A fluorescent probe for simultaneously detecting cysteine and hypochlorous acid, and a preparation method and application thereof
Patent Information
- Application Number
- CN202610977515.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-02
- Publication Date
- 2026-09-25
AI Technical Summary
[0027](1) 实现半胱氨酸与次氯酸的双通道无串扰并行检测：半胱氨酸可特异性激活红色荧光通道，次氯酸可特异性激活绿色荧光通道，二者信号完全独立无交叉干扰，有效攻克了双响应探针普遍存在的通道信号串扰技术难题；
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Figure CN122810106A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a fluorescent probe that uses hemicyanine as a matrix to simultaneously detect cysteine (Cys) and hypochlorous acid (HClO), its preparation method, and its application in the fields of biological detection and fluorescence imaging. Background Technology
[0002] Cysteine is a core sulfur-containing α-amino acid in organisms, participating in key physiological processes such as protein synthesis, glutathione production, cellular redox homeostasis regulation, and heavy metal chelation. Abnormal intracellular cysteine concentration is closely related to various diseases such as growth retardation, liver damage, cardiovascular disease, Alzheimer's disease, and cancer.
[0003] Hypochlorous acid is an important reactive oxygen species (ROS) in organisms. It is mainly generated by the reaction of hydrogen peroxide and chloride ions catalyzed by myeloperoxidase (MPO) and plays a core role in killing pathogens in innate immunity. However, excessive accumulation of hypochlorous acid can cause irreversible oxidative damage to proteins, lipids, and DNA, and is highly correlated with pathological processes such as inflammation, tumors, rheumatoid arthritis, neurodegenerative diseases, and atherosclerosis.
[0004] The concentration changes of Cys and HClO in organisms are highly correlated in time and space. Both participate in the regulation of cellular redox homeostasis and the inflammatory and immune processes. Therefore, achieving simultaneous, in situ, and accurate detection of both is of great scientific significance and application value for revealing the pathogenesis of related diseases and early diagnosis.
[0005] Traditional methods for detecting Cys and HClO include high-performance liquid chromatography (HPLC), electrochemical methods, spectrophotometry, and titration. While these methods can achieve quantitative detection, they suffer from drawbacks such as complex sample pretreatment and the inability to perform in-situ dynamic imaging of live cells and organisms. Organic small-molecule fluorescent probes, with their advantages of high sensitivity, high selectivity, non-invasiveness, excellent spatiotemporal resolution, and strong structural designability, have become the mainstream tool for detecting and imaging active molecules in biological systems.
[0006] Hemicyanine dyes are classic D-π-A type near-infrared fluorophores, possessing core advantages such as high molar extinction coefficient, high fluorescence quantum yield, emission wavelength in the near-infrared region, excellent photostability, and easy structural modification, making them ideal matrix for constructing bioimaging probes. Therefore, designing a fluorescent probe based on hemicyanine matrix that simultaneously achieves cross-detection of Cys and HClO channels, and developing such high-performance dual-response fluorescent probes, has significant research value and application prospects. Summary of the Invention
[0007] The purpose of this invention is to provide a dual-channel fluorescent probe with simple structure, convenient synthesis, and excellent performance, which can simultaneously detect Cys and HClO, and to provide its preparation method and application.
[0008] This invention provides a fluorescent probe for the simultaneous detection of Cys and HClO, the structure of which is shown in formula (II):
[0009]
[0010] (Ⅱ).
[0011] The preparation route of the fluorescent probe of this invention is as follows:
[0012]
[0013] .
[0014] The method for preparing the fluorescent probe of the present invention includes the following steps:
[0015] Step 1: Dissolve cyanine dye (1) and 3,5-dihydroxybenzyl alcohol (2) separately in acetonitrile. After dissolving, mix them evenly. Add anhydrous potassium carbonate to the mixture and react at room temperature for 10-20 minutes under nitrogen protection. Then, raise the temperature to 40-50℃ and react for 30-60 minutes. Then, raise the temperature to 50-55℃ and react for 2-4 hours. The crude product is purified by 200-300 mesh silica gel chromatography column using dichloromethane and ethanol with a volume ratio of 100:1 as eluent to obtain the intermediate compound shown in formula (Ⅰ).
