Double-targeting probe as well as preparation method and application thereof

Abstract

The invention discloses a double-targeting probe and a preparation method and application thereof.The structure of the double-targeting probe is shown in the formula I. The double-targeting probe has the beneficial effects that SO2 can be monitored in the cell apoptosis process, the dynamic change of SO2 between mitochondria and lipid droplets in cells can be tracked, the core structure of the probe MLR is based on a coumarin part, and the core structure of the probe MLR can be used for detecting the apoptosis of the cells. The probe MLR is connected with a benzopyran cation structure specially designed for detecting SO2, SO2 generated in the death process of copper enables charges of a cation part in the probe MLR to be redistributed, a Michael addition reaction of SO2 and carbon-carbon double bonds is triggered, a fluorescence signal of the probe is changed, and the fluorescence signal is detected. The design of the probe provides a new tool for monitoring key biochemical changes in the copper death process, and is helpful for deeply understanding the molecular mechanism of copper death.

Description

Technical Field

[0001] This invention relates to the field of biochemistry. More specifically, this invention relates to a dual-targeting probe, its preparation method, and its uses. Background Technology

[0002] Copper death manifests as copper-dependent cell death through interconnected mechanisms: excess Cu 2+ Binding to lipid-bound tricarboxylic acid cycle (TCA) proteins induces aggregation and instability of iron-sulfur clusters, while ferroredoxin-1 (FDX1) mediates Cu... + Copper death amplifies protein toxicity through pyruvate dehydrogenase esterification. Copper overload can trigger Fenton-like reactions, generating reactive oxygen species (ROS) that, in conjunction with protein toxicity stress, disrupt the hub of ROS metabolism and sulfur antioxidant synthesis in mitochondria (MITs). Mitochondrial dysfunction depletes SO2 (a key ROS scavenger), thereby exacerbating oxidative damage and disrupting redox balance. Simultaneously, mitochondrial damage makes copper death metabolism dependent on lipid droplets (LDs), leading to lipid droplet accumulation and lipotoxicity, revealing the close mitochondrial-lipid droplet relationship during copper death. While sulfur antioxidants such as SO2 can mitigate oxidative stress, their depletion during cell death can trigger a self-sustaining cycle of redox collapse. Therefore, tools are needed to dynamically map organelle interactions and sulfur metabolism processes during copper death.

[0003] Given the complexity of this mechanism, fluorescent probes have become an indispensable tool for dynamically tracking copper ions and their metabolites due to their non-invasiveness, real-time sampling capability, high sensitivity, and superior spatiotemporal resolution. Recently, a large number of SO2 fluorescent probes targeting mitochondria or lipid droplets have been developed. For example, bifunctional probes such as TPA-SO2 for dual-parameter imaging of lipid droplet polarity and SO2 during ferroptosis, and AND-logic-gate probes for the synergistic detection of mitochondrial bisulfite and viscosity, have all demonstrated high specificity and ultra-low detection limits. It is necessary to investigate the relationship between copper death and organelles. To our knowledge, the use of fluorescent probes monitoring SO2 kinetics to explore the mitochondrial-lipid droplet interaction during copper death has not been reported. To effectively track these interactions during copper death, an ideal probe should possess the following characteristics: (i) dual targeting capability for mitochondria and lipid droplets; (ii) rapid SO2 responsiveness; and (iii) high specificity and sensitivity to SO2. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0005] Another objective of this invention is to provide a dual-targeting probe, its preparation method, and its uses. The dual-targeting probe is a probe MLR, which can monitor SO2 during apoptosis and track the dynamic changes of SO2 between mitochondria and lipid droplets within the cell. The core structure of the probe MLR is based on a coumarin moiety, which is linked to a benzopyran cation structure specifically designed for SO2 detection. The SO2 generated during copper death causes a redistribution of charge in the cation moiety of the probe MLR, triggering a Michael addition reaction between SO2 and carbon-carbon double bonds, resulting in a change in the probe's fluorescence signal. The design of this probe provides a new tool for monitoring key biochemical changes during copper death and helps to deepen the understanding of the molecular mechanism of copper death.

[0006] To achieve these objectives and other advantages according to the present invention, a dual-targeting probe is provided, the structure of which is shown in Formula I:

[0007]

[0008] The preparation method of the dual-targeting probe includes the following steps:

[0009] S1. Reaction of 7-(diethylamino)-2-oxo-4-(phenylethynyl)-2H-benzopyran-3-carboxaldehyde with resorcinol yields the first compound;

[0010] S2. React 4-(diethylamino)salicylaldehyde with p-piperazine phenylacetyl to obtain the second compound;

[0011] S3. Take the first compound and react it with the second compound to obtain the dual-targeting probe.

[0012] Preferably, 7-(diethylamino)-2-oxo-4-(phenylethynyl)-2H-benzopyran-3-carboxaldehyde and resorcinol are dissolved in a solvent, and sodium hypochlorite aqueous solution and sodium dihydrogen phosphate aqueous solution are slowly added dropwise at 0°C. After stirring in an ice bath for 10-20 minutes, a reaction solution is obtained. The reaction solution is then poured into ice water to precipitate the target product, which is the first compound. The molar ratio of 7-(diethylamino)-2-oxo-4-(phenylethynyl)-2H-benzopyran-3-carboxaldehyde, resorcinol, sodium hypochlorite, and sodium dihydrogen phosphate is 0.1:1.97:0.35:0.45.

[0013] Preferably, step S2 specifically involves: dissolving 4-(diethylamino)salicylaldehyde and p-piperazine phenylacetyl in concentrated sulfuric acid, reacting at 90°C for 6 hours, cooling to room temperature to obtain a cooling solution; pouring the cooling solution into ice water at 0°C, adding a 70% perchloric acid solution by mass and stirring, observing the precipitation of a green solid, filtering and drying to obtain a crude solid product, and separating the crude solid product by column chromatography to obtain the second compound; wherein the molar ratio of 4-(diethylamino)salicylaldehyde to p-piperazine phenylacetyl is 1:1.

[0014] Preferably, step S3 specifically involves dissolving the first compound, the second compound, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in a solvent, reacting under nitrogen protection for 24 hours, removing the solvent under reduced pressure to obtain a crude product, and separating the crude product by column chromatography to obtain a purple solid, which is the dual-targeting probe; wherein the molar ratio of the first compound, the second compound, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.5:0.5:0.05:1.

[0015] The dual-targeting probe is used to monitor the dynamic changes of SO2 during copper death in living cells in real time.

[0016] The dual-targeting probe is used for real-time monitoring of the interaction between mitochondria and lipid droplets in living cells.

[0017] A test strip comprising the aforementioned dual-targeting probe.

[0018] The present invention has at least the following beneficial effects:

[0019] First, this invention provides a dual-targeting probe, namely a probe MLR, which can monitor SO2 during apoptosis and track the dynamic changes of SO2 between mitochondria and lipid droplets in the cell. The core structure of the probe MLR is based on the coumarin moiety and is linked to a benzopyran cation structure designed specifically for SO2 detection. The SO2 generated during copper death causes a redistribution of charge in the cation moiety of the probe MLR, triggering a Michael addition reaction between SO2 and carbon-carbon double bonds, resulting in a change in the probe's fluorescence signal. The design of this probe provides a new tool for monitoring key biochemical changes during copper death and helps to deepen the understanding of the molecular mechanism of copper death.

[0020] Secondly, the dual-targeting probe provided by this invention has a detection limit of 0.34 μM for SO2, a response time of less than 10 seconds, and fluorescence stability of better than 95% in the pH range of 6-8.

[0021] Third, the dual-targeting probe provided by this invention has many application scenarios. For example, it can be made into test strips, fluorescent hydrogels and other products for the detection and identification of SO2. It is convenient to use and has good detection effect, and has good market application prospects.

[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the mechanism of SO2 specific detection by the probe MLR.

[0024] Figure 2 Figure A shows the molecular orbital diagram of fluorescence changes caused by the fluorescence resonance energy transfer (FRET) mechanism, and Figure B shows the HOMO and LUMO band gaps of MLR and MLR-SO2. In the figure, bound-form represents the orbital state of the molecule in the interaction or excited state, apo-form represents the frontier orbital state of the molecule when it is isolated, wavelength represents wavelength, fluorescence intensity represents fluorescence intensity, abs represents absorbance, acceptor (D) represents electron acceptor, donor (A) represents electron donor, and energy represents energy.

[0025] Figure 3 The ESP plots of MLR and MLR-SO2 were obtained through DFT calculation;

[0026] Figure 4 The graph shows the UV absorption changes in response of probe MLR (10 μM) and SO2 (100 μM); where wavelength is wavelength and absorbance is absorbance.

[0027] Figure 5 Figure A shows the fluorescence emission spectra of MLR before and after reaction with SO2 in HEPES buffer; Figure B shows the time-dependent fluorescence changes of MLR in response to SO2 in HEPES buffer at 37℃; Figure C shows the fluorescence intensity changes of SO2 (0–100 μM) on MLR (10 μM); Figure D shows the linear relationship of the fluorescence intensity ratio (I575 / I637) of SO2 concentration (1.0–100 μM); where wavelength is wavelength, absorbance is absorbance, FL. Intensity is fluorescence intensity, concentration is concentration, and I575 is fluorescence intensity. 575 I represents the fluorescence intensity at 575 nm. 637 The value represents the fluorescence intensity at 637 nm; Probe is the probe.

[0028] Figure 6 The graph shows the fluorescence intensity results of the probe MLR (10 μM) and different analytes (100 μM) after 1 min.

