Fluorescent probe for tracking microenvironment change before and after treatment of ischemia-reperfusion injury as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for tracking microenvironment change before and after ischemia reperfusion injury treatment as well as a preparation method and application of the fluorescent probe. The fluorescent probe generates cascade response to H2S, polarity and viscosity; the structural formula of the fluorescent probe is # imgabs0 #. The invention also specifically discloses a preparation method of the fluorescent probe and an application of the fluorescent probe in cell imaging or in-vivo cell imaging. In a selective experiment, fluorescence enhancement of the fluorescent probe can only be induced by hydrogen sulfide and cannot be induced by other metals and biological mercaptan. Therefore, the fluorescent probe shows good selectivity on hydrogen sulfide. On the basis, a rapid, simple and convenient fluorescence detection method for simultaneously detecting microenvironment changes, namely H2S, polarity and viscosity before and after ischemia reperfusion injury treatment is established.

Description

Technical Field

[0001] The invention belongs to the technical field of H2S, polarity and viscosity responsive fluorescent probes, and specifically relates to a fluorescent probe for tracking microenvironment changes before and after ischemia-reperfusion injury treatment, and a preparation method and application thereof. Background Art

[0002] Ischemia-reperfusion injury (IRI) is a common pathological condition in diseases and surgical procedures such as stroke, thrombolytic therapy, myocardial infarction, organ transplantation, and coronary angioplasty. Current studies have shown that the restoration of oxygen supply during reperfusion will lead to the generation of a large amount of ROS. The continued generation of ROS will lead to phospholipid and protein oxidation, which will in turn lead to damage to plasma, mitochondrial membranes, and DNA, and ultimately lead to cellular oxidative stress, mitochondrial dysfunction, and cell death. In order to reduce ischemia-reperfusion injury and maximize the protection of organ function, people have developed methods such as reducing Ca 2+ Ion overload, removal of excess ROS, regulation of inflammatory response, implementation of ischemic preconditioning, postconditioning and remote treatment, etc. Among them, the use of antioxidants to remove excess ROS is an important way to reduce ischemia-reperfusion injury.

[0003] Hydrogen sulfide (H2S) is a colorless, foul-smelling gas that is irritating and asphyxiating. It has traditionally been considered an environmental pollutant and a toxic gas. Long-term exposure to hydrogen sulfide can cause headaches and nausea, and severe acute exposure can cause sudden loss of consciousness, coma, and suffocation. , Even death. Clinical studies have shown that H2S may cause direct damage to blood vessels after entering the human body, which can cause coronary vasospasm and myocardial ischemia in patients, increasing the risk of heart disease. However, H2S has also been found to be the third endogenous gaseous transmitter (signaling molecule) after carbon monoxide and nitric oxide. Endogenous H2S is produced through enzymatic or non-enzymatic pathways in mammalian tissues. The concentration of H2S varies in tissues, with the highest concentration observed in the heart (6.89-8.45 μg / g). In recent years, a number of studies have revealed that H2S has a significant protective effect on the heart in MIRI. As an antioxidant, it can play an important protective role in attenuating MIRI through mechanisms such as attenuating oxidative stress, restoring mitochondrial function, regulating miRNA, interacting with NO, and S-sulfidation.

[0004] Cell polarity refers to the asymmetric shape and uneven distribution of cell components (such as organelles and macromolecules). Polarity is a universal characteristic of cells, which can be observed in almost all cells and is necessary for the development of most multicellular organisms. As an important indicator of the cell microenvironment, polarity directly reflects the health of cells. In addition, viscosity is another important parameter of the cell microenvironment and is the main factor in controlling the diffusion process within the cell. In organisms, abnormal changes in viscosity have a great impact on the transport and signal transduction of intracellular substances, the interaction between biomolecules, and the diffusion of active metabolites. Therefore, the execution of apoptosis, autophagy, ferroptosis, oxidative stress and other cellular functions is inseparable from viscosity.

[0005] Ischemia-reperfusion injury also represents a pathological state involving processes such as inflammation, excessive ROS production, autophagy dysregulation, and mitochondrial dysfunction, and its relationship with factors such as the cellular microenvironment (polarity or viscosity) is less documented in the literature. Hydrogen sulfide, as a reducing gas protective molecule, can inhibit ischemia-reperfusion injury and promote the recovery of damaged cell function. Real-time detection of changes in the cellular microenvironment (polarity or viscosity) during treatment is of great significance for a deeper understanding of the pathology and repair mechanisms of reperfusion injury. Therefore, there is an urgent need to develop a probe that responds to H2S, polarity, and viscosity to help achieve this goal.

[0006] Traditional methods such as gas chromatography, colorimetric detection, and electrochemical analysis have been used to detect H2S, but these methods cannot selectively detect H2S in living systems in a non-invasive manner. In contrast, fluorescence technology, as a non-destructive detection method, is very attractive due to its high sensitivity and real-time detection capability. Many fluorescent probes that can respond to hydrogen sulfide, polarity, or viscosity alone have been reported, all of which show excellent performance, such as rapid excretion ability, low toxicity in organisms, and easy structural modification, but there are still some unresolved issues, such as low sensitivity, single detection target, poor solubility, and inevitable biological toxicity. To the best of our knowledge, there are currently few reports on the technology of integrating hydrogen sulfide, polarity, and viscosity detection into one fluorescent probe through a cascade reaction to detect microenvironmental changes during ischemia-reperfusion injury and treatment. Therefore, these unfavorable characteristics limit its application in biological systems.