[0016] Step 2: Dissolve the intermediate compound shown in formula (I) in anhydrous dichloromethane, add anhydrous triethylamine, stir in an ice bath for 15-25 minutes under nitrogen protection, add acryloyl chloride (3) dropwise, continue stirring for 25-35 minutes, then heat to 20-30℃ under nitrogen protection and stir for 1-2 hours.
[0017] After the reaction was complete, the solution was diluted with dichloromethane, washed with water, the organic phase was collected, dried with anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by silica gel column chromatography with dichloromethane / ethanol = 50:1 as the eluent to obtain the probe shown in formula (II).
[0018] In step 1, the molar ratio of cyanine dye to 3,5-dihydroxybenzyl alcohol is 1:5, and the molar ratio of cyanine dye to potassium carbonate is 1:3.
[0019] In step 2, the molar ratio of the intermediate compound to acryloyl chloride is 1:4, and the molar ratio of the intermediate compound to triethylamine is 1:2.
[0020] This invention also discloses the application of fluorescent probes in the detection of cysteine and hypochlorous acid in diagnosis and treatment.
[0021] Specifically, in the application described above, the detection of cysteine is performed using fluorescence detection at a wavelength of 730 nm, and the detection of hypochlorous acid is performed using fluorescence detection at a wavelength of 570 nm; cysteine only causes an increase in fluorescence intensity at 730 nm, and hypochlorous acid only causes an increase in fluorescence intensity at 570 nm, and the two do not interfere with each other.
[0022] Furthermore, the application of the fluorescent probe in monitoring changes in cysteine and hypochlorous acid levels during oxidative stress, inflammation progression, liver injury, and cellular redox imbalance is disclosed.
[0023] Furthermore, the monitoring includes assessing the dynamic redox interaction between cysteine and hypochlorous acid through changes in dual-channel fluorescence signals.
[0024] Furthermore, the application of the fluorescent probe as a tool molecule in screening candidate drugs for liver injury is disclosed.
[0025] Furthermore, the application was performed in the MCF-7 cell model.
[0026] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:
[0027] (1) Achieve parallel detection of cysteine and hypochlorous acid in two channels without crosstalk: Cysteine can specifically activate the red fluorescent channel, and hypochlorous acid can specifically activate the green fluorescent channel. The two signals are completely independent and have no cross interference, which effectively overcomes the technical problem of channel signal crosstalk that is common in dual-response probes.
[0028] (2) It can realize real-time visualization and dynamic tracking of cysteine and hypochlorous acid level fluctuations in physiological and pathological processes such as oxidative stress, inflammation progression, liver injury and cell oxidative damage at the living cell and living body level.
[0029] (3) The fluorescent probe of the present invention can also be used as a functional tool molecule for screening candidate drugs for inflammation, liver injury and oxidative stress-related diseases. Attached Figure Description
[0030] Figure 1 It is intermediate compound 1 1 H NMR spectrum; Figure 2 It is intermediate compound 1 13 C NMR spectrum; Figure 3 It is fluorescent probe compound 2 1 H NMR spectrum; Figure 4 It is fluorescent probe compound 2 13 C NMR spectrum; Figure 5 This is the kinetic curve of the fluorescent probe's response to Cys; Figure 6 It is the kinetic curve of the response of the fluorescent probe to HClO; Figure 7 These are the fluorescence spectra of the fluorescent probe under different concentrations of Cys (A) and HClO (B); Figure 8 It is the fluorescence spectrum of the fluorescent probe in the presence of both Cys and HClO; Figure 9 It refers to the selectivity and anti-interference properties of fluorescent probes for amino acids and ions; Figure 10 It is the selectivity and anti-interference ability of the fluorescent probe to Cys and HClO; Figure 11 This is an imaging diagram showing the changes in Cys and HClO levels in MCF-7 cells by a fluorescent probe; Figure 12 This is an image showing the changes in Cys and HClO levels by a fluorescent probe in an doxorubicin oxidative stress model. Detailed Implementation
[0031] The following describes the embodiments and appendices. Figure 1-12 The present invention will be further described below, but the present invention is not limited to the following embodiments.