[0029] Figure 7 Figure A shows the response of probes MLR (10 μM) and SO2 (100 μM) under different pH conditions, and Figure B shows the fluorescence stability of probes MLR and MLR-SO2 within 5 min. (I) 575 I represents the fluorescence intensity at 575 nm. 637 This indicates the fluorescence intensity at 637 nm;

[0030] Figure 8 The image shows the intracellular photostability of MLR (10 μM) after 25 min of continuous laser irradiation.

[0031] Figure 9 Figure A shows the fluorescence imaging results of HeLa cells incubated with different concentrations of exogenous and endogenous SO2, scale bar: 50μm; Figure B, from left to right, shows the first and second figures quantified data from the two fluorescence channels in Figure A (the vertical axis of the first and second figures is the measured fluorescence intensity), and the third figure shows the cell viability results of HeLa and MCF-10A cells incubated with different concentrations of the MLR probe, with error bars representing mean ± standard deviation (n=3); where Orange Channel represents the orange fluorescence channel, Red Channel represents the red fluorescence channel, and CellViability represents cell viability;

[0032] Figure 10 Figure A shows the results of tracking SO2 fluorescence changes in cells using MLR, scale bar: 5μm; Figure B quantifies the data from the two fluorescence channels in Figure A, with error bars representing mean ± standard deviation (n=3); Red Channel represents the red fluorescence channel, and Orange Channel represents the orange fluorescence channel; the vertical axis of both graphs in Figure B represents fluorescence intensity.

[0033] Figure 11 Figure A shows Cu observed using a fluorescence confocal laser microscope. 2+Figure B shows the co-staining results of HeLa cells treated with +ES and MLR, MTB (Ai, left), and BODIPY 630 / 650X (A-ii, right). Scale bar: 2μm. Figure B shows the changes in subcellular organelle targeting co-localization coefficients and the quantitative results of fluorescence channel data. From left to right in Figure B, the figures correspond to Figure Ai at different time points and show the co-localization curves and fluorescence intensity quantification of commercial mitochondrial dye (Mito) and MLR, and Figure A-ii at different time points and show the co-localization curves and fluorescence intensity quantification of commercial lipid droplet dye (LD) and MLR+SO2. Error bars represent mean ± standard deviation (n=3). Among them, Mito represents the fluorescence channel for mitochondrial targeting, MLR represents the fluorescence channel for the targeting probe, Merge represents the combined fluorescence channel diagram, Correlation represents the Pearson correlation coefficient, MLR-SO2 represents the channel after the probe reacts with SO2, and LD represents the fluorescence channel for the targeting lipid droplets. Figure 11 In Figure B, the vertical axis of each of the four graphs from left to right represents fluorescence intensity.

[0034] Figure 12 Figure A shows the distribution of ROS and its corresponding fluorescence intensity in DCFH-DA stained cells of different treatment groups detected by laser confocal microscopy (scale bar: 20 μm); Figure B shows the Western blot detection of DLAT, FDX1, and LIAS proteins; Figure C shows the assessment of cell viability in different treatment groups by calcein-AM (green, live) / PI (red, dead) co-staining (scale bar: 50 μm); Figure D shows the immunofluorescence image of HeLa cells stained with anti-DLAT antibody (green) and Hoechst 33342 (blue); Figure E shows the quantification of ROS levels in different cell treatment groups by flow cytometry (flow cytometry x-axis represents fluorescence intensity); Figure F shows the detection of ROS levels under different conditions (Cu) by Annexin V-FITC / PI method. 2+ ES, Cu 2+ +ES、Cu 2+ The apoptosis / necrosis ratio under (+ES+TTM) conditions is shown in the first four figures from left to right, representing HeLa cells under different conditions (from left to right: Cu...). 2+ ES, Cu 2+ +ES、Cu 2+ The fifth figure shows the apoptosis rate of the four treatment groups (+ES+TTM) using flow cytometry analysis.

[0035] Figure 13 Figure A shows the fluorescence changes in zebrafish monitored by MLR in response to exogenous / endogenous SO2, and Figure B shows the fluorescence intensity analysis of the fluorescence channels in Figure A. The error bars represent the mean ± standard deviation (n = 3).

[0036] Figure 14Figure A shows the quantitative detection of SO2 production in zebrafish under different copper death-inducing / inhibiting treatment groups using MLR; Figure B shows the fluorescence intensity analysis of the fluorescence channels in Figure A, with error bars representing the mean ± standard deviation (n = 3).

[0037] Figure 15 A represents Cu 2+ +ES treatment: dynamic changes in SO2 production in zebrafish. Figure B shows the fluorescence intensity analysis of the fluorescence channels in A. Scale bar: 500 μm (red channel: λ) ex =570nm, λ em =600-700nm; Orange channel: λ ex =455nm, λ em =500-600nm). The error bars represent the mean ± standard deviation (n=3);

[0038] Figure 16 Figure A shows the relationship between MLR concentration and RGB color information; Figure B shows the functional relationship between MLR concentration and photographic R / G value information; Figure C shows the relationship between SO2 concentration and RGB color information; Figure D shows the functional relationship between SO2 concentration and photographic R / G value information. The error bars represent the mean ± standard deviation (n = 3); where Probe represents a probe.

[0039] Figure 17 Figure A shows the colorimetric images of MLR test strips treated with different concentrations of MLR and SO2 under natural and ultraviolet (365nm) light. Figure B shows the CIE colorimetric R / G values ​​of the corresponding test strips in Figure A, which are analyzed using the Color recognition app and smartphone. Figure C shows the colorimetric images of MLR (10μM) and MLR test strips treated with different concentrations of SO2 under natural and ultraviolet (365nm) light. Figure D shows the CIE colorimetric R / G values ​​of the corresponding test strips in Figure C, which are analyzed using the Color recognition app and smartphone. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0041] <Preparation of Dual-Target Probes>

[0042] 1. Synthesis of the first compound

[0043] 7-(diethylamino)-2-oxo-4-(phenylethynyl)-2H-benzopyran-3-carboxaldehyde (34.5 mg, 0.1 mmol) and compound 2 (217 mg, 1.97 mmol) were dissolved in 5 mL of acetonitrile. Sodium hypochlorite (42 mg, 0.35 mmol) and sodium dihydrogen phosphate (41 mg, 0.45 mmol) aqueous solutions were slowly added dropwise at 0 °C, and the mixture was stirred in an ice bath for 15 min. After the reaction was complete, the reaction solution was poured into 20 mL of ice water, and the target product, the first compound, precipitated. The product from this step can be used directly in the next reaction without purification. Yield: 95%. The synthetic route of the first compound is shown below: Wherein, the compound above number 1 is 7-(diethylamino)-2-oxo-4-(phenylethynyl)-2H-benzopyran-3-carboxaldehyde, the compound above number 2 is resorcinol, and the compound above number 3 is the first compound.

[0044]

[0045] The characterization data of the first compound obtained by nuclear magnetic resonance (NMR) of the proton spectrum are as follows: 1 H NMR (600MHz, CDCl3) δ10.40(s,1H),7.94(d,J=9.1Hz,1H),7.75(d,J=7.1Hz,2H),7.49(d,J=7.3Hz,1H),7.45(t ,J=7.4Hz,2H),6.71(dd,J=9.2,2.3Hz,1H),6.46(d,J=2.3Hz,1H),3.49(q,J=7.1Hz,4H),1.27(t,J=7.1Hz,6H).

[0046] The characterization data of the first compound obtained by nuclear magnetic resonance carbon spectrum are as follows: 13 C NMR (600MHz, CDCl3)δ

[0047] 189.78,187.70,160.70,157.43,153.20,132.84,131.46,130.72,130.38,128.75,128.17,126.88,121.49,113.89,110.15,108.97,96.99,83.40,45.30,12.52.

[0048] The calculated value C of the first compound was obtained by high-resolution electrospray ionization mass spectrometry. 22 H 19 NO4[M+Na] + 384.1206; Measured value: 384.1202. (HRMS(ESI)m / z: calculated for C)22 H 19 NO4[M+Na] + :384.1206.;found:384.1202)

[0049] 2. Synthesis of the second compound

[0050] Compound 4-(diethylamino)salicylaldehyde (193 mg, 1 mmol) and p-piperazine phenylacetyl (204 mg, 1 mmol) were dissolved in 5 mL of concentrated sulfuric acid and refluxed at 90 °C for 6 h. After the reaction was completed, the mixture was cooled to room temperature, poured into 30 mL of ice water, and 0.5 mL of perchloric acid (70% by mass) was added and stirred. A green solid precipitated out, which was filtered and dried to obtain a crude solid product. The crude solid product was then purified by silica gel (200-300 mesh, eluent: V). DCM :V MeOH The second compound was obtained by column chromatography (20 / 1). The second compound was a purple solid, 185.16 mg, yield 80.46%. The synthetic route of the second compound is shown below: where the compound above number 4 is 4-(diethylamino)salicylaldehyde, the compound above number 5 is p-piperazine phenylacetyl, and the compound above number 6 is the second compound.

[0051]

[0052] The characterization data of the second compound obtained by nuclear magnetic resonance (NMR) of the proton spectrum are as follows: 1 H NMR (600MHz, DMSO) d6 )δ8.81(s,1H),8.65(d,J=8.4Hz,1H),8.28(d,J=8.7Hz,2H),7.98(d,J=8.3Hz,1H),7.91(d,J=9.3Hz,1H),7.38(d,J=9.3 Hz,1H),7.30(s,1H),7.21(d,J=8.6Hz,2H),3.77(d,J=4.2Hz,4H),3.68(d,J=6.6Hz,4H),3.32–3.27(m,4H),1.25(s,6H).

[0053] The characterization data of the second compound obtained by nuclear magnetic resonance carbon spectrum are as follows: 13 C NMR (600MHz, DMSO) d6 )δ167.08,158.70,155.65,154.26,148.49,132.35,130.96,118.31,117.27,114.65,108.55,96.36,45.76,43.86,42.98,12.92.