[0007] Based on the above background, the present invention designs and prepares a fluorescent probe that can respond to multiple stimuli (such as H2S, polarity and viscosity). The fluorescent probe is formed by coupling tetraphenylethylene (TPE) with classic aggregation-induced emission (AIE) properties and coumarin with good photon quantum yield and stability through double bonds. , The TPEC-OH intermediate was obtained. Subsequently, the classical responsive group 2 of H2S was ,4-Dinitrobenzenesulfonyl (DNBS) was connected as a quencher to synthesize the probe TPEC-DNBS with weak fluorescence emission. The presence of H2S can cleave the DNBS fragment to produce the TPEC-OH precursor, which has a classic D-π-A molecular structure and whose spectrum changes significantly with changes in external environmental parameters (polarity, viscosity, etc.). In addition, due to the AIE characteristics of the TPE molecule, it exhibits weak green fluorescence at low polarity and strong red fluorescence at high polarity, which is exactly the opposite of the characteristics of low polarity strong fluorescence and high polarity weak fluorescence of traditional polar fluorescent probes. Therefore, the fluorescent probe designed by the present invention is a multifunctional fluorescent probe that can respond to H2S, polarity and viscosity at the same time. By constructing an ischemia-reperfusion cell model, it is further proved that the fluorescent probe can detect H2S, polarity and viscosity under physiological conditions, which also proves that polarity or viscosity is an important variable parameter before and after ischemia-reperfusion injury, and H2S can induce the recovery of polarity or viscosity after ischemia-reperfusion injury. However, there are currently no relevant reports in this regard. Summary of the invention

[0008] The technical problem solved by the present invention is to provide a fluorescent probe that responds to H2S, polarity and viscosity and is used to track the changes in the microenvironment before and after ischemia-reperfusion injury treatment, and a preparation method thereof. The fluorescent probe prepared by this method can be used for the detection of endogenous or exogenous hydrogen sulfide in living LO2 cells, and can be used to track the changes in the cell microenvironment (polarity and viscosity) in the ischemia-reperfusion injury model, and can also be used for cell imaging.

[0009] The present invention adopts the following technical solution to solve the above technical problems: a fluorescent probe for tracking microenvironment changes before and after ischemia-reperfusion injury treatment, characterized in that: the fluorescent probe can produce a cascade response to H2S, polarity and viscosity, and the structural formula of the fluorescent probe is:

[0010]

[0011] The method for preparing the fluorescent probe for tracking microenvironment changes before and after ischemia-reperfusion injury treatment of the present invention is characterized by the following specific steps:

[0012] Step S1: adding 7-hydroxycoumarin-4-acetic acid and compound TPE-CHO to methanol, then adding piperidine and reflux reaction, monitoring the reaction process by thin layer chromatography, and after the reaction is completed, separating the obtained mixed solution by column chromatography to obtain a yellow solid compound TPEC-OH;

[0013] Step S2: Add compound TPEC-OH and triethylamine to dichloromethane and stir, then add 2 ,4-dinitrobenzenesulfonyl chloride, the resulting solution was stirred at room temperature, and the reaction process was monitored by thin layer chromatography. After the reaction was completed, the resulting mixed solution was separated by column chromatography to obtain orange solid TBEC-DNBS;

[0014] The corresponding synthetic route in the preparation process is:

[0015]

[0016] The invention discloses an application of the fluorescent probe for tracking microenvironment changes before and after ischemia-reperfusion injury treatment in the preparation of a selective detection preparation for endogenous or exogenous hydrogen sulfide in LO2 living cells.

[0017] The fluorescent probe for tracking microenvironment changes before and after ischemia-reperfusion injury treatment of the present invention is used in preparing a detection preparation for tracking cell microenvironment changes in an ischemia-reperfusion injury cell model.

[0018] It is further defined that the cell microenvironment changes are specifically changes in cell polarity and / or viscosity.

[0019] Furthermore, the fluorescent probe used to track the changes in the microenvironment before and after the treatment of ischemia-reperfusion injury has its own maximum absorption peak at 385nm in a PBS solution containing 20% ​​THF by volume. After the addition of H2S, the maximum absorption wavelength does not change, and the absorption intensity is slightly enhanced. Under excitation at 385nm, the fluorescent probe exhibits extremely weak fluorescence emission. After the addition of H2S, it emits obvious yellow-green fluorescence at 563nm, and the peak intensity is significantly improved relative to the fluorescent probe. The Stocks shift between its excitation and emission peaks reaches 178nm. The TPEC-OH generated after the fluorescent probe reacts with H2S has an obvious solvation effect. In solvents of different polarities, its emission wavelength is significantly red-shifted with the increase of solvent polarity, and the emission intensity is enhanced with the increase of polarity. When the viscosity of the solvent is increased, the fluorescence wavelength of TPEC-OH remains almost unchanged, and the fluorescence intensity is significantly enhanced with the increase of viscosity.

[0020] The invention discloses an application of the fluorescent probe for tracking microenvironment changes before and after ischemia-reperfusion injury treatment in the preparation of cell or in vivo cell imaging preparations.

[0021] The present invention has the following advantages and beneficial effects: the present invention uses tetraphenylethylene aldehyde as a starting material, and 7-hydroxycoumarin-4-acetic acid is reacted by condensation reaction to prepare TPEC-OH intermediate, and then reacts with the classical response group 2 of H2S. ,4-Dinitrobenzenesulfonyl coupling constitutes the fluorescent probe TPEC-DNBS. In this fluorescent probe, tetraphenylethylene has a classic AIE effect, and coumarin fluorescent probes have good photostability and quantum yield. The two are combined through double bonds to obtain a fluorescent probe with AIE characteristics whose emission spectrum is further red-shifted. The prepared fluorescent probe reacts with H2S to generate TPEC-OH with a D-π-A structure, which can further respond to polarity and viscosity. In vitro pH dependence and selectivity experiments show that this fluorescent probe can detect H2S under physiological conditions. Cell fluorescence imaging and colocalization experiments further prove that this fluorescent probe can be used to detect endogenous or exogenous H2S in lipid droplets and mitochondria in living LO2 cells, and can also be used to monitor changes in ischemia-reperfusion injury cell models (H2O2 models or glucose-oxygen deprivation OGD / R models). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the response of the fluorescent probe TPEC-DNBS to H2S, polarity and viscosity.

