased from 0 μM to 50 μM, the fluorescence increased by approximately 3.5 times; Figure 8 As shown in C, within the range of 0–20 μM (R 2 =0.9959), QX-DP fluorescence intensity (I 752nm ) and ONOO - There was an excellent linear relationship between concentrations, and the limit of detection (3 / slope) was 127.4 nM;

[0052] Furthermore, embodiments of the present invention also tracked QX-DP for ONOO. - Time-dependent fluorescence response, such as Figure 5 As shown, in ONOO - In the presence of [a specific substance], the fluorescence intensity of QX-DP gradually increased and reached a plateau within 60 seconds, indicating that this probe can effectively monitor ONOO. - Rapid fluctuations in biological systems;

[0053] The embodiments of this invention also demonstrate that QX-DP can inhibit ONOO in complex biological environments. - It has a high degree of selectivity, such as Figure 6 As shown; and under physiological conditions, it exhibits significant fluorescence enhancement at pH 7.4, such as Figure 7 As shown; these results indicate that QX-DP can monitor endogenous ONOO under physiological / pathological conditions. - It has enormous potential in terms of volatility.

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

[0055] To determine whether QX-DP responds to viscosity, absorption and emission spectra were tested in systems with different viscosities (η), such as... Figure 8 As shown in Figure D, the maximum absorption wavelength of QX-DP in water is 540 nm (ε). 540nm =1.61×10 4 M –1 ·cm –1 In glycerol, it redshifts to 574 nm (ε). 574nm =1.70×10 4 M –1 ·cm –1 );like Figure 8 As shown in E, the fluorescence intensity of QX-DP at 668 nm increased by approximately 24-fold with increasing glycerol volume fraction, which may be due to the suppression of the TICT effect in high-viscosity systems; 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 is between 0.89 and 5.04 cP and between 5.04 and 945 cP, respectively, there is a good linear relationship between fluorescence intensity (logI) and viscosity (logη);

[0056] Furthermore, the absorption and emission spectra of QX-DP in solvents of different polarities are as follows: Figure 9 As shown, the viscosity-related fluorescence lifetime characteristics are as follows: Figure 10 As shown, the effect of pH on fluorescence intensity in 0% glycerol and 90% glycerol is as follows: Figure 11 As shown, this further demonstrates that QX-DP exhibits high selectivity and sensitivity to viscosity;

[0057] In summary, QX-DP exhibits excellent fluorescence performance and a large Stokes shift of 94 nm, making it a promising candidate for widespread application in the field of bioimaging.

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

[0059] Cell culture and cytotoxicity assays:

[0060] Before cell fluorescence imaging, the cytotoxicity of QX-DP was first assessed using the MTT assay. The cytotoxicity of QX-DP against live HeLa cells was determined using a standard MTT assay in 96-well microplates, with approximately 1 × 10⁻⁶ cells per well. 4Cells were seeded per well in 200 μL of cell culture medium, and then the original medium was replaced with medium containing different concentrations of QX-DP (0, 0.1, 0.5, 1, 2, 5, 8, 10 μM) for 12 hours. Six replicates were used for each control and test concentration. After washing the cells three times with fresh medium, 20 μL of MTT and 180 μL of medium were added to each well, and the cells were cultured for another 4 hours. The MTT solution was discarded, and 200 μL of DMSO was added to each well, and the cells were shaken at low speed for 10 min. Each well was then analyzed using an ELISA microplate reader, measuring absorbance at 570 nm. Cell viability was expressed as a baseline of 100% metabolic activity of control cells.

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

[0062] The results are as follows Figure 12 As shown, according to Figure 12 As can be seen, 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. The effects of QX-DP on viscosity and ONOO were analyzed. - The significant dual-channel sensing characteristics of QX-DP were used to monitor viscosity and ONOO in live HeLa cells. - Changes in monensin (Mon) or nystatin (Nys), which act as intracellular viscosity stimulants, can cause mitochondrial dysfunction and an increase in viscosity levels, resulting in... Figure 13 As shown, cells treated with QX-DP alone emitted negligible fluorescence signals, while cells pretreated with Mon- or Nys- were found to have significantly enhanced fluorescence emission. This phenomenon indicates 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 - Further analysis of QX-DP levels under different conditions reveals exogenous / endogenous ONOO. -The fluorescence response of the imaging changes was analyzed using four treatment methods: Pretreatment with SIN-1 (3-morpholinoenoimide, 100 μM) for 30 min, followed by incubation with QX-DP (5 μM) for 30 min; Pretreatment with minocycline (100 μM) for 30 min, followed by treatment with SIN-1 (100 μM) for 30 min, followed by incubation with QX-DP (5 μM) 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 incubation with QX-DP (5 μM) for 30 min; Pretreatment with AG (aminourea, a drug that reduces intracellular ONOO). - Pretreatment with a nitrite oxidase inhibitor (5 mM) for 30 min, followed by treatment with LPS (100 μg / mL) for 4 h, PMA (10 nM) and IFN-γ (50 ng / mL) for 30 min, and then incubation with QX-DP (5 μM) for 30 min. Fluorescence images were obtained on ONOO. - Channel (λ) ex =633nm,λ em Obtained using a confocal laser scanning microscope (700-754 nm); results are as follows Figure 14 As shown, compared to the weak fluorescence signal in control cells, the fluorescence signal in cells treated with SIN-1 (3-morpholinoimide hydrochloride, an ONOO...) was significantly higher. - Brighter fluorescence was observed in cells pretreated with donor (a type of fluorescein); [the text abruptly ends here, likely due to an incomplete sentence or missing information.] - After stimulation of HeLa cells with a scavenger, intracellular fluorescence disappeared, demonstrating that QX-DP can monitor exogenous ONOO. - Changes; 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, enhanced fluorescence was detected, and AG (aminoguanidine, a nitric oxide synthase inhibitor, reduced intracellular ONOO) was observed. - This can effectively suppress fluorescence enhancement. The above results indicate that QX-DP can image the exogenous / endogenous ONOO of live cells. - .