[0054] The calculated value C of the second compound was obtained by high-resolution electrospray ionization mass spectrometry. 23 H 28 N3O + [M+H] + :362.2227.;Measured value:362.2234 (HRMS(ESI)m / z: calculated for C 23 H 28 N3O + [M+H] + :362.2227.;found:362.2234).

[0055] 3. Synthesis of dual-targeting probes (i.e., probe MLR)

[0056] The first compound (18 mg, 0.05 mmol), the second compound (198 mg, 0.05 mmol), 4-dimethylaminopyridine (0.61 mg, 0.005 mmol, DMAP), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (19 mg, 0.1 mmol, EDCI) were dissolved in 2 mL of anhydrous dichloromethane and stirred at room temperature for 24 h under N2 protection. The solvent was removed under reduced pressure to obtain the crude product. The crude product was separated by column chromatography to obtain silica gel (200-300 mesh). (Leaching: V) DCM :V MeOH =20 / 1) Dual-targeting probe MLR, the probe MLR is a purple solid, 213 mg, yield 85.14%. The structural formula of the probe MLR is as follows:

[0057]

[0058] The characterization data of the proton spectrum obtained by probe MLR via nuclear magnetic resonance are as follows: 1 H NMR (600MHz, DMSO) d6)δ8.63(d,J=8.4Hz,1H),8.25(d,J=9.0Hz,2H),7.96(d,J=8.4Hz,1H),7.90(d,J=9.3Hz,1H) ,7.78(d,J=9.0Hz,1H),7.62(d,J=7.1Hz,2H),7.52–7.48(m,1H),7.46(t,J=7.5Hz,2H),7.36 (d,J=9.3Hz,1H),7.29(s,1H),7.16(d,J=9.1Hz,2H),6.84(d,J=8.9Hz,1H),6.63(d,J=2.0Hz ,1H),3.67(d,J=6.8Hz,8H),3.59–3.42(m,8H),1.23(t,J=6.0Hz,6H),1.16(t,J=7.0Hz,6H).

[0059] The characterization data of the carbon spectrum obtained by probe MLR via nuclear magnetic resonance are as follows: 13 C NMR (600MHz, DMSO) d6 )δ167.45,163.51,158.68,158.43,156.02,155.51,154.79,151.80,148 .41,133.33,132.60,132.26,131.68,131.17,129.56,129.12,128.46,12 6.88, 120.73, 118.44, 117.56, 117.04, 116.94, 114.39, 110.19, 108.66, 106.76, 104.28, 82.36, 47.32, 46.27, 45.67, 44.74, 32.01, 29.89, 12.79.

[0060] The calculated value of C obtained by probe MLR via high-resolution electrospray ionization mass spectrometry 45 H 45 N4O4 + [M+H] + :705.3435.;Measured value:705.3439 (HRMS(ESI)m / z: calculated for C 45 H 45 N4O4 + [M+H] + :705.3435.;found:705.3439)

[0061] <Design Principles of MLR Probes>

[0062] Coumarin nuclei possess superior optical properties, including high quantum yield and ease of structural modification. The cationic structure can effectively target mitochondria, while the neutral lipophilic molecule can trace LDs (lipid droplets). Utilizing these properties, we developed a dual-targeting organelle fluorescent probe, MLR, for SO2 detection. MLR is primarily based on a donor-acceptor (D-π-A) structure: a quinoline group acts not only as a FRET (fluorescence resonance energy transfer) acceptor but also as a mitochondrial (mitochondrial) targeting group; a benzopyranium cation interacts with the negatively charged surface of the lipid droplets for targeting, and the pyran ring serves as a specific recognition site for SO2. Figure 1 As shown, the probe itself exhibits red fluorescence. Due to FRET, upon response to SO2, the double bond breaks, the cation transforms into a neutral lipophilic structure, and after a Michael addition reaction, it emits the yellow fluorescent compound MLR-SO2. Figure 1 In this context, "Mitochondria targeting" refers to mitochondrial targeting, and "Lipid droplet targeting" refers to lipid droplet targeting.

[0063] <Study on the Response Mechanism of Probe MLR and SO2>

[0064] I. To confirm the interaction mechanism between MLR and SO2, HRMS and density functional theory (DFT) calculations were used. HRMS analysis confirmed that the primary product of the reaction between MLR and SO2 is MLR-SO2, with a mass-to-charge ratio of 770.3138. Figure 2 The MLR shown is based on the FRET principle, using the coumarin core as the energy donor and the benzopyran salt structure as the energy acceptor and SO2 reaction site. Upon reaction with SO2, the acceptor structure is disrupted, FRET ceases, and a change in radiofluorescence at two wavelengths is induced, thus enabling the radiometric detection of SO2. The excited-state fluorophore donor transfers an electron to the lowest unoccupied molecular orbital (LUMO) of the acceptor. Due to its electron-rich nature, the SO2 ion undergoes a Michael addition reaction with the electron-deficient benzopyran cation, leading to intramolecular charge transfer and a shift in the fluorescence signal. DFT calculations show that compared to MLR-SO2, the HOMO (highest occupied molecular orbital)-LUMO band gap in the MLR is smaller, resulting in a significant shift in the emission wavelength. Furthermore, the electrostatic potential (ESP) plot shows that charge separation is more pronounced in MLR-SO2, with a lower ESP value at the reaction site, indicating higher affinity for the reaction site and confirming the proposed mechanism. Figure 3 ).

[0065] II. Spectroscopic Testing of Probe MLR

[0066] 1. Solution preparation

[0067] Preparation of HEPES buffer (10mM, pH=7.4, containing 10% DMSO): Prepare 10mM HEPES buffer (pH=7.4), mix HEPES buffer and DMSO at a volume ratio of 9:1 (e.g., 9mL buffer + 1mL DMSO) to obtain HEPES buffer containing 10% DMSO.

[0068] Preparation of MLR probe stock solution: Dissolve the MLR probe in dimethyl sulfoxide (DMSO) to prepare a 1M solution, and store it in a refrigerator at 4°C for later use.

[0069] Preparation of analyte solutions: Weigh appropriate amounts of homocysteine ​​(Hcy), cysteine ​​(Cys), glutathione (GSH), lysine (Lys), alanine (Ala), glycine (Gly), isoleucine (Ile), proline (Pro), glutamic acid (Glu), arginine (Arg), aspartic acid (Asp), KNO3, MgCl2, KI, NaAc, ZnCl2, HClO, NaNO2, 30% H2O2, Na2S2O3, H2S, Na3PO4, and NaHSO3 in sequence and dissolve them in ultrapure water to prepare a 1M solution for later use.

[0070] Preparation of the test system: Measure 20 μL of MLR probe stock solution with a pipette, add 100 μL of 100 μM analyte solution, and then add HEPES buffer to 2 mL. Mix well and then perform spectral determination.

[0071] 2. Spectral testing

[0072] (1) Measurement of absorption and fluorescence spectra

[0073] NaHSO3 (as a SO2 donor) was dissolved in ultrapure water to prepare a 1M NaHSO3 solution. The MLR probe stock solution (1M) was diluted to 10μM in HEPES buffer. After adding 100μL of the 100μM NaHSO3 solution, UV and fluorescence spectroscopy measurements were performed. In the spectral testing experiment, λ... ex =455nm, λ em =490-590nm. See the UV absorption variation graph. Figure 4 The results of fluorescence property detection are shown in [the table below]. Figure 5 .

[0074] Results analysis: such as Figure 4 As shown, the probe MLR has a strong absorption band centered at 575 nm, and the addition of HSO3... - Subsequently, the maximum absorption peak at 575 nm gradually decreases, while a new absorption band centered at 455 nm rises significantly. This absorption change from 575 nm to 455 nm can be attributed to HSO3.- Changes in the FRET effect caused by the double bond addition reaction.

[0075] like Figure 5 As shown in Figure A, the probe itself exhibits red fluorescence, which is altered by FRET. (This is followed by a seemingly unrelated sentence about 100 μM HSO3.) - After the reaction, the absorption rate of MLR decreased at 637 nm, while a new absorption band centered at 575 nm increased significantly.

[0076] The reaction kinetics of MLR and SO2 were determined by monitoring fluorescence intensity at different time intervals. The reaction reached a steady-state concentration within 30 seconds. Figure 5 B). To determine the optimal detection range and limit of detection (LOD), the MLR was titrated with different concentrations of SO2 (from NaHSO3 solution), and it increased with increasing SO2 concentration. Figure 5 C). The fluorescence intensity of MLR is in the range of 1-100 μM SO2(R 2 =0.996, Figure 5 The fluorescence exhibits a linear response within the range of D), with a LOD of 0.34 μM (from the formula LOD = 3σ / s, where σ is the standard deviation of the blank measurement (n = 10), and s is the slope between the corresponding fluorescence intensity and the analyte concentration).

[0077] (2) Selective testing (specificity study of probe MLR and SO2 response)

[0078] Methods: The prepared analyte solution was diluted with HEPES buffer to a 10 mM solution. 3 μL of the LMLR probe stock solution was added to 3 mL of HEPES buffer to prepare the working probe solution. Then, 9 μL of different analyte solutions were added to the working probe solution to prepare 30 μM selective analyte solutions. After standing for 1 min, a series of selective spectral measurements were performed. ex =455nm. See details in [link / reference]. Figure 6 .

[0079] like Figure 6 As shown, in λ ex HSO3 at 455nm - The induced MLR fluorescence was significantly increased, while the fluorescence response to other amino acids (Lys, Arg, Gly, Ile, Ala, Pro, Arg, Glu, Asp, GSH, and Hcy, where GSH is glutathione and Hcy is homocysteine) and biothiols (H2S, Na2S2O3, and Cys) was negligible. The experimental results confirm that the probe MLR can specifically recognize SO2.