[0023] Figure 2 The fluorescent probe TPEC-DNBS (10 μM) was prepared in PBS buffer (10 mM , pH=7.4 , (A) UV-visible absorption spectra before and after adding H2S (20 equivalents) to 20wt% THF; (B) fluorescence spectra comparison; (C) fluorescence enhancement at 563nm after adding H2S (20 equivalents) over time; (D) fluorescence emission spectrum after adding H2S (0-90μM); (E) fluorescence intensity (563nm) at different pH values; (F) different analytes (H2S is 200μM, GSH is 1mM, and other analytes are 0.5mM) (1-15: Hcy, Cys, GSH, Val, Thr, Gln, Tyr, Asp, Lys, ClO4 - 、SO4 2- 、SO3 2- , ClO - 、S2O3 2- 、HCO3 - , 16-26: F - Br - 、NO3 - 、CO3 2- , Ca 2+ , K + Cr 3+ 、Na + Mg 2+ 、Al 3+ 、Co 2+ , 27: HS -) after addition of the fluorescent probe (563 nm). The black bars represent the probe response to various analytes. The red bars represent the response after further addition of NaHS (200 μM, 20 equivalents) in the presence of the above analytes.

[0024] Figure 3 The sensing mechanism of the fluorescent probe TPEC-DNBS for selective detection of H2S.

[0025] Figure 4 (A) Normalized fluorescence emission spectra of TPEC-DNBS (10 μM) after adding NaHS (20 equivalents) in different solvents; (B) Linear fitting plot of the maximum emission wavelength of TPEC-DNBS (10 μM) after adding NaHS (20 equivalents) in different solvents and the empirical solvent polarity parameter ET (30).

[0026] Figure 5 (A) UV-visible spectrum of the precursor TPEC-OH (10 μM) in PBS / THF mixed solvent (PBS fraction (fw) 0-100%, v / v); (B) fluorescence spectrum; (C) changes in the maximum emission wavelength and corresponding fluorescence intensity under each PBS component. λex: 385nm; (D) when the PBS fraction (fw) is 20wt%-60wt%, the maximum fluorescence emission wavelength is linearly fitted with the corresponding polarity parameter Δf (0.27-0.30); (E) fluorescence photograph of TPEC-OH under 365nm UV light in THF / PBS solvent system.

[0027] Figure 6 (A) Fluorescence spectrum changes of precursor TPEC-OH (10 μM) in THF / MeOH mixed solvent with changes in methanol content; (B) Linear fitting of the maximum fluorescence emission peak and emission wavelength of TPEC-OH with changes in the polarity of the mixed solvent; (C) Fluorescence photograph of TPEC-OH under 365 nm UV light in THF / MeOH solvent system.

[0028] Figure 7 (A) Fluorescence emission spectra of TPEC-OH (10 μM) in different ratios of ethanol and glycerol; (B) Linear fitting curves of Log(η) and Log(I623).

[0029] Figure 8Co-localization fluorescence imaging of the fluorescent probe TPEC-DNBS after incubating LO2 cells with Nile Red, Mitto-Tracker Red and Lyso-Tracker Red and adding 10 times NaHS; (a1-a3) green channel (λex=405nm, λem=500~550nm), (b1-b3) red channel (λex=561nm, λem=663~738nm); (c1-c3) bright field images; (d1-d3) merged images; (e1-e3) intensity scatter plots of TPEC-DNBS+NaHS and various commercial probes; (f1-f3) gray value distribution of LO2 cells, where the red line is the gray value distribution of commercial probes, and the black line is the gray value distribution of TPEC-DNBS+NaHS, scale bar: 10μm.

[0030] Fig. 9 A) Fluorescence imaging of exogenous H2S in cells; control group: cells cultured with TPEC-DNBS (10μM); NaHS group: cells treated with different concentrations of NaHS (100μM, 300μM, 500μM) and TPEC-DNBS (10μM) simultaneously; B) The bar graph on the right represents the relative pixel intensity values ​​of the image on the left. λex=405nm,λem=570~620nm; scale bar: 50μm.

[0031] Fig.10 Middle A) Endogenous H2S fluorescence imaging of LO2 cells. Control group: cells cultured with TPEC-DNBS (10μM). Cys group: cells pretreated with 400μM Cys for 1h and 2h, then treated with 10μM TPEC-DNBS. Cys+AOAA group: cells were incubated with AOAA (1mM) and Cys (400μM) for 2h, then treated with Probe (10μM). B) The bar graph on the right represents the relative pixel intensity values ​​of the left image. λex=405nm,λem=570~620nm; Scale bar: 50μm.

[0032] Fig.11 Fluorescence imaging of cell viscosity: control group: cells incubated with TPEC-OH (10 μM); nystatin group: cells induced with nystatin (10 μM) for 30 min and then co-incubated with TPEC-OH (10 μM). Green channel: λex = 405 nm, λem = 500-550 nm; red channel: λex = 405 nm, λem = 570-620 nm. Scale bar: 50 μm.

[0033] Fig.12(A) Fluorescence imaging of the microenvironment (polarity or viscosity) of LO2 cells. Control group: cells cultured with TPEC-OH (10 μM); H2O2 group: cells treated with H2O2 (400 μM) and then co-incubated with TPEC-OH (10 μM); NaHS group: LO2 cells treated with H2O2 (400 μM) and co-incubated with TPEC-OH (10 μM), and then treated with NaHS (100 μM) for different time periods (30 min, 2.5 h); (B) Bar graph of relative pixel intensity on the right. Green channel: λex = 405 nm, λem = 500-550 nm; Red channel: λex = 405 nm, λem = 570-620 nm. Scale bar: 50 μm.