[0032] Example 1: Synthesis of fluorescent probes:
[0033] 1. Synthesis of Compound 1 (Formula I intermediate)
[0034] ;
[0035] Cyanide dye 1 (51 mg, 0.1 mmol) and 3,5-dihydroxybenzyl alcohol 2 (69 mg, 0.5 mmol) were dissolved separately in acetonitrile. After dissolution, the two were mixed evenly. Anhydrous potassium carbonate (38 mg, 0.3 mmol) was added to the mixture. The mixture was reacted at room temperature under nitrogen protection for 15 minutes, then heated to 42.5 °C and reacted for 45 minutes, and then heated to 52 °C and reacted for 3 hours to obtain the crude product. Finally, the crude product was purified by silica gel column chromatography using dichloromethane / ethanol = 100 / 1 as the eluent to obtain compound 1 of formula I.
[0036] Compound 1 1 H NMR spectrum as follows Figure 1 As shown, 1H NMR (600 MHz, DMSO) δ 8.57 (d, J =14.7 Hz, 1H), 7.76 – 7.62 (m, 3H), 7.53 (d, J = 1.2 Hz, 1H), 7.43 (d, J = 0.9Hz, 1H), 6.93 (d, J = 2.3 Hz, 1H), 6.82 (d, J = 2.3 Hz, 1H), 6.49 (d, J =14.8 Hz, 1H), 5.45 (s, 1H), 4.71 (d, J = 5.2 Hz, 2H), 4.40 (d, J = 7.3 Hz,2H), 2.72 (dt, J = 38.9, 6.2 Hz, 4H), 1.87 – 1.81 (m, 2H), 1.74 (s, 6H), 1.38– 1.35 (m, 3H).
[0037] Compound 1 13 C NMR spectra as follows Figure 2 As shown, 13 C NMR (151 MHz, DMSO) δ 175.45,160.70, 160.50, 153.97, 141.28, 141.12, 140.56, 131.14, 128.26, 128.06,125.99, 124.79, 122.18, 113.06, 112.47, 112.01, 111.35, 102.29, 99.93, 64.42, 59.19, 49.49, 29.40, 27.87, 26.86, 19.44, 18.05.
[0038] 2. Synthesis of Compound 2 (Probe II)
[0039] ;
[0040] 26 mg (0.06 mmol) of compound 1 of formula I was dissolved in 3 mL of anhydrous dichloromethane until completely dissolved. Then, 50 μL (0.36 mmol) of anhydrous triethylamine was added, and the mixture was stirred in an ice bath under nitrogen protection for 20 min. Under stirring, 19.7 μL (0.24 mmol) of acryloyl chloride 3 was added dropwise to the mixture, and stirring continued for 30 min. The mixture was then heated to 25 °C under nitrogen protection and stirred for 1 hour. After the reaction was complete, the reaction solution was diluted with 10 mL of dichloromethane, washed three times with 15 mL of water, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography using dichloromethane / ethanol = 50 / 1 as the eluent to obtain compound 2 of formula II, a fluorescent probe.