[0080] (3) pH stability of probe MLR

[0081] Methods: Two 2 mL solvent systems (1 / 9 volume ratio) were prepared by mixing DMSO and 10 mM HEPES solution. These systems were then adjusted to pH values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 by adding appropriate amounts of NaOH / HCl. One system was mixed with 10 μL of 10 μM probe MLR solution, and the fluorescence intensity of the mixed solution was measured at each pH. The other system was mixed with 10 μL of 10 μM probe MLR stock solution and 50 μL of freshly prepared 100 μM NaHSO3 solution. After standing for 10 min, the fluorescence intensity of the mixed solution at 575 nm and 637 nm was measured again at each pH. Results are shown below. Figure 7 .

[0082] like Figure 7 As shown in Figure A, the red fluorescence of the MLR probe itself remained essentially unchanged within the pH range of 1-14, indicating that the probe has good stability. Furthermore, the fluorescence intensity of the MLR-SO2 reactant with added SO2 increased significantly within the pH range of 6-8, indicating that the MLR probe can detect SO2 under physiological conditions.

[0083] (4) Test of light stability

[0084] Add 5 μL of 1M MLR probe stock solution to 5 mL of HEPES buffer, then add 25 μL of 100 μM NaHSO3 solution. Test continuously for 5 min under dual excitation conditions at 455 nm and 575 nm, and record the changes in fluorescence intensity. Figure 7 As shown in B, Figure 7 The ordinate of B represents I. 575 / I 637 The ratio of fluorescence intensity of probes MLR and MLR-SO2 in I 575 / I 637 The fact that the fluorescence intensity remained relatively stable at different ratios indicates its potential for detecting SO2 in vivo.

[0085] III. Fluorescence Imaging of SO2 by MLR in HeLa Cells

[0086] 1. Cytotoxicity test

[0087] HeLa cells were cultured in DMEM (Dupuyet modified medium) containing 10% FBS (Fetal Bovine Serum) and 1% P / S (penicillin-streptomycin mixture). MCF-10A cells were cultured in DMEM / F12 containing 5% horse serum (HS), 1% P / S, 20 ng / mL epidermal growth factor (EGF), 0.5 μg / mL hydrocortisone, 10 μg / mL insulin, and 1% non-essential amino acids (NEAA). All cell lines were cultured at 37°C in an incubator containing 5% CO2.

[0088] The toxicity to different cell lines was investigated using the CCK-8 assay. The seeding density was 1 × 10⁶ cells per well. 4 Different cell types (HeLa, MCF-10A) were evenly seeded onto 96-well cell culture plates. The plates were gently shaken to ensure uniform cell distribution, and then incubated for 24 hours. After discarding the old culture medium, cells were cultured for another 24 hours in complete medium containing different concentrations of the probe MLR (2μM, 4μM, 6μM, 10μM, 15μM, 20μM, 25μM). Cell viability was then assessed using the CCK-8 assay. 100μL of 10% CCK-8 in DMEM complete medium was added to each well, and the plates were incubated in the dark for 1-4 hours. The absorbance of cells at 450nm was measured using a microplate reader, and the cell viability was calculated as follows: Cell viability = (Abs) / (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs)) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs) * (Abs))))) 样本 -Abs 空白 ) / (Abs 对照 -Abs 空白 ), where Abs 样本 Abs represents the absorbance values ​​measured after incubation with probes of different concentrations. 空白 Abs represents the absorbance value measured during normal incubation of untreated cells. 对照 The absorbance values ​​represent those of cells without MLR treatment after incubation. Results are shown below. Figure 9 The rightmost image in diagram B.

[0089] from Figure 9 As shown in Figure B, after culturing cells with different concentrations of probe MLR, the survival rate of HeLa cells and MCF-10A cells treated with probe MLR remained above 85% even when the probe concentration was as high as 25 μM, demonstrating the low cytotoxicity of MLR.

[0090] 2. Cell imaging experiments

[0091] 2.1 Solution preparation:

[0092] Cu 2+ Solution: Dissolve CuCl2 in water to prepare a 1mM Cu solution. 2+Solution;

[0093] ES solution: Ilisimor (ES, a reactive oxygen species inducer) was dissolved in DMSO to prepare a 1 mM ES solution;

[0094] Copper death-inducing inhibitor solution: Dissolve the copper chelating agent (Ammonium tetrathiomolybdate, TTM) in DMSO to prepare a 1 mM copper death-inducing inhibitor solution;

[0095] 2.2 Experimental Cells: HeLa cells were cultured normally in DMEM complete medium under the following incubator conditions: temperature 37℃, humidified environment containing 5% CO2. For imaging experiments, the seeding density was 1×10⁶ cells per well. 4 HeLa cells were evenly seeded onto cell culture dishes, and the plates were gently shaken to ensure uniform distribution of cells. After culturing in a cell incubator for 24 hours, subsequent cell imaging experiments were performed.

[0096] 2.3 Cell Imaging Stability Experiment

[0097] HeLa cells cultured for 24 h were continuously imaged for 25 min under laser confocal microscopy with 1 mL of DMEM complete medium containing MLR (10 μM). Fluorescence imaging was performed using an Olympus FV300 confocal microscope. Observations were made under 20× magnification. The fluorescence channel parameters were as follows: Orange fluorescence channel: λ ex =455nm, λ em =490-590nm. Red fluorescence channel: λ ex =575nm, λ em =600-700nm. See details in the attached image. Figure 8 .

[0098] from Figure 8 As can be seen, the fluorescence signal of the cells remained almost identical under continuous laser irradiation for 25 minutes, proving its applicability for cell imaging.

[0099] 2.4 Cell imaging experiments of HeLa cells with different concentrations of SO2 (exogenous and endogenous)

[0100] Control group: HeLa cells cultured for 24 h were incubated in a cell culture incubator with 1 mL of DMEM complete medium containing MLR (10 μM) for 30 min. The old medium was discarded and the cells were washed three times with PBS buffer. After adding 1 mL of HBSS buffer, imaging was performed.

[0101] Exogenous SO2 experimental groups: In the first group, HeLa cells cultured for 24 h were co-incubated with 1 mL of DMEM complete medium containing NaHSO3 (50 μM) in a cell culture incubator for 1 h (to provide exogenous SO2 to the cells). The old medium was discarded, and the cells were washed three times with PBS buffer. Then, 1 mL of DMEM complete medium containing MLR (10 μM) was added, and the cells were incubated in a cell culture incubator for 30 min. The old medium was discarded, and the cells were washed three times with PBS buffer. Then, 1 mL of HBSS buffer was added, and imaging was performed. In the second group, HeLa cells cultured for 24 h were co-incubated with 1 mL of DMEM complete medium containing NaHSO3 (200 μM) in a cell culture incubator for 1 h (to provide exogenous SO2 to the cells). The old medium was discarded, and the cells were washed three times with PBS buffer. Then, 1 mL of DMEM complete medium containing MLR (10 μM) was added, and the cells were incubated in a cell culture incubator for 30 min. The old medium was discarded, and the cells were washed three times with PBS buffer. Then, 1 mL of HBSS buffer was added, and imaging was performed.

[0102] Endogenous SO2 experimental groups: In the first group, HeLa cells cultured for 24 h were co-incubated with 1 mL of DMEM complete medium containing Cys (200 μM) in a cell culture incubator for 1 h (to stimulate cells to produce endogenous SO2). The old medium was discarded, and the cells were washed three times with PBS buffer. Then, 1 mL of DMEM complete medium containing MLR (10 μM) was added, and the cells were incubated in a cell culture incubator for 30 min. The old medium was discarded, and the cells were washed three times with PBS buffer. Then, 1 mL of HBSS buffer was added, and imaging was performed. In the second group, HeLa cells cultured for 24 h were co-incubated with 1 mL of DMEM complete medium containing Cys (400 μM) in a cell culture incubator for 1 h (to stimulate cells to produce endogenous SO2). The old medium was discarded, and the cells were washed three times with PBS buffer. Then, 1 mL of DMEM complete medium containing MLR (10 μM) was added, and the cells were incubated in a cell culture incubator for 30 min. The old medium was discarded, and the cells were washed three times with PBS buffer. Then, 1 mL of HBSS buffer was added, and imaging was performed.

[0103] Fluorescence imaging was performed using an Olympus FV300 confocal microscope. Observations were made at 20x magnification. The parameters for each fluorescence channel group are as follows: Orange fluorescence channel: λ ex =455nm, λ em =490-590nm. Red fluorescence channel: λ ex =575nm, λ em =600-700nm. See details in the attached image. Figure 9 A, where the control group is shown in Figure A. Figure 9 Figure i in A shows the imaging of the two groups of cells in the exogenous SO2 experiment. Figure 9Figures A and B (NaHSO3 (50 μM) and iii (NaHSO3 (200 μM)) show the images of the two groups of cells in the endogenous SO2 experiment. Figure 9 Figure A contains Figure 4 (including Cys (200 μM)) and Figure V (including Cys (400 μM)).

[0104] from Figure 9 As can be seen from the AI, in the control group, HeLa cells incubated with 10 μM MLR showed strong red fluorescence, while a negligible yellow fluorescence signal was observed. From Figure 9 A-ii and Figure 9 As shown in A-iii, after treatment with exogenous SO2, the red fluorescence gradually weakened, while the yellow fluorescence significantly increased. Existing research indicates that sulfur-containing amino acids in mammals, such as L-cysteine ​​(L-Cys) and sulfonamides, can produce endogenous SO2. To further verify the probe's ability to detect endogenous SO2, this invention used Cys as an endogenous SO2 stimulant to treat HeLa cells. From... Figure 9 A-iv and Figure 9 As can be seen in Av, treatment of HeLa cells with 200 μM Cys and 400 μM Cys to stimulate intracellular SO2 production resulted in a significant enhancement of fluorescence in the yellow channel. Furthermore, as... Figure 9 Statistical analysis of fluorescence intensity (B) showed that MLR can detect exogenous SO2 in living cells. Conversely, the inventors observed a gradual decrease in fluorescence in the red channel. These results demonstrate that MLR can visualize SO2 produced by intracellular Cys metabolism in both channels, proving the probe's specific recognition of intracellular SO2.