[0034] Fig.13 This is the 1H-NMR spectrum of compound TPEC-OH in DMSO-d6 at 298K.

[0035] Fig.14 This is the 13C-NMR spectrum of compound TPEC-OH at 298K in DMSO-d6.

[0036] Fig.15 This is the ESI-MS spectrum of compound TPEC-OH.

[0037] Fig.16 This is the 1H-NMR spectrum of compound TPEC-DNBS at 298K in DMSO-d6.

[0038] Fig.17 This is the 13C NMR spectrum of compound TPEC-DNBS at 298K in DMSO-d6.

[0039] Fig.18 This is the ESI-MS spectrum of compound TBEC-DNBS.

[0040] Fig.19 The linear fitting graph of the fluorescence emission intensity (563 nm) of TPEC-DNBS (10 μM) titrated with NaHS (10-70 μM) in PBS buffer (10 mM, pH=7.4, 20 wt% THF).

[0041] Fig. 20 To compare TPEC-DNBS (10 mM) by HPLC, NaHS was added to TPEC-DNBS and TPEC-OH, eluent: CH3OH:H2O=9:1, v / v.

[0042] Fig.21 This is the negative ion mode ESI-MS spectrum of the reaction between compound TPEC-DNBS (10.0 mM) and NaHS (10 equiv).

[0043] Fig. 22 (A) Normalized fluorescence emission spectra of TPEC-OH in different solvents; (B) Maximum emission wavelength and empirical parameter E of TPEC-OH (10 μM) in different solvents T (30), excitation wavelength: 385 nm.

[0044] Fig.23 The UV-visible spectrum of the precursor TPEC-OH (10 μM) in THF / MeOH mixed solvent changes with the methanol content.

[0045] Fig.24 UV-visible spectra of (A) TPEC-OH and (B) TPEC-DNBS (10 μM) in different ratios of ethanol and glycerol.

[0046] Fig.25 (A) Fluorescence spectra of the fluorescent probe TPEC-DNBS after adding 20 times NaHS to different proportions of ethanol and glycerol; (B) Log(I 623 ) and Log(η) linear fitting curve.

[0047] Fig.26 Cytotoxicity experiment of LO2 cells incubated with different concentrations of TPEC-DNBS (left) and TPEC-OH (right) for 24 hours.

[0048] Fig. 27 (A) Fluorescence imaging of the microenvironment (polarity or viscosity) of LO2 cells. Control group: cells cultured with TPEC-OH (10 μM); OGD / R group: OGD / R cells cultured with TPEC-OH (10 μM); NaHS group: OGD / R cells co-incubated with TPEC-OH (10 μM) and then treated with NaHS (100 μM) for different time periods (30 min, 2.5 h); (B) Bar graph of relative pixel intensity on the right. Green channel: λex = 405 nm, λem = 500-550 nm; Red channel: λex = 405 nm, λem = 570-620 nm, scale bar: 50 μm.

[0049] Fig.28(A) Fluorescence imaging of the microenvironment (polarity or viscosity) of H9C2 cells. Control group: cells were cultured with TPEC-DNBS (10 μM) and then treated with NaHS (100 μM) for different time periods (30 min, 2.5 h); H2O2 group: H9C2 cells were treated with H2O2 (400 μM) and then co-incubated with TPEC-DNBS (10 μM), and then treated with NaHS (400 μM, 700 μM) for different time periods (30 min, 2.5 h); Green channel: λex = 405 nm, λem = 500-550 nm; Red channel: λex = 405 nm, λem = 570-620 nm, scale bar: 50 μm. DETAILED DESCRIPTION

[0050] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0051] Example 1

[0052] Unless otherwise specified, the instruments and reagents used in the present invention were obtained from commercial sources.

[0053] 1 Experimental part

[0054] 1.1 Materials and reagents

[0055] All chemicals were purchased from Anhui Energy Chemical Co., Ltd., China. All other reagents were of analytical grade and used without further purification unless otherwise specified.

[0056] 1.2 Main instruments

[0057] In addition, NMR spectra of organic compounds were obtained on a Bruker Ascend TM Electron spray ionization (ESI) mass spectra were recorded on a Bruker Microtof QII spectrometer. UV-visible absorption spectral data were obtained by Shimadzu UV2600 spectrophotometer. Fluorescence spectra were recorded using an Edinburgh InstruMents FS-5 fluorescence spectrophotometer. Cell imaging was recorded using a NIKON A1R confocal fluorescence microscope.

[0058] 1.3 Methods

[0059] 1.3.1 Preparation of fluorescent probes

[0060] Synthetic route

[0061]

[0062] The fluorescent probe is synthesized according to the synthesis route shown above. The specific synthesis process is:

[0063] The specific preparation method of the compound TPEC-OH is as follows: take a 50 mL round-bottom flask, add 7-hydroxycoumarin-4-acetic acid (110 mg, 0.5 mmol) and TPE-CHO (180 mg, 0.5 mmol) to 10 mL of methanol, and then add piperidine (46 μL), and the resulting solution is refluxed for 8.0 hours, and the reaction process is monitored by thin layer chromatography. After the reaction is completed, the obtained mixed solution is subjected to column chromatography (dichloromethane / methanol=50:1, v / v) to obtain a yellow solid TPEC-OH (103.6 mg, yield 40%).