[0041] Compound 2 1 H NMR spectrum as follows Figure 3 As shown, 1 H NMR (600 MHz, DMSO) δ 8.57 (d, J =15.3 Hz, 1H), 8.20 (s, 1H), 8.09 (s, 1H), 7.84 – 7.77 (m, 3H), 7.71 (d, J =2.3 Hz, 1H), 7.62 – 7.57 (m, 1H), 7.54 (t, J = 7.4 Hz, 1H), 6.80 – 6.58 (m,3H), 6.50 (dd, J = 17.3, 10.4 Hz, 2H), 6.28 (d, J = 10.4 Hz, 1H), 5.76 (s,1H), 5.32 (t, J = 4.6 Hz, 1H), 4.54 (q, J = 7.2 Hz, 2H), 2.79 (t, J = 6.1 Hz, 2H), 2.71 (t, J = 6.1 Hz, 2H), 1.88 – 1.83 (m, 2H), 1.77 (s, 6H), 1.41 (t, J= 7.2 Hz, 3H).
[0042] Compound 2 13 C NMR spectra as follows Figure 4 As shown, 13C NMR (151 MHz, DMSO) δ 192.44,179.12, 164.15, 157.66, 153.58, 151.77, 145.77, 143.35, 141.28, 135.26,133.41, 131.74, 130.12, 129.96, 129.54, 128.59, 127.61, 125.75, 123.39,123.08, 119.58, 115.59, 115.26, 114.42, 107.84, 56.50, 51.57, 41.42, 31.76, 29.50, 27.46, 22.57, 13.48.
[0043] Example 2: Response kinetics of the fluorescent probe to Cys and HClO, referring to... Figure 5-6 :
[0044] Figure 5 To demonstrate the time response of the fluorescent probe to Cys, fluorescence spectral changes at different time gradients were measured by mixing 40 μM Cys with the probe. Figure 5 It can be seen that once the probe molecule is mixed with Cys, the reaction is completed within 150 seconds, and the fluorescence intensity at 730 nm no longer changes; when 40 μM HClO is added to the system that has responded to Cys, as... Figure 6 It can be seen that the reaction is completed within 15 minutes, and the fluorescence intensity at 570 nm no longer changes. This indicates that the probe provided by this invention can detect HClO and Cys in real time.
[0045] Example 3: The responsiveness of the fluorescent probe to different concentrations of Cys and HClO, referring to... Figure 7-8 :
[0046] 7A shows the fluorescence spectra of the fluorescent probe responding to different concentrations of Cys, and 7B shows the fluorescence spectra of the probe responding to different concentrations of HClO. Figure 8 The fluorescence spectra are shown for the response of the fluorescent probe to Cys followed by the addition of HClO. The probe concentration was 10 μM, the Cys concentration varied from 0 to 15 μM, and the HClO concentration varied from 0 to 20 μM. The solution used was a PBS solution containing 30% DMSO (pH = 7.3).
[0047] The testing method was as follows: Different concentrations of cysteine were sequentially added to a PBS buffer system and mixed thoroughly. The fluorescence spectrum changes within the wavelength range of 680-840 nm were then measured using a fluorescence spectrophotometer to obtain a graph showing the relationship between fluorescence spectrum and cysteine concentration. Subsequently, different concentrations of hypochlorous acid were added to the system after the cysteine response was completed. After mixing thoroughly, the fluorescence spectrum changes within the wavelength range of 500-650 nm were measured using a fluorescence spectrophotometer.
[0048] from Figure 7 and Figure 8 It can be seen that the fluorescence intensity at 730 nm is positively correlated with the cysteine concentration as the cysteine concentration increases sequentially; similarly, the fluorescence intensity at 570 nm is positively correlated with the HClO concentration as the HClO concentration increases sequentially. Adding HClO after the Cys response leads to a decrease in fluorescence intensity at 570 nm, indicating that HClO oxidizes and consumes Cys. This demonstrates that the probe provided by this invention can efficiently respond to Cys and HClO, exhibiting good concentration dependence. The fluorescence intensity of the probe shows a good linear relationship with the concentrations of Cys and HClO within a certain range, and the dynamic interaction between Cys and HClO can be monitored in real time.