[0105] In summary, MLR can simultaneously monitor exogenous and endogenous SO2 in living cells.

[0106] 2.5 Mitochondrial-Lipid Droplet Colocalization Experiment

[0107] To confirm that MLR can specifically stain mitochondria and lipid droplets in both SO2-free and SO2-present conditions, we used the commercial fluorescent probe MitoLite. TM Blue FX490 (MTB, 1 μM, targeting mitochondria) and BODIPY 630 / 650X (1 μM, targeting lipid droplets) markers in Cu 2+ Colocalization studies were conducted in cells treated with ES. ES was used as a copper death inducer to observe changes in mitochondrial-lipid droplet contact during copper death. The specific methods are as follows:

[0108] HeLa cells cultured for 24 hours were mixed with 1 mL of Cu. 2+ +ES(Cu2+ Both ES and DMEM were 1 μM, diluted to 1 μM using DMEM medium (the same applies below). The cells were incubated together in a cell culture incubator for 2 hours. After discarding the old medium, the cells were washed three times with PBS. Then, 1 mL of MitoLite containing 1 μM was added to the cells. TM Cells were cultured in DMEM complete medium containing Blue FX490 (MTB, a commercial dye for mitochondrial targeting) and 1 μM BODIPY 630 / 650X (BODIPY, a commercial dye for lipid droplet targeting) for 15 minutes. After discarding the old medium, cells were washed three times with PBS and then 1 mL of HBSS buffer was added for imaging experiments. Fluorescence imaging was performed using an Olympus FV300 confocal microscope. Observation was performed at 20× magnification. The fluorescence channel parameters for each group are as follows: Orange fluorescence channel: λ ex =455nm, λ em =490-590nm. Red fluorescence channel: λ ex =575nm, λ em =600-700nm. See details in the attached image. Figure 11 .

[0109] from Figure 11 It can be seen from this that in Cu 2+ In cells treated with +ES, the initial Pearson correlation coefficient between the red light channel (MLR) and the blue light channel (MITO) was 0.84, while that between the orange light channel (MLR-SO2) and the deep red light channel (LD, i.e., Figure 11 The initial Pearson correlation coefficient between the channels in the column containing LD (lipid droplets) in Figures A-II is 0.60. This indicates that MLR initially localizes within the mitochondria. As induction time increases, intracellular SO2 levels rise. At this point, the overlap coefficient between the red and blue channels decreases to 0.47, while the overlap coefficient between the orange and dark red channels increases to 0.93. This corresponds to the MLR reacting with accumulated SO2 to form lipophilic neutral adducts, which subsequently localize to lipid droplets. As the induced copper apoptosis process deepens, excessive reactive oxygen species lead to SO2 depletion, and the correlation coefficient between the red and blue channels decreases to 0.21, while the correlation coefficient between the orange and dark red channels also decreases to 0.56. The simultaneous fluorescence decay of both fluorescent channels corroborates this dynamic process. This co-localization trajectory and fluorescence change indicate that mitochondrial-lipid droplet contact is enhanced during early apoptosis, followed by organelle dissociation during late apoptosis, as evidenced by the dual-channel ratio response of the MLR.

[0110] 2.6 Tracking SO2 fluorescence changes in cells using MLR

[0111] First use 1 mL of 1 μM Cu 2++ES DMEM complete medium was co-incubated with HeLa cells cultured for 24 hours for 2 hours, and the Cu-containing medium was discarded. 2+ After adding ES medium, the cells were washed three times with PBS buffer, and then co-incubated with 1 mL of DMEM complete medium containing MLR (5 μM) for imaging.

[0112] Fluorescence imaging was performed using an Olympus FV300 confocal microscope. Observations were made at 20x magnification. The parameters for each fluorescence channel group are as follows: Orange fluorescence channel: λ ex =455nm, λ em =490-590nm. Red fluorescence channel: λ ex =575nm, λ em =600-700nm. See results below. Figure 10 .

[0113] from Figure 10 As shown in Figure A, HeLa cells were co-incubated with MLR to visualize the dynamic fluctuations of intracellular SO2 during apoptosis within the biological system. Initially, HeLa cells incubated with mitochondrial-targeting MLR (10 μM) showed enhanced fluorescence in the red fluorescence channel, while almost no fluorescence was observed in the orange fluorescence channel. A time-dependent fluorescence shift occurred after treatment with irismo. The intensity of the orange channel gradually increased over 30 min, reflecting an increase in intracellular SO2 levels. After 60 min of incubation with ES, Cu... 2+ +ES further triggered a surge in intracellular ROS. Simultaneously, intracellular SO2 levels decreased, leading to a gradual weakening of fluorescence in the red and orange channels. After 90 minutes of incubation, the cells became increasingly blurred, eventually leading to apoptosis. Simultaneously, apoptosis resulted in a decrease in ROS levels, and the red and orange fluorescence almost disappeared. Quantitative fluorescence analysis confirmed the ability of MLR to monitor SO2 content fluctuations during the accumulation process. Figure 10 B, Figure 10 In B, "intensity" refers to the measured fluorescence intensity.

[0114] 2.7 Cell live / dead staining

[0115] ES was used as a copper death inducer, and the copper chelator TTM was used to inhibit the generation of ROS / RSS (reactive oxygen species) in cells during copper death. Details are as follows:

[0116] After 24 hours of culture, HeLa cells were seeded into 6-well cell culture plates (1 × 10⁶ cells per well). 4 (100 cells), place the culture plate in a 37°C constant temperature cell culture incubator containing 5% CO2, and after the cells adhere, add 1 mL of 1 μM different treatments (Cu) to each well of the culture plate.2+ ES, Cu 2+ +ES、Cu 2+ +ES+TTM, where 1μM refers to the concentration of each substance being 1μM, for example, Cu. 2+ Cu in +ES processing group 2+ Both DMEM complete medium (containing 1 μM of ES) and DMEM complete medium alone (as a control group) were used. The culture plates were incubated in an incubator for 2 hours. The old medium was removed, and the cells were washed three times with PBS buffer. Then, 1 mL of DMEM complete medium containing 5 μL of 1 μM Calcein-AM (calcein acetoxymethyl ester) was added, and the cells were incubated for 15 minutes. The old medium was discarded, and the cells were washed three times with PBS. Then, 5 μL of DMEM complete medium containing 1 μM PI (propidium iodide) was added, and the cells were incubated for 5 minutes. The old medium was discarded, and the cells were washed three times with PBS buffer. 1 mL of HBSS buffer was added for imaging experiments. The ratio of live to dead cells in each group was observed under a laser confocal microscope (FV300) (PI-labeled dead cells showed red fluorescence, and Calcein-AM-labeled live cells showed green fluorescence). The entire process was performed in the dark and repeated three times. The fluorescence channel parameters for each group were as follows, observed under 20× magnification: Red fluorescence channel: λ ex =535nm, λ em =550-650nm. Green fluorescence channel: λ ex =494nm, λ em =500-550nm. The result is as follows: Figure 12 As shown in C.

[0117] from Figure 12 As can be seen from C, Cu 2+ The cell viability of the +ES group was significantly lower than that of other cell treatment groups.

[0118] 2.8 DLAT Oligomer Detection

[0119] Inoculate 1×10 per well 4 To determine the optimal seeding density, HeLa cells were evenly seeded onto cell culture plates. The plates were gently shaken to ensure uniform cell distribution, and then cultured in a cell culture incubator for 24 hours.

[0120] Subsequently, 1 mL of different treatments (Cu) containing 1 μM were added to the cell culture wells. 2+ ES, Cu 2+ +ES、Cu 2+Cells were cultured in DMEM complete medium containing ES+TTM and 1 mL PBS (as a control group) for 2 hours. The old medium was discarded, and cells were washed three times with PBS buffer. Cells were then fixed with 0.5 mL of 4% paraformaldehyde for 10 minutes. After discarding the old medium and washing with PBS buffer, 1 mL of DLAT antibody (1:2000) was added, and the cells were incubated at 4°C for 24 hours. The DLAT antibody was recovered, and cells were washed three times with 1×TBST solution for 10 minutes each time. Then, 1 mL of Alexa Fluor 488 conjugated fluorescent secondary antibody (1:3000) was added to each cell treatment group, and the cells were incubated at room temperature for 1 hour. Cells were then washed three times with 1×TBST solution for 10 minutes each time. Finally, 0.5 mL of Hoechst 33452 (1 μM) was added to each treatment group to stain the cell nuclei for 10 minutes. After washing three times with PBS buffer, 1 mL of HBSS buffer was added for cell imaging. Fluorescence imaging was performed using an Olympus FV300 confocal microscope. Under a 20x microscope, the fluorescence channel parameters for each group were as follows: Alexa Fluor 488 conjugated with secondary antibody was excited with a 488nm laser, collecting 500-600 fluorescence channels; Hoechst 33452 was excited with a 346nm wavelength, collecting 400-500 fluorescence channels. The results are as follows... Figure 12 As shown in D.

[0121] from Figure 12 As can be seen from D, compared to pure Cu 2+ Compared to the group, ES+Cu 2+ The presence of individual DLAT aggregates suggests DLAT oligomerization.