[0064] Structural characterization of compound TPEC-OH: 1 H NMR (400MHz , DMSO)δ10.60(s , 1H) , 7.99(d , J=8.9Hz , 1H) , 7.54(dt , J=24.1 , 12.2Hz , 4H) , 7.19–7.09(m , 9H) , 7.03–6.96(m , 8H) , 6.80(dd , J=8.8 , 2.4Hz , 1H) , 6.74(d , J=2.4Hz , 1H) , 6.50(s , 1H). 13 C NMR (101MHz , DMSO-d6)δ161.35 , 160.74 , 155.33 , 150.23 , 144.43 , 143.11 , 143.06 , 142.93 , 141.18 , 140.07 , 137.14 , 134.00, 131.17 , 130.76 , 130.70 , 130.66 , 127.99 , 127.91 , 127.84 , 127.48 , 126.81 , 126.71 , 126.66 , 120.27 , 112.88 , 110.60 , 104.73 , 102.56. ESI-MS , (m / z) , Calcd for [C 37 H 27 O3] + :519.1954; found , 519.1942.

[0065] The specific preparation method of compound TPEC-DNBS is as follows: In a 50 mL round-bottom flask, TPEC-OH (45 mg, 0.087 mmol) and triethylamine (26 uL) were added to 8 mL of dichloromethane and stirred for 10 min, and then 2 , 4-Dinitrobenzenesulfonyl chloride (46.4 mg, 0.174 mmol), the resulting solution was stirred at room temperature for 5.0 hours, and the reaction process was monitored by thin layer chromatography. After the reaction was completed, the resulting mixed solution was subjected to column chromatography (eluent: pure dichloromethane) to obtain an orange solid compound TBEC-DNBS (57 mg, yield 86%).

[0066] Structural characterization of compound TPEC-DNBS: 1 H NMR (400MHz , DMSO-d6)δ9.13(d , J=2.3Hz , 1H) , 8.61(dd , J=8.7 , 2.3Hz , 1H) , 8.32(d , J=8.7Hz , 1H) , 8.28(d , J=8.9Hz , 1H), 7.57(d , J=7.0Hz , 4H) , 7.38(d , J=2.4Hz , 1H) , 7.23–7.11(m , 10H) , 7.03–6.947(m , 8H) , 6.84(s , 1H). 13 C NMR (101MHz, DMSO) δ = 159.62, 153.99, 151.64, 150.09, 149.18, 148.17, 144. 73,143.11,143.04,142.90,141.29,140.04,138.23,133.83,133.73,131.2 2,130.77,130.72,130.67,130.53,128.01,127.94,127.87,127.65,127.55 , 126.85, 126.75, 126.71, 121.29, 119.44, 118.27, 118.02, 110.81, 109.43. ESI-MS(m / z),Calcd for[C 43 H 29 N2O9S] + :749.1588; found:749.1518. Calcd for[C 43 H 28 N2NaO9S] + :771.1407;found:771.1342.

[0067] 1.3.2 Cell culture

[0068] Culture: LO2 cells were cultured in DMEM medium with 10wt% fetal bovine serum (FBS) and 1wt% penicillin-streptomycin. The cells were incubated at 37°C with 5% H2S and the cells were passaged when the cell saturation exceeded 70%.

[0069] Inoculation: Add DMEM complete medium containing 10 wt % FBS to a 24-well plate and culture in a 37° C., 5% H 2 S incubator for 12 h.

[0070] Counting and photography: The number and status of cells in the 24-well plate were observed using an inverted biological microscope.

[0071] 2 Results and discussion

[0072] 2.1 Design and synthesis of TPEC-DNBS

[0073] In this experiment, tetraphenylethylene aldehyde was used as the starting material, and 7-hydroxycoumarin-4-acetic acid was used to prepare the TPEC-OH intermediate through a condensation reaction, and then coupled with the classic response group of H2S, 2,4-dinitrobenzenesulfonyl, to form the fluorescent probe TPEC-DNBS. In this fluorescent probe, tetraphenylethylene has a classic AIE effect, and coumarin fluorescent probes have good photostability and quantum yield. After the two are combined through a double bond, it is possible to obtain a fluorescent probe with AIE characteristics whose emission spectrum is further red-shifted. After the fluorescent probe reacts with H2S, TPEC-OH with a D-π-A structure is generated, which can further respond to polarity or viscosity.

[0074] 2.2 Spectral response of TPEC-DNBS to H2S

[0075] After obtaining the fluorescent probe, the corresponding spectral experiments were carried out. First, the UV-vis absorption spectrum of the fluorescent probe TPEC-DNBS in PBS buffer (10mM, pH 7.4, 20wt% THF, v / v) showed that for the fluorescent probe TPEC-DNBS, its own maximum absorption peak was at 385nm. After reacting with H2S (with NaHS as the H2S source), the maximum absorption wavelength remained unchanged, but the absorption intensity increased slightly. The change in the absorption spectrum indicated that a new H2S-mediated TPEC-OH reaction product was generated.

[0076] Fluorescence experiments on the reaction of the fluorescent probe TPEC-DNBS with H2S were also carried out. When excited at 385nm, the free fluorescent probe TPEC-DNBS showed very weak fluorescence emission. After the addition of H2S (20 equivalents), the fluorescent probe emitted obvious yellow-green fluorescence at 563nm, and the peak intensity increased by 95 times compared with the single probe. The Stokes shift between the excitation peak and the emission peak reached 178nm. The results showed that the fluorescent probe can respond to H2S while reducing the influence of background excitation light. In addition, time-resolved kinetic studies showed that after adding H2S (20 equivalents) to PBS buffer (10mM, pH=7.4, 20wt% THF), the fluorescence intensity (563nm) of the fluorescent probe reached equilibrium within 8 minutes, indicating that the fluorescent probe has a rapid response characteristic to H2S. Concentration-dependent fluorescence titration experiments showed that the fluorescence intensity of the fluorescent probe at 563nm increased with the increase of H2S concentration, and showed a good linear relationship in the range of 10-70μM (R 2=0.991) Based on 3σ / slope (σ=0.00883), the corresponding detection limit of the fluorescent probe TPEC-DNBS for H2S is 0.0657μM. The results show that TPEC-DNBS can achieve rapid and highly sensitive quantitative analysis of H2S.