[0049] Example 4: Selectivity and anti-interference properties of the fluorescent probe, refer to... Figure 9-10 :
[0050] Figure 9 , Figure 10 The selectivity and anti-interference properties of the fluorescent probe for Cys and HClO were investigated. The probe (10 μM) was measured in relation to amino acids (GSH, Cys, Arg, Tyr, Lys) and cations (Fe). 3+ Na + K + Ca 2+ Cu 2+ ), anions (NO3) - Cl - S 2- SO4 2- SO3 2- The fluorescence spectrum changes were observed, and bar charts were obtained showing the fluorescence intensity at 730 nm and 570 nm versus different analytes. Figure 9It is known that the probe exhibits strong fluorescence emission at 730 nm in the presence of Cys. When the aforementioned amino acids, anions, and cations are added to the system after the Cys response, only the presence of HClO induces strong fluorescence emission at 570 nm. Other amino acids, anions, and cations do not cause significant changes in the fluorescence signal, nor do they interfere with the responses of Cys and HClO. Therefore, the probe can achieve highly selective detection of Cys and HClO.
[0051] Example 5: Fluorescence imaging experiment of Cys and HClO in MCF-7 cells:
[0052] MCF-7 cells were selected for confocal microscopy imaging. The specific experimental procedure was as follows: Logarithmic growth phase MCF-7 cells were seeded into confocal culture dishes, with 2 mL of culture medium added to each dish. The dishes were then incubated for 24 hours to allow cell adhesion and growth. After culture, the original culture medium was aspirated, and the cells were gently washed three times with PBS. The cells were then treated according to the following groups.
[0053] (1) Probe assembly: Add only the target fluorescent probe (final concentration 10 μM) and incubate for 30 min;
[0054] (2) Probe + Cys group: First, add cysteine (final concentration 100 μM) for pretreatment for 30 min, then add 10 μM probe and incubate for 30 min;
[0055] (3) Probe + Hypochlorous Acid Group: First, add hypochlorous acid (final concentration 50 μM) for pretreatment for 30 min, then add 10 μM probe and incubate for 30 min;
[0056] (4) Probe + Cys + HClO: First, add 100 μM cysteine for pretreatment for 30 min, then add 50 μM hypochlorous acid for incubation for 30 min, and finally add 10 μM probe for incubation for 30 min.
[0057] (5) Probe + Hypochlorous Acid + Cysteine group: First, add 50 μM hypochlorous acid for pretreatment for 30 min, then add 100 μM cysteine for incubation for 30 min, and finally add 10 μM probe for incubation for 30 min.
[0058] After all groups were treated, the cells were washed three times with PBS buffer to remove unbound probes and reagents, and the changes in fluorescence signal were monitored by confocal fluorescence microscopy.
[0059] Experimental results are as follows Figure 11 As shown, a certain intensity of basal fluorescence signal can be observed after incubating cells with the probe alone. This is because endogenous Cys exists at physiological levels within the cells. After adding exogenous Cys, the fluorescence intensity of the red channel in the cells is significantly enhanced, while the fluorescence signal of the green channel does not change significantly. This indicates that the probe of the present invention can specifically recognize exogenous Cys in cells, and Cys does not interfere with the HClO detection channel. When the probe is co-incubated with HClO, the fluorescence signal of the red channel in the cells does not change significantly, while the fluorescence signal of the green channel is significantly enhanced. The ratio of the green channel to the red channel is significantly higher than that of the control group, indicating that the probe can specifically respond to HClO in the cells, induce the green channel fluorescence signal to open, and achieve specific fluorescence detection of HClO. For the treatment group where Cys is added first and then HClO is added, the fluorescence signal of the green channel is significantly higher than that of the group where only HClO is added, and the ratio of the green to red channel signals is further increased. This indicates that even if Cys is present in advance, the subsequently added HClO can still effectively trigger the probe response and produce a strong green fluorescence signal. In the treatment group where HClO was added first, followed by Cys, a strong green fluorescence signal was also produced. This indicates that the probe exhibits a strong green fluorescence signal after responding to Cys and hypochlorous acid. Changing the order of addition does not affect the detection of HClO, reflecting the probe activation effect under the dynamic interaction of the two substances within the cell. In summary, this probe can achieve visualized detection of the dynamic interaction between Cys and HClO in MCF-7 cells through dual-channel fluorescence ratio imaging. It can clearly distinguish the differences in HClO response under the presence or absence of Cys, providing a reliable imaging tool for studying intracellular redox balance and related physiological and pathological processes.