[0122] 2.9 Research on the mechanism of copper death in cells

[0123] Existing research indicates that copper death is a novel regulated cell death mechanism characterized by copper overload, iron-sulfur (Fe-S) cluster protein deficiency, and DLAT oligomerization. ES effectively removes Cu from the culture medium. 2+ Transported into cancer cells, it acts as a copper ion carrier, where FDX1 reduces it to the more toxic Cu. + FDX1 promotes protein lipidation by directly binding to lipoic acid synthase (LIAS) and functions as a key upstream regulator in the lipidation process of dihydrolipoamide acetyltransferase (DLAT). Subsequently, Cu + Direct binding to the disulfide bond on the terminal cysteine ​​residue of sulfonated DLAT leads to the aggregation of sulfonated DLAT and the loss of Fe-S cluster proteins, resulting in protein toxicity stress and ultimately cell death.

[0124] To further investigate the mechanism of copper death, the inventors conducted the following experiment:

[0125] 2.9.1 Flow cytometry analysis:

[0126] (1) Apoptosis detection

[0127] After normal culture, HeLa cells were digested, collected, centrifuged, and counted, then seeded into 24-well plates (1×10⁻⁶ cells / well). 4 Cells were collected (number per well), then cultured in a cell culture incubator until the cells covered the bottom of the plate. The culture medium was aspirated, and the cells were washed three times with PBS buffer. Five cell treatment groups were set up, with three replicates per group. The groups were, in order: blank group, negative control group, Cu... 2+ Stimulation group, ES drug administration group, Cu 2+ +ES-induced group, Cu 2+ +ES+TTM induced inhibition group, in which the blank group was not treated in any way, and the negative control group was supplemented with the negative control reagent from the DCFH-DA kit;

[0128] Subsequently, 1 mL of the corresponding treatment substance (Cu) containing 1 μM was added to each well cell. 2+ ES, Cu 2+ +ES、Cu 2+ Cells were cultured in DMEM complete medium (+ES+TTM) for 2 hours. The old medium was discarded, and cells were washed three times with PBS buffer, then digested with trypsin and collected. Cells were centrifuged at 300g for 5 minutes, the supernatant was discarded, and cells were washed again with PBS buffer. After centrifugation, the supernatant was discarded again. Cells were resuspended in 195 μL of Annexin V-FITC working binding solution for each group, followed by 5 μL of Annexin V-FITC reagent stock solution. The mixture was gently pipetted and mixed. 10 μL of PI staining solution was added to each group, and the mixture was incubated at room temperature in the dark for 15 minutes. Cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and cells were resuspended in 50 μL of Annexin V-FITC binding solution and placed on ice. Cell apoptosis was detected within 1 hour using flow cytometry under dark conditions. The fluorescence channel parameters for each group were as follows: Annexin V-FITC (green fluorescence channel: B525); PI (red fluorescence channel: B585). See [link to results] for details. Figure 12 F( Figure 12 From left to right in F are Cu 2+ ES, Cu 2+ +ES、Cu 2+ +ES+TTM group).

[0129] Figure 12 F shows that Cu 2+The +ES group showed the brightest red and the weakest green fluorescence, respectively, indicating the highest number of dead cells and the lowest number of live cells. Figure 12 As shown in F, Cu 2+ +ES induced higher ROS production and a 42.5% apoptosis rate during apoptosis induction (11.2-fold higher than ES alone (apoptosis rate 3.82%)). The apoptosis rate corresponds to the F-plot. The synergistic downregulation of DLAT / LIAS by the TCA cycle, coupled with increased ROS and DLAT oligomerization, mechanistically confirms the role of Cu in apoptosis. 2+ +ES induces copper death through iron-sulfur cluster depletion and a protein toxicity cascade.

[0130] (2) Detection of total intracellular reactive oxygen species level

[0131] Healthy HeLa cells were seeded into 6-well cell culture plates (1×10⁻⁶ cells / well). 4 Cells were cultured in a 37°C cell culture incubator containing 5% CO2 for 24 hours. Reactive oxygen species (ROS) detection probe preparation: 10 μM DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) working solution was prepared using serum-free HBSS buffer according to the manufacturer's instructions. After the cells adhered and filled the culture plate, 1 mL of 1 μM of different treatments (PBS, Cu) was added to each well. 2+ ES, Cu 2+ +ES、Cu 2+ Incubate the cells in DMEM complete medium (+ES+TTM, with PBS as a control) for 2 hours. After removing the old culture medium and washing three times with PBS buffer, add 1 mL of 1 μM DCFH-DA working solution diluted with DMEM. Incubate at 37°C for 20 minutes. Wash the cells three times with PBS to remove any unbound DCFH-DA working solution. Observe the green fluorescence intensity of each treatment group directly using an Olympus FV300 confocal microscope to reflect the total reactive oxygen species production. Under 20× magnification, observe the DCFH-DA fluorescence channel: λ ex =502nm, λ em =510-610nm. See results below. Figure 12 A.

[0132] from Figure 12 As can be seen in A, the CLSM image shows that, compared to Cu... 2+ Compared to the treatment group alone, Cu 2+ +ES-treated HeLa cells showed stronger green fluorescence, which was attributed to the fact that ES was able to transport more copper ions, indicating that the ES-mediated pro-apoptotic effect was enhanced.

[0133] In addition, HeLa cells were subjected to the above-mentioned different stimuli (PBS, Cu...2+ ES, Cu 2+ +ES、Cu 2+ After treatment with +ES+TTM (PBS is used as a control), the cells were washed three times with PBS buffer, then digested with 1 mL of trypsin containing EDTA. After centrifugation, the cells were collected, and 1 mL of diluted 1 μM DCFH-DA working solution was added to HeLa cells. The 6-well cell culture plates were incubated in a cell culture incubator for 30 min under light. The cells were mixed every 5 min to ensure complete contact between the DCFH-DA probe and the cells. After incubation, the cells were washed three times with PBS buffer, and total ROS production was detected by flow cytometry. DCFH-DA is a green fluorescent channel: B525. Results are shown below. Figure 12 E.

[0134] from Figure 12 As can be seen from E, Cu 2+ HeLa cells treated with +ES showed the highest proportion of ROS-positive cells, significantly exceeding other groups.

[0135] (3) Detection of copper death-related proteins

[0136] Healthy HeLa cells were seeded into 6-well cell culture plates (1×10⁻⁶ cells / well). 4 Cells per well were placed in a 37°C cell culture incubator containing 5% CO2 and cultured for 24 hours.

[0137] Cell treatment groups included PBS, Cu 2+ ES, Cu 2+ +ES, the concentration of each treatment in each group was 1 μM, with PBS serving as the control group; the levels of intracellular DLAT, LIAS, and FDX1 proteins were studied using Western blotting. The specific experimental procedures are as follows:

[0138] 1) Sample preparation: Mix RIPA (Radio Immunoprecipitation Assay Lysis buffer), phosphatase inhibitor, and PMSF (phenylmethylsulfonyl fluoride) in a ratio of 100:1:1 to form a protein lysis buffer, and pre-cool it in a 4°C refrigerator for later use.

[0139] Cells were then collected and washed with pre-chilled PBS to remove residual culture medium. Cells from different treatment groups were collected into 1.5 mL centrifuge tubes, and 1 mL of PBS was added. After centrifugation at 1000 rpm for 5 min at room temperature, the cell pellet was resuspended in 1 mL of PBS and centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was then discarded. Next, 200 μL of protein lysis buffer was added to the cell pellets from each treatment group. After mixing by pipetting on ice, the mixture was sonicated three times for 10 seconds each time, followed by lysis on ice for 40 min. After lysis, the mixture was centrifuged at 14000 rpm for 15 min using a refrigerated ultracentrifuge to remove unlysed cell debris and organelles. The supernatant was collected as protein. Finally, the protein concentration in the lysis buffer was determined using a BCA kit. The supernatant samples were aliquoted and stored at -80°C for subsequent experiments.

[0140] 2) SDS-PAGE Gel Preparation: First, prepare a 10% separating gel (lower layer). The preparation process is as follows: Using a pipette, pipette 4 mL of ultrapure water, 3.3 mL of 30% acrylamide gelling solution, 2.5 mL of 1.5 M Tris-HCl buffer (pH 8.8), 100 μL of 10% sodium dodecyl sulfate (SDS), 100 μL of 10% ammonium persulfate solution, and 4 μL of TEMED and mix them. Then pour the mixture between two glass plates, filling it to about 2 cm from the top of the glass plates. Next, fill the glass plates with ultrapure water to seal the surface of the separating gel. When a boundary appears between the ultrapure water and the separating gel, discard the ultrapure water and then add the upper layer gel. The specific procedure is as follows: Add 2.7 mL of ultrapure water, 670 μL of 30% acrylamide gel solution, 500 μL of 1.5 M Tris-HCl (pH 6.8), 40 μL of 10% SDS, 40 μL of 10% ammonium persulfate solution, and 4 μL of TEMED solution. After thorough mixing, the supernatant gel solution is ready for further experimental procedures. For electrophoresis buffer preparation, accurately weigh out 30.2 g of tris-hydroxymethylaminomethane (Tris-base). Tris-hydroxymethylaminomethane is a commonly used pH adjuster that maintains a stable pH value in the buffer during electrophoresis. Then, weigh out 188 g of glycine. Glycine, as a non-essential amino acid, acts as an ionic strength regulator in the electrophoresis buffer, helping to improve protein migration conditions and resolution. Next, weigh out 10 g of sodium dodecyl sulfate (SDS). SDS is an anionic surfactant that denatures proteins and imparts a negative charge, causing all proteins to migrate at approximately the same speed in the electric field. Add the weighed tris(hydroxymethyl)aminomethane, glycine, and SDS to approximately 800 mL of ultrapure water and stir thoroughly with a glass stir bar until completely dissolved. Ultrapure water is used to prepare the buffer solution; it is free of impurities and can avoid interfering with the electrophoresis process. Adjust the solution volume to 1 liter to obtain a 10-fold concentrated electrophoresis buffer. Typically, for ease of storage and use, we prepare a 10-fold concentrated buffer instead of directly preparing the working concentration buffer. Before use, dilute the 10-fold concentrated electrophoresis buffer 10 times with ultrapure water as needed to obtain the required concentration of working buffer. Finally, adjust the pH to the required range. The pH for SDS-PAGE electrophoresis is set at approximately 8.8. Measure the pH using a pH meter. After completing these steps, a 1× buffer suitable for SDS-PAGE electrophoresis is prepared. Before starting the experiment, it is also necessary to check the pH and transparency of the buffer to ensure they meet the experimental requirements.