[0077] In addition, by detecting various potential interfering substances, such as Hcy, Cys, GSH, Val, Thr, Gln, Tyr, Asp, Lys, ClO4 - 、SO4 2- 、SO3 2- , ClO - 、S2O3 2- 、HCO3 - 、16-26F - Br - 、NO3 - 、CO3 2- , Ca 2+ , K + Cr + 、Na + Mg 2+ 、Al 3 + 、Co 2+ , HS - It can be seen that the above analytes have no obvious interference with the detection of H2S. In addition, a pH-dependent fluorescence experiment was conducted. The fluorescence emission intensity of the fluorescent probe TPEC-DNBS itself was weak, and the intensity fluctuation was small in the range of pH = 3 to 12, indicating that the fluorescent probe has good stability at different pH levels. After adding excess H2S (20 equivalents), the fluorescent probe showed a robust response characteristic in the pH range of 4 to 9. This shows that the fluorescent probe TPEC-DNBS can detect H2S within the physiological range, laying the foundation for the fluorescent probe to detect H2S in cells or in vivo.

[0078] 2.3 Response mechanism of TPEC-DNBS to H2S

[0079] According to literature reports, the response mechanism of the fluorescent probe TPEC-DNBS to H2S may be as follows: Figure 3 As shown, in order to further prove this reaction mechanism, HPLC and MS experiments were performed to verify it. Fig. 20 ), the emission peak of the fluorescent probe is at 7.6min. With the addition of H2S, the signal peak of the fluorescent probe TPEC-DNBS gradually disappears, and a new peak appears at 8.7min. The position of this new peak is consistent with the peak of the precursor compound TPEC-OH. In addition, after TPEC-DNBS reacts with H2S, the reaction solution is taken for ESI-MS experiment. From the results, we can see that ( Fig.21 ), there is a peak of fluorescent probe TPEC-DNBS (783.1214, attributed to [M+Cl] - , calculated value is 783.1209), the precursor TPEC-OH peak (517.1833, assigned to [MH] - The above results show that there is a positive relationship between the fluorescent probe and H2S. Figure 3 The response mechanism is shown.

[0080] 2.4 Response of TPEC-DNBS to polarity

[0081] When investigating the response of the probe to H2S, it was found that H2S induced an obvious solvation effect when the probe fluorescence was enhanced, that is, the emission wavelength showed obvious differences in solvents of different polarity. Figure 4 As shown in the figure, from the intensity normalized fluorescence spectrum change data of the fluorescent probe TPEC-DNBS in different solvents after adding NaHS, it can be seen that with the change of solvent polarity (E T 30 ) is enhanced, and the wavelength corresponding to the maximum emission peak is red-shifted. T 30 ) have a good linear relationship. This is the classic solvation effect characteristic of polar probes. According to the reaction mechanism proposed above, it is speculated that the precursor TPEC-OH generated after the fluorescent probe reacts with H2S may have an obvious solvation effect; the fluorescence of the precursor TPEC-OH may change with the change of solvent polarity. In order to further verify this speculation, the changes in the fluorescence of the precursor TPEC-OH in solvents of different polarities were tested, such as Fig. 22 As shown. The results further confirmed that TPEC-OH does have the characteristic of responding to polarity. Therefore, the above results show that the fluorescent probe can achieve polarity response to different solvents in the presence of H2S.

[0082] In order to further study the response of fluorescent probes to polarity ( Figure 5 ), the UV and fluorescence changes of TPEC-OH were studied in the dual solvent system THF / PBS with a large polarity difference (Δf: 0.21-0.32). From the results, the UV absorption of TPEC-OH did not change significantly with polarity, and only the absorption intensity changed slightly ( Figure 5 However, the fluorescence intensity and maximum emission wavelength changed significantly with the change of polarity. w) From 0-70%, the wavelength gradually red-shifts from 529nm to 615nm, and the fluorescence intensity shows a trend of slow enhancement, and its intensity increases from 3.3 times to 29.1 times relative to the probe. When the PBS content is in the range of 70%-90%, the wavelength rapidly blue-shifts to around 563nm, and the fluorescence intensity shows a trend of dramatic enhancement, reaching more than 100 times that of the fluorescent probe. When the PBS content exceeds 90%, the fluorescence intensity is almost completely quenched. The reason for the above spectral changes may be attributed to the fact that the TPEC-OH generated by the probe after the reaction with NaHS has a typical TICT effect and AIE dual effect. TPEC-OH is a D-π-A structure composed of tetraphenylethylene as an electron donor and coumarin as an electron acceptor. Tetraphenylethylene has a classic AIE effect, and the D-π-A structure of the entire molecule is easy to produce a TICT effect. It is well known that highly polar solvents can stabilize and promote the TICT state, while non-polar solvents are not conducive to the TICT state. In the low polarity range (0-70%), as the polarity of the solvent increases, the intramolecular rotation of the probe D / A unit transforms the molecule from the local excitation (LE) state to the twisted intramolecular charge transfer (TICT) state, which promotes the formation of the probe's TICT state. At this time, the highest occupied molecular orbital energy level increases and the energy level band gap narrows, so the probe emission peak red-shifts. In addition, the TICT state is usually very sensitive to various non-radiative quenching, which traditionally leads to a decrease in emission intensity. For TPEC-OH, since its (π,π*) energy level is much lower than the (n,π*) energy level, it produces a proximity effect suppression (SOPE), which is an effect that leads to fluorescence enhancement. The two opposite effects of SOPE and TICT make TPEC-OH show very weak luminescence enhancement in this range. When the PBS content exceeds 70%, due to the increase in water content, molecular aggregation inhibits the intramolecular rotation that plays a decisive role in the TICT state, and the AIE effect occurs, resulting in a sharp increase in fluorescence intensity in this range. When the PBS content exceeds 90%, the rapid aggregation of TPEC-OH molecules will form amorphous substances with low emission and small size, and their fluorescence emission is more easily affected by the surrounding solvent environment, resulting in a sharp decrease in the fluorescence intensity of high water fractions. In addition, a linear fit was performed between the maximum emission wavelength of TPEC-OH and the polarity parameter Δf (0.27-0.30) of the solvent in the range of 20% to 60%, which produced a good linear relationship (R 2 =0.9705). These results all indicate that within a certain polarity range, the fluorescent probe can be used to quantitatively analyze polarity in the presence of H2S.