[0060] Example 6: Application of fluorescent probes in monitoring changes in Cys and HClO levels during oxidative stress:
[0061] MCF-7 cells were selected for confocal microscopy imaging. An oxidative stress model was established by inducing oxidative stress in MCF-7 cells with doxorubicin. NAC was used as a Cys supplement, and ABAH (myeloperoxidase inhibitor) was used to inhibit HClO production. The specific experimental procedure was as follows: Logarithmic growth phase MCF-7 cells were seeded in confocal culture dishes, with 2 mL of culture medium added to each dish. The dishes were incubated for 24 hours to allow cell adhesion. After culture, the original culture medium was discarded, and the cells were gently washed three times with PBS. The cells were then treated according to the following groups:
[0062] (1) Control group: Normal MCF-7 cells, without drug treatment, only probe incubation.
[0063] (2) Dox-10 μM group: MCF-7 cells were first incubated with 10 μM doxorubicin, and then incubated with the probe.
[0064] (3) Dox-30 μM group: MCF-7 cells were first incubated with 30 μM doxorubicin, and then incubated with the probe.
[0065] (4) Dox + NAC group: MCF-7 cells were first incubated with doxorubicin, then with N-acetylcysteine (NAC, Cys supplement), and finally with probe.
[0066] (5) Dox + ABAH group: MCF-7 cells were first incubated with doxorubicin, then incubated with ABAH (myeloperoxidase inhibitor, which inhibits the generation of HClO), and finally incubated with probe.
[0067] (6) NAC group: Normal MCF-7 cells were incubated with NAC only, and then incubated with probe.
[0068] (7) ABAH group: Normal MCF-7 cells were incubated with only ABAH (which inhibits HClO generation), and then incubated with the probe.
[0069] (8) Dox + NAC + ABAH group: MCF-7 cells were first incubated with doxorubicin, then NAC and ABAH were added in sequence, and finally the probe was added for incubation.
[0070] After all groups were treated, the cells were washed three times with PBS buffer to remove unbound probes and reagents, and the changes in fluorescence signal were monitored by confocal fluorescence microscopy.
[0071] Experimental results are as follows Figure 12 As shown, in the control group, the probe mainly exhibited fluorescence in the red channel, with very weak fluorescence in the green channel. As the Dox concentration increased (10 μM → 30 μM), the fluorescence in the red channel gradually weakened, while the fluorescence in the green channel significantly increased. This indicates that doxorubicin-induced oxidative stress increases the intracellular HClO level, triggering the red-to-green fluorescence conversion of the probe, and the response intensity is positively correlated with the Dox concentration.
[0072] In the Dox + NAC group, the green channel fluorescence was significantly weakened compared to the Dox group at the same concentration; while in the NAC alone group, the red channel fluorescence was enhanced, and the green channel showed no obvious fluorescence. This indicates that the Cys supplemented by NAC enhances the red fluorescence response of the probe, and at the same time, it can reduce the intracellular HClO level through reducing action, inhibit the green fluorescence response of the probe, and reverse the Dox-induced increase in fluorescence ratio.
[0073] In the Dox + ABAH group, the green channel fluorescence was significantly weakened; in the ABAH alone group, the green channel fluorescence was also extremely weak. This indicates that ABAH effectively inhibits doxorubicin-induced HClO generation, reduces the green fluorescence trigger source of the probe, and thus reduces the ratiometric fluorescence response of the probe.