[0141] 3) Sample loading: After the gel has fully polymerized, place it in the electrophoresis tank and add the inner electrophoresis buffer. Then, slowly and evenly pull out the comb to ensure the sample wells are intact. Pour 1×SDS-PAGE electrophoresis buffer into the electrophoresis tank. The sample loading order is: 5μL Loading Buffer, Marker, Sample, 5μL Loading Buffer.

[0142] 4) Electrophoresis: After setting up the electrophoresis tank, set the voltage to 90V for 30 minutes, then switch to 120V for 60 minutes. Observe the position of the protein bands at the bottom continuously, and adjust the time as needed. After electrophoresis, turn off the power to prepare for gel transfer.

[0143] 5) Wet Transfer: First, cut an appropriate amount of 0.45mm PVDF membrane according to the strip size, and activate it by soaking it in methanol for 5 minutes. Then, assemble the "sandwich" structure in the transfer buffer in the following order: sponge pad-filter paper-gel-membrane-filter paper-sponge pad. Gently remove air bubbles between the gel and membrane with a glass rod to ensure tight adhesion. Place the assembled transfer clamp into the transfer tank and add enough transfer buffer to ensure complete immersion. Set the transfer conditions according to the molecular weight of the target protein. Generally, small molecular weight proteins (<20kDa) are transferred at 200mA for 30 minutes, medium molecular weight proteins (20-50kDa) are transferred at 300mA for 45 minutes, and large molecular weight proteins (>50kDa) are transferred at 350mA for 50-90 minutes.

[0144] 6) Blocking: After the transfer is complete, remove the PVDF membrane and wash away the transfer solution on the surface with ultrapure water. Then, perform a blocking process using a blocking solution composed of 5% skim milk. After adding the blocking solution to the membrane, shake on a shaker for 1 hour to achieve proper sealing. After blocking, wash with 1×TBST solution for 10 minutes each time, for a total of three washes.

[0145] 7) Antibody Incubation and Development: Dilute DLAT and LIAS primary antibodies to appropriate ratios according to the instructions. Add 5 mL of the diluted primary antibody to the PVDF membrane and incubate at 4°C for 24 h. After incubation, recover the primary antibody solution, wash the membrane three times with 1×TBST solution for 10 min each time, then discard the liquid. Add 5 mL of DyLight 800 fluorescent secondary antibody diluted according to the kit instructions to the PVDF membrane and incubate on a shaker at room temperature for 1 h. After incubation, recover the secondary antibody solution, wash the membrane three times with 1×TBST solution for 10 min each time. After washing, use a membrane scanner to image and analyze the PVDF membrane.

[0146] 8) Results Analysis: The results are shown in [link to results]. Figure 12 B, Immunoblotting showed that in Cu 2+In the +ES group, the levels of DLAT monomer and LIAS protein were significantly reduced, while the level of FDX1 protein was slightly increased, and DLAT showed fluorescent oligomerization.

[0147] 2.10. Zebrafish imaging of SO2 using MLR probes

[0148] The zebrafish used in the experiment were hatched at 28°C and 80% humidity, and the experiment was conducted three days after hatching.

[0149] 2.10.1 SO2 Zebrafish Imaging Experiment

[0150] (1) SO2 exogenous zebrafish imaging experiment

[0151] In the first group of experiments, zebrafish were incubated with 1 mL of E3 medium containing 10 μM MLR (available for purchase) in a constant temperature and humidity incubator (25°C, 60% humidity) for 30 min. The old medium was then discarded, and the zebrafish were washed three times with PBS solution. After adding 2 mL of PBS buffer, fluorescence imaging was performed. Each group of experiments contained three zebrafish in a glass culture dish (the same applies below).

[0152] In the second group of experiments, zebrafish were first incubated with 1 mL of E3 medium containing NaHSO3 (50 μM) in a constant temperature and humidity incubator (25℃, 60% humidity) for 1 h (to provide exogenous SO2 for zebrafish), and then incubated with 1 mL of E3 medium containing MLR (10 μM) in a cell culture incubator for 30 min. After the medium was removed, the zebrafish were washed 2-3 times with PBS buffer, and then 2 mL of HBSS buffer was added again before imaging.

[0153] In the third group of experiments, zebrafish were first incubated with 1 mL of E3 medium containing 200 μM NaHSO3 in a constant temperature and humidity incubator (25℃, 60% humidity) for 1 h (to provide exogenous SO2 for zebrafish), and then incubated with 1 mL of E3 medium containing 10 μM MLR for 30 min. The old medium was discarded and the zebrafish were washed 2-3 times with PBS buffer before adding 2 mL of HBSS buffer for imaging experiments.

[0154] Before imaging, zebrafish were transferred to glass culture dishes using a disposable sterile dropper. The zebrafish were anesthetized with an appropriate amount of EM222 (depending on size) before imaging. Fluorescence imaging was performed using an Olympus FV-300 laser confocal microscope at 20x magnification. The parameters for each fluorescence channel are as follows: Orange fluorescence channel: λ ex =455nm, λ em =490-590nm. Red fluorescence channel: λ ex =575nm, λ em =600-700nm. See details in the attached image. Figure 13 .

[0155] (2) SO2 endogenous zebrafish imaging experiment

[0156] In the first group of experiments, zebrafish were incubated with 1 mL of E3 medium containing MLR (10 μM) in a constant temperature and humidity incubator (25℃, 60% humidity) for 30 min. The old medium was then discarded, and the zebrafish were washed three times with PBS buffer before being re-incubated with 1 mL of HBSS buffer for imaging.

[0157] In the second group of experiments, zebrafish were first incubated with 1 mL of E3 medium containing Cys (200 μM) in a constant temperature and humidity incubator (25℃, 60% humidity) for 1 h (to stimulate zebrafish to produce endogenous SO2), and then incubated with 1 mL of E3 medium containing MLR (10 μM) in a cell culture incubator for 30 min. After the medium was removed, the zebrafish were washed 2-3 times with PBS and then 1 mL of HBSS buffer was added again before imaging.

[0158] In the third group of experiments, zebrafish were first incubated with 1 mL of E3 medium containing Cys (400 μM) in a cell culture incubator for 1 h (to stimulate zebrafish to produce endogenous SO2), and then incubated with 1 mL of E3 medium containing MLR (10 μM) in a constant temperature and humidity incubator (25℃, 60% humidity) for 30 min. The old medium was discarded and the zebrafish were washed 2-3 times with PBS before adding 1 mL of HBSS buffer for imaging experiments.

[0159] Before imaging, zebrafish were transferred to glass culture dishes using a disposable sterile dropper. The zebrafish were anesthetized with an appropriate amount of EM222 (depending on size) before imaging. Fluorescence imaging was performed using an Olympus FV-300 laser confocal microscope at 20x magnification. The parameters for each fluorescence channel are as follows: Orange fluorescence channel: λ ex =455nm, λ em =490-590nm. Red fluorescence channel: λ ex =575nm, λ em =600-700nm. See details in the attached image. Figure 13 .

[0160] (3) Results Analysis

[0161] like Figure 13As shown in Figure A, imaging was performed after pre-incubating zebrafish with the probe MLR (10 μM) for 1 h. Under excitation at 455 nm and 570 nm, the red channel showed significant fluorescence changes, while the yellow channel lacked fluorescence signal, indicating effective uptake of the probe by the zebrafish. When zebrafish were pretreated with exogenous SO2 (50 μM) for 60 min, followed by incubation with MLR (10 μM) for 30 min, a weak yellow fluorescence signal was observed. With increasing SO2 concentration, the red fluorescence signal gradually weakened until it disappeared, while the yellow fluorescence signal gradually increased. Similarly, Cys was used to stimulate the production of endogenous SO2 in zebrafish. With increasing endogenous SO2 concentration, the red fluorescence channel gradually weakened, while the orange fluorescence channel gradually increased. The quantitative PCR plot visually shows the fluorescence intensity (…). Figure 13 Changes in B). These results demonstrate the effectiveness of MLR in detecting SO2 in zebrafish.

[0162] 2.10.2 Zebrafish Copper Death Imaging Experiment

[0163] (1) Imaging experiment of SO2 produced during the copper death process of zebrafish

[0164] Normally cultured zebrafish were divided into 4 groups: Cu 2+ Stimulation group, ES stimulation group, Cu 2+ +ES-induced group, Cu 2+ +ES+TTM induced inhibition group, three zebrafish in each group; 1 mL of 1 μM of different treatments (Cu) was added to each of the four groups of zebrafish. 2 + ES, Cu 2+ +ES、Cu 2+ The zebrafish were incubated with E3 medium containing ES+TTM for 2 hours, then the old medium was discarded, and the zebrafish were washed three times with PBS buffer. Then, 1 mL of E3 medium containing MLR (5 μM) was added to each zebrafish, and the zebrafish were incubated for 30 minutes. After washing three times with PBS buffer, imaging was performed. Before imaging, the zebrafish were transferred to glass culture dishes using a disposable sterile dropper, anesthetized with EM222, and then imaged using an Olympus FV300 confocal microscope. Observations were made under 20× magnification. The fluorescence channel parameters for each group were as follows: Orange fluorescence channel: λ ex =455nm, λ em =490-590nm. Red fluorescence channel: λ ex =575nm, λ em =600-700nm. See details in the attached image. Figures 14-15 .