[0083] In order to eliminate the interference of AIE effect and possible viscosity on the quantitative analysis of polarity, corresponding UV and fluorescence spectroscopy experiments were carried out in THF / MeOH solvent system. THF and MeOH have similar viscosity coefficients (0.53cp vs. 0.59cp) and large polarity differences (Δf0.21 vs. 0.31). At the same time, TPEC-OH has good solubility in THF and MeOH, avoiding the AIE effect caused by poor solvents. Figure 6 As shown in the figure, in the entire range of methanol content from 0-100%, as the amount of methanol increases, the polarity of the mixed solvent gradually increases, and the fluorescence intensity of TPEC-OH increases from the initial 3.3 times (in THF) to 17.2 times (in MeOH) relative to the fluorescence intensity of the fluorescent probe; at the same time, the maximum emission peak gradually increases from 530nm to 620nm. This change shows that in the THF / MeOH solvent system, the AIE effect is significantly suppressed, and the suppression of the proximity effect (SOPE) that leads to fluorescence enhancement is always stronger than the TICT effect that leads to fluorescence attenuation, so as the solvent polarity increases, the emission peak red shifts and the intensity increases. In addition, the wavelength and intensity are linearly fitted with the solvent polarity parameter Δf (0.21-0.31), and it is found that both show a good linear relationship with the polarity (wavelength R 2 =0.977, strength R 2 =0.933). However, the UV absorption is only slightly affected in the entire polarity range ( Fig.23 ). Therefore, the above experimental results show that the fluorescent probe can further respond to polarity after responding to H2S.

[0084] 2.5 Response of TPEC-DNBS to viscosity

[0085] Viscosity is also an important microenvironmental parameter, and the effect of the fluorescent probe on the viscosity response in the presence of H2S was also studied. Figure 7 As shown in Figure 2, the response of the fluorescence emission of TPEC-OH to viscosity was studied in the ethanol / glycerol system. Ethanol and glycerol have similar polarity parameters (E T (30):51.9vs 57.0) and a large viscosity difference (viscosity (η):1.2vs 1390), which can effectively avoid the interference of polarity when studying the effect on viscosity. In pure ethanol solvent, TPEC-OH produces a maximum emission peak at 601nm. As the glycerol content continues to increase, the fluorescence intensity gradually increases. From pure ethanol to ethanol solution containing 90% glycerol, the fluorescence intensity increases by 3.95 times ( Figure 7 In addition, the fluorescence intensity (log I) and viscosity (logη) parameters of TPEC-OH at the maximum emission wavelength of 623 nm were calculated according to -Hoffmann equation was used for linear fitting and it was found that there was a good linear relationship (R 2 =0.9694)( Figure 7 The above results show that TPEC-OH can produce a significant response to viscosity, and viscosity only affects the change of fluorescence intensity, and has no effect on the absorption and emission wavelengths. When the fluorescent probe TPEC-DNBS was used directly to study the change of fluorescence intensity with viscosity after adding NaHS in the ethanol / glycerol system, similar results were produced (such as Fig.25 ). Therefore, the above results show that the fluorescent probe can achieve quantitative detection of viscosity in the presence of H2S.

[0086] 2.6 Cytotoxicity and colocalization experiments of TPEC-DNBS

[0087] After obtaining the in vitro response results of the fluorescent probe to parameters such as H2S, polarity and viscosity, we attempted to further study the application of the fluorescent probe in vivo. First, the cytotoxicity of the fluorescent probe was tested and the MTT experiment was performed. According to the experimental results, the cell survival rate of the fluorescent probe at 20μM was still above 80%, indicating that the cytotoxicity of the fluorescent probe was relatively low, laying the foundation for further in vivo application. Subsequently, co-localization experiments were carried out, and commercially available lysosomal red, mitochondrial red, Nile red and other dyes were co-incubated with the probe. The fluorescence co-localization experiment of the fluorescent probe co-incubated with Nile red dye after adding NaHS (10 times) showed good co-localization phenomenon, and its co-localization coefficient reached 0.87, the co-localization coefficient with mitochondrial red was 0.81, and the co-localization coefficient with lysosomal red was 0.55. This result shows that the probe is mainly distributed in cell lipid droplets and mitochondria, rather than in lysosomes.

[0088] 2.7 TPEC-DNBS imaging of H2S in living cells

[0089] Encouraged by the results of the in vitro fluorescent probe's highly sensitive and selective detection of H2S, we further conducted experiments on the probe's response to H2S in living cells. First, we detected exogenous H2S ( Fig. 9 ), when TPEC-DNBS was added alone to LO2 cells, there was almost no fluorescence in the control group. After the cells were treated with 100μM, 300μM and 500μM NaHS for 30 minutes, and then incubated with TPEC-DNBS, obvious fluorescence enhancement was observed, and the fluorescence intensity gradually increased with the increase of H2S concentration. These research results show that TPEC-DNBS can be used to monitor changes in exogenous H2S concentration in living cells.