[0074] In the Dox + NAC + ABAH group, the green channel fluorescence almost disappeared, indicating that simultaneous supplementation of Cys and inhibition of HClO generation can completely block the doxorubicin-induced probe green fluorescence response. This further verifies that the specificity of the probe's green fluorescence signal depends on the intracellular HClO level, and that Cys can antagonize the HClO response through reducing action.
[0075] The results showed that the probe can specifically recognize intracellular Cys and HClO, and its ratiometric fluorescence signal can reflect the dynamic changes of the two substances during doxorubicin-induced oxidative stress, providing a visual detection tool for studying the mechanism of chemotherapeutic drug-induced oxidative stress.
[0076] Finally, it should be noted that the above embodiments do not limit the present invention in any way. Those skilled in the art can make modifications and improvements based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.
Claims
1. A fluorescent probe for simultaneous detection of cysteine and hypochlorous acid, characterized in that, Its structure is shown in equation (II): (Ⅱ) 。 2. A method for preparing the fluorescent probe of claim 1, characterized in that, The preparation route is as follows: ； ； The preparation method includes the following steps: Step 1: Dissolve cyanine dye (1) and 3,5-dihydroxybenzyl alcohol (2) separately in acetonitrile. After dissolving, mix the two evenly. Add anhydrous potassium carbonate to the mixture and react at room temperature for 10-20 minutes under nitrogen protection. Then, raise the temperature to 40-50℃ and react for 30-60 minutes. Then, raise the temperature to 50-55℃ and react for 2-4 hours. Purify by silica gel column chromatography to obtain the intermediate compound shown in formula (Ⅰ). Step 2: Dissolve the intermediate compound shown in formula (I) in anhydrous dichloromethane, add anhydrous triethylamine, stir in an ice bath for 15-25 minutes under nitrogen protection, add acryloyl chloride (3) dropwise, continue stirring for 25-35 minutes, then heat to 20-30℃ under nitrogen protection and stir for 1-2 hours. After post-treatment and purification by silica gel column chromatography, obtain the fluorescent probe shown in formula (II). In step 1, the molar ratio of cyanine dye to 3,5-dihydroxybenzyl alcohol is 1:5, and the molar ratio of cyanine dye to potassium carbonate is 1:
3. In step 2, the molar ratio of the intermediate compound to acryloyl chloride is 1:4, and the molar ratio of the intermediate compound to triethylamine is 1:
2.
3. The preparation method according to claim 2, characterized in that, The silica gel column chromatography purification in step 1 uses dichloromethane and ethanol at a volume ratio of 100:1 as eluents, and the silica gel is 200-300 mesh.
4. The preparation method according to claim 2, characterized in that, In step 2, the silica gel column chromatography purification uses dichloromethane and ethanol at a volume ratio of 50:1 as the eluent, and the silica gel is 200-300 mesh.
5. The application of the fluorescent probe according to claim 1 in the detection of cysteine and hypochlorous acid during diagnosis or treatment.
6. The application according to claim 5, characterized in that, The cysteine was detected by fluorescence at a wavelength of 730 nm, and the hypochlorous acid was detected by fluorescence at a wavelength of 570 nm. Cysteine only caused an increase in fluorescence intensity at 730 nm, and hypochlorous acid only caused an increase in fluorescence intensity at 570 nm. The two do not interfere with each other.
7. The application of the fluorescent probe according to claim 1 in monitoring changes in cysteine and hypochlorous acid levels during oxidative stress, inflammation progression, liver injury, and cellular redox imbalance.
8. The application of the fluorescent probe according to claim 1 as a tool molecule in screening candidate drugs for liver injury.
9. The application according to any one of claims 5-8, characterized in that, The application was performed in the MCF-7 cell model.
10. The application according to claim 7, characterized in that, The monitoring includes assessing the dynamic redox interaction between cysteine and hypochlorous acid through changes in dual-channel fluorescence signals.