[0165] (2) Imaging experiment of total reactive oxygen species during copper death in zebrafish

[0166] Preparation of reactive oxygen species (ROS) detection probe: Prepare 10 μM MCFH-DA working solution using serum-free HBSS buffer according to the instructions.

[0167] Add 1 mL of 1 μM Cu (containing different treatment groups) to glass culture dishes incubating zebrafish. 2+ ES, Cu 2+ +ES、Cu 2+ The zebrafish were incubated in E3 medium (+ES+TTM) at 25°C and 60% RH for 2 hours. The old culture medium was removed, and the zebrafish were washed 3-4 times with PBS buffer, followed by the addition of 1 mL of diluted 1 μM DCFH-DA working solution. The zebrafish were then incubated in the dark at 25°C and 60% RH for 20 minutes. The zebrafish were washed 3 times with PBS to remove any unbound DCFH-DA working solution. Before imaging, the zebrafish were transferred to glass culture dishes using a disposable sterile dropper, anesthetized with an EM222 microscope, and then imaged. The intensity of green fluorescence in each zebrafish treatment group was directly observed using an Olympus FV300 confocal microscope to reflect the total reactive oxygen species production. Under 20× magnification, the DCFH-DA fluorescence channel was observed as follows: λ ex =502nm, λ em =510-610nm. See details in the attached image. Figures 14-15 .

[0168] (3) Results Analysis

[0169] Figure 14 A indicates that only Cu 2+ The +ES group showed a distinct orange fluorescence. These observations indicate that copper ions induce endogenous oxidative stress in zebrafish, leading to the production of trace amounts of SO2, which is detected by MLR and emits orange fluorescence. Figure 14 B). For example Figure 15 As shown in A, in Cu 2+ Following +ES stimulation, a distinct green fluorescence gradually appeared over time, then disappeared. Simultaneously, due to ROS production, zebrafish exhibited shortened body length, enlarged yolk sac, dilated cardiac chambers, and edema of other organs, ultimately leading to death. Subsequently, fluorescence quantification of the fluorescent channels during copper death was performed to further analyze its effects on zebrafish (…). Figure 15 The impact of B).

[0170] These findings indicate that ES-Cu 2+ The complex significantly impairs the tissue structure and antioxidant system of zebrafish, and induces the production of inflammatory mediators such as SO2, exerting toxic effects on zebrafish development and metabolism. The MLR probe can be used for dynamic monitoring of SO2 production during oxidative stress in zebrafish.

[0171] <Preparation, Colorimetric Experiment and Application of MLR Test Strips>

[0172] Sulfites pose a significant risk to human health, and there is an urgent need for on-site real-time detection of SO2. Based on this, the inventor has developed a fluorescence colorimetric test strip based on the fluorescent probe MLR for point-of-care testing (POCT), which can be integrated with a smartphone to quickly and conveniently respond to SO2.

[0173] 1. Screening of Probe Concentration

[0174] Preparation of test strips with different concentrations: Immerse multiple cellulose papers into solutions of the probe MLR with different concentrations respectively. After 5 minutes, take out the test strips and then dry them to obtain test strips with different concentrations of the MLR probe.

[0175] Immerse the test strips with different concentrations of the MLR probe into a 100 μM NaHSO3 solution. After 2 minutes, take out the test strips. Observe the color of the test strips and take photos under natural light and ultraviolet light, and use the "Color Picker" software on the mobile phone to analyze the R, G, B values of the test strips.

[0176] See the specific results in Figure 16 A, Figure 16 B, Figure 17 A, Figure 17 B. By analyzing the MLR probe solutions with different concentrations, the relationship between the RGB color and the MLR probe concentration was established. As shown in Figure 16 A and 16B, the concentration of the probe MLR has a linear relationship with the signal of the R / G channel (R / G represents the ratio of the two channels, and R / G / B represents the three channels existing in the figure). Finally, 50 μM of the probe MLR was selected to measure SO2.

[0177] 2. Experiment of the MLR Probe Test Strip in Environments with Different SO2 Concentrations

[0178] Preparation of test strips: Immerse the cellulose paper into a 50 μM solution of the probe MLR. After 5 minutes, take out the test strip and then dry it to obtain the test strip with the MLR probe.

[0179] Colorimetric experiment: Immerse the obtained test strips into NaHSO3 solutions with different concentrations (0, 5, 10, 20, 30, 40, 60, 80, 100 μM). After 2 minutes, take out the test strips. Observe the color of the test strips and take photos under natural light and ultraviolet light, and use the "Color Picker" software on the mobile phone to analyze the R, G, B values of the test strips. See the specific results in Figure 16 C, Figure 16 D, Figure 17 C, Figure 17 D.

[0180] From Figure 16As can be seen from D, using the R / G ratio as the fitting parameter can achieve semi-quantitative measurement of SO2; Figure 17 These are illustrations of specific test strip applications, with CIE colorimetric diagrams used to further explain the color change process of the test strips. Figure 17 As can be seen from the results, the color change of the MLR test paper and the CIE colorimetric diagram change with the increase of MLR probe and SO2 concentration.

[0181] 3. Summary

[0182] Sample collection, photography, and data analysis are the three main steps in SO2 detection using a smartphone (such as the "Color Assistant" app in the Android app store). Cellulose paper was chosen as the solid-phase carrier due to its excellent porous structure and load-bearing capacity. When using the app to extract color information and measure RGB values, a uniformly colored area is selected to eliminate edge effects in other color areas and improve the accuracy of image analysis.

[0183] In summary, this invention successfully constructed a mitochondrial-lipid droplet dual-targeting fluorescent probe (MLR) based on the fluorescence resonance energy transfer (FRET) mechanism, enabling real-time monitoring of the dynamic changes in sulfite (SO2) and organelle interactions during copper death in living cells. Compared with existing commercial organelle probes, MLR has the following advantages: (1) dual organelle dynamic targeting capability, which can simultaneously track the conversion process of mitochondria to lipid droplets; (2) SO2-responsive fluorescence switching mechanism, enabling dynamic visualization of organelle interactions; and (3) efficient one-step synthesis process. Experiments confirmed that the detection limit of MLR for SO2 is 0.34 μM, and the response time is <10 s. It has been successfully applied to the quantitative analysis of the dynamic correlation between mitochondrial dysfunction and lipid droplet formation during copper death, providing a novel research tool for elucidating the molecular mechanism of copper-induced cell death.

[0184] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A dual-targeting probe, characterized in that, Its structure is shown in Equation I:

2. The method for preparing the dual-targeting probe as described in claim 1, characterized in that, Includes the following steps: S1. Reaction of 7-(diethylamino)-2-oxo-4-(phenylethynyl)-2H-benzopyran-3-carboxaldehyde with resorcinol yields the first compound; S2. React 4-(diethylamino)salicylaldehyde with p-piperazine phenylacetyl to obtain the second compound; S3. React the first compound and the second compound to obtain the dual-targeting probe.

3. The method for preparing the dual-targeting probe as described in claim 2, characterized in that, Step S1 is as follows: 7-(diethylamino)-2-oxo-4-(phenylethynyl)-2H-benzopyran-3-carboxaldehyde and resorcinol are dissolved in a solvent. Sodium hypochlorite aqueous solution and sodium dihydrogen phosphate aqueous solution are slowly added dropwise at 0°C. After stirring in an ice bath for 10-20 minutes, a reaction solution is obtained. The reaction solution is then poured into ice water to precipitate the target product, which is the first compound. The molar ratio of 7-(diethylamino)-2-oxo-4-(phenylethynyl)-2H-benzopyran-3-carboxaldehyde, resorcinol, sodium hypochlorite, and sodium dihydrogen phosphate is 0.1:1.97:0.35:0.

45.

4. The method for preparing the dual-targeting probe as described in claim 3, characterized in that, Step S2 is as follows: 4-(diethylamino)salicylaldehyde and p-piperazine phenylacetyl are dissolved in concentrated sulfuric acid and reacted at 90°C for 6 hours. The mixture is then cooled to room temperature to obtain a cooling solution. The cooling solution is poured into ice water at 0°C, and a 70% perchloric acid solution is added and stirred. A green solid is observed to precipitate. The solid is filtered and dried to obtain a crude solid product. The crude solid product is separated by column chromatography to obtain the second compound. The molar ratio of 4-(diethylamino)salicylaldehyde to p-piperazine phenylacetyl is 1:

1.

5. The method for preparing the dual-targeting probe as described in claim 4, characterized in that, Step S3 specifically involves dissolving the first compound, the second compound, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in a solvent, reacting under nitrogen protection for 24 hours, removing the solvent under reduced pressure to obtain a crude product, and separating the crude product by column chromatography to obtain a purple solid, which is the dual-targeting probe; wherein the molar ratio of the first compound, the second compound, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.5:0.5:0.05:

1.

6. The use of the dual-targeting probe as described in any one of claims 1 to 5, characterized in that, The dual-targeting probe is used to monitor the dynamic changes of SO2 during copper death in living cells in real time.

7. The use of the dual-targeting probe as described in any one of claims 1 to 5, characterized in that, The dual-targeting probe is used to monitor the interaction between mitochondria and lipid droplets in living cells in real time.

8. A test strip, characterized in that, It includes a dual-targeting probe as described in any one of claims 1 to 5.

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