[0090] In order to further verify whether the fluorescent probe can respond to endogenous H2S, cysteine ​​(Cys) was used as an inducer to enhance the production of endogenous H2S in cells. According to literature reports, Cys can desulfurize to generate endogenous H2S under the direct action of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). After LO2 cells were pretreated with Cys (400μM) for 1 hour, the fluorescent probe (10μM) was added for co-incubation. Subsequently, it was found that there was a significant fluorescence enhancement compared with cells not treated with Cys. The reason may be that Cys induces the production of endogenous H2S and then reacts with the fluorescent probe, resulting in fluorescence enhancement. When the cells were co-treated with Cys (400μM) and the inhibitor AOAA (1mM, CBS synthase inhibitor), and then co-incubated with the probe, it was found that the fluorescence signal was significantly reduced, indicating that the process of converting Cys to generate endogenous H2S was significantly inhibited by AOAA, and endogenous H2S could not be generated to react with the probe, resulting in a decrease in fluorescence. These fluorescence imaging pictures can be seen in Fig.10 The above experimental results show that fluorescent probes can be used to detect exogenous and endogenous H2S in living cells.

[0091] 2.8 TPEC-DNBS imaging of living cell viscosity

[0092] Inspired by the probe's response to viscosity in the presence of hydrogen sulfide in in vitro experiments, we further studied whether the fluorescent probe could perform cellular imaging of viscosity in living cells. Nystatin is often used to construct a high-viscosity cellular environment. In the experimental group, 10 μM nystatin was added to the LO2 cells for 30 minutes, and then TPEC-OH (10 μM) was added for co-incubation for 30 minutes; in the control group, TPEC-OH was used alone to co-incubate with LO2 cells. The fluorescence changes of the green and red fluorescence channels when excited at 405 nm were observed under a laser confocal microscope. The results showed that only weak red and green fluorescence was observed when TPEC-OH was co-incubated with the cells alone. After the cells were first incubated with nystatin and then co-incubated with TPEC-OH, the fluorescence intensity of the cells was significantly enhanced ( Fig.11 ), the possible reason is that nystatin induces an increase in cell viscosity, which leads to an increase in the fluorescence of TPEC-OH, which is consistent with the results of previous in vitro studies. These results show that TPEC-DNBS can monitor changes in cell viscosity in the presence of H2S.

[0093] 2.9 Detection of changes in the microenvironment before and after H2S treatment in the ischemia-reperfusion injury model

[0094] An important cause of ischemia-reperfusion injury is the production of excessive ROS. Many literatures have reported that H2S can be used to treat ischemia-reperfusion injury. One of the main reasons is that it can act as a reducing agent to remove ROS and thus reduce oxidative stress. Changes in polarity or viscosity at the cellular level in this process have not been studied in depth. Here, we used LO2 hepatocytes and H9C2 cardiomyocytes to construct an ischemia-reperfusion cell model using the H2O2 method and the OGD / R method to simulate the process of liver ischemia-reperfusion injury and myocardial ischemia-reperfusion injury, and studied whether there are changes in the cellular microenvironment before and after injury or during treatment in these models. According to literature reports, H2O2 can be used to simulate the reactive oxygen burst during ischemia-reperfusion to construct an ischemia-reperfusion injury model. Here, we added H2O2 to LO2 cells and H9C2 cardiomyocytes to construct a myocardial ischemia-reperfusion injury model to verify our speculation. Fig.12 As shown in the figure, the control group LO2 cells co-incubated with TPEC-OH showed strong fluorescence in the red channel and the green channel, while the fluorescence intensity of the two channels of the LO2 cells treated with H2O2 and TPEC-OH decreased by about 4-5 times. In the H2S treatment group, as the hydrogen sulfide treatment time prolonged, the fluorescence intensity of the two channels of the LO2 cells treated with H2O2 recovered to varying degrees. These results show that normal LO2 hepatocytes are a high-polarity environment under appropriate viscosity, so the red channel (corresponding to viscosity) and the green channel (corresponding to polarity) both show a certain intensity of fluorescence signal. After ischemia-reperfusion injury, the hepatocytes become a low-viscosity and weakly polar pathological environment, showing the phenomenon of weakened fluorescence intensity of both channels. After different treatment times with hydrogen sulfide, the polarity and viscosity of the cells gradually recovered, so the fluorescence intensity of the red channel and the green channel gradually increased. After about 1 hour, judging from the fluorescence intensity, the polarity and viscosity of the cells returned to the level of normal cells.

[0095] In addition, OGD / R is a common method to construct an ischemia-reperfusion injury model. Fig. 27 ), LO2 cells co-incubated with TPEC-OH showed similar phenomena to those in the H2O2 model. In order to further verify the proposed viewpoint, H2O2 was also used to construct an H9C2 cardiomyocyte ischemia-reperfusion injury model. Fig.28As shown, in this model, the fluorescent probe TPEC-DNBS was directly co-incubated with H9C2 cells. Under normal cell conditions, due to high polarity or appropriate viscosity, the fluorescent probe brightened both the red and green channels 0.5 hours after the addition of NaHS (100 μM). However, under the pathological environment of ischemia-reperfusion injury, due to the low polarization and low viscosity of the microenvironment, even if a higher dose of NaHS (400 μM, 700 μM) than the control group was added, no fluorescence enhancement was observed in the two channels within the same time (0.5h). Only when the incubation time was longer at a high dose (700 μM, 2.5h), fluorescence enhancement was observed in both channels. Therefore, the above experiments prove that the probe TPEC-DNBS can track the changes in the cell microenvironment (polarization or viscosity) during H2S treatment of ischemia-reperfusion injury in real time. It was further clarified that ischemia-reperfusion injury induces a pathological environment of weak polarity and low viscosity in cells. When treated with H2S reducing gas, the polarity and viscosity of the cells will be restored to the normal cell state in real time while removing excess ROS.

[0096] 3 Conclusion

[0097] The present invention designs and prepares a fluorescent probe that can respond to multiple stimuli (such as H2S, polarity and viscosity). The fluorescent probe is obtained by coupling tetraphenylethylene