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

[0067] In this embodiment of the invention, an acute epilepsy mouse model was established by subcutaneous injection of PTZ (pentylenetetrazole, a clinical acute epilepsy drug). Control mice were subcutaneously injected with an equal volume of physiological saline. Brain tissue was then isolated, and frozen sections were stained using QX-DP. The results are as follows: Figure 15 As shown, the brain tissue of the control group exhibited weak fluorescence, while the epileptic brain tissue emitted strong fluorescence signals in both channels, indicating the viscosity and ONOO of the brain in mice with acute epilepsy. - Elevated levels were observed for the first time, with PTZ-induced acute seizures accompanied by increased viscosity and ONOO. - The significant upregulation of levels demonstrates the great potential of QX-DP in the visual diagnosis and pathological research of epilepsy.

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

[0069] Analyzing whether QX-DP can simultaneously detect viscosity and ONOO - To diagnose non-alcoholic fatty liver disease (NAFL), a mouse model of NAFL was established by feeding mice a high-fat diet and administering dexamethasone intraperitoneally for 9 days. Control mice were fed a normal diet and received intraperitoneal injections of saline. Based on the advantages of NAC (N-acetyl-L-cysteine) in effectively reducing ROS and acting as a mucolytic agent to reduce in vivo viscosity, the treatment group achieved NAFL treatment by simultaneously administering NAC intragastricly. Subsequently, all three groups of mice were intravenously injected with QX-DP for 30 minutes. The livers were then dissected and fluorescence imaging was performed. The results are as follows: Figure 16 As shown, the dual-channel fluorescence intensity in the NAFL model group was significantly higher than that in the control group, indicating that the viscosity and ONOO in the liver of NAFL mice were significantly higher. - Levels of expression were elevated; simultaneously, as expected, the fluorescence signal in the NAC-treated group was significantly weakened, further indicating that the dual-channel fluorescence enhancement was indeed due to viscosity and ONOO induced by NAFL. - Elevated levels; in addition, similar phenomena were observed in NAFL liver tissue sections (e.g. Figure 17 As shown), this confirms the viscosity and ONOO in NAFL. - The levels also increased simultaneously, as did the sensitivity of QX-DP to drugs for early diagnosis or screening of NAFL.

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

[0071] First, a cancer ferroptosis model was constructed by pretreating HeLa cells with erastin (a typical ferroptosis inducer). Figure 18As shown, compared with the control group, HeLa cells treated with erastin showed improvements in viscosity and ONOO. - Enhanced fluorescence emission in the channels was observed, and Fer-1 (Ferrostatin-1, a ferroptosis inhibitor) effectively reduced the fluorescence intensity of both channels, confirming that the increased fluorescence was due to viscosity and ONOO generated during ferroptosis. - Due to the rise in level;

[0072] To further demonstrate QX-DP's ability to visualize ferroptosis in vivo, dual-channel fluorescence imaging of ferroptosis within tumors in live mice was analyzed, such as... Figure 19 As shown in Figure A, a strong fluorescence signal was detected in the control mice (intratumoral injection of QX-DP) compared to the fluorescence in blank mice (treated with saline only). This was attributed to the viscosity within the tumor and the presence of ONOO. - Horizontal adjustment (e.g.) Figure 19 B) After 24 hours of erastin pretreatment, QX-DP injection significantly enhanced the fluorescence intensity of both channels. Subsequently, after 24 hours of erastin pretreatment, followed by 4 hours of Fer-1 treatment and QX-DP injection, the dual-channel fluorescence intensity within the tumor significantly decreased, further demonstrating that intratumoral ferroptosis is accompanied by viscosity and ONOO. - Elevated levels. Viscosity and ONOO were observed non-invasively and directly in live mice during ferroptosis. - Simultaneous changes make QX-DP a highly promising dual-channel imaging tool that can be used to track pathological processes associated with ferroptosis.

[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 within the protection scope of the present invention.

Claims

1. A dual-channel fluorescent probe QX-DP for simultaneous detection of ONOO − and viscosity, characterized in that, The chemical formula of the QX-DP is shown as formula 1: ； Formula 1 The simultaneous detection of ONOO − A preparation method of a two-channel fluorescent probe for viscosity and viscosity, and a chemical reaction formula is shown as formula 2: Formula 2 Specifically comprising the following steps: Dissolve QX-OH in a solvent, then add triethylamine, after stirring at room temperature, add diphenyl phosphinic chloride, carry out reaction, after the reaction is completed, evaporate the solvent, and purify the residue to obtain black solid QX-DP.

2. The simultaneous detection of ONOO − and viscosity dual-channel fluorescent probe QX-DP, characterized in that, The solvent is acetonitrile, tetrahydrofuran, N,N - one of dimethylformamide, dichloromethane.

3. The simultaneous detection of ONOO according to claim 1 − and viscosity dual-channel fluorescent probe QX-DP, characterized in that, The temperature of the reaction is 0-80 DEG C, and the time is 1-24 h.

4. The simultaneous detection of ONOO according to claim 1 − and viscosity dual-channel fluorescent probe QX-DP, characterized in that, The molar ratio of the QX-OH and diphenyl phosphinic chloride is 1:1-1:

20.

5. The simultaneous detection of ONOO according to claim 1 − and viscosity dual-channel fluorescent probe QX-DP, characterized in that, The purification is carried out by silica gel chromatography, and one or more of the following solvents is used: ethyl acetate, dichloromethane, methanol, acetone and petroleum ether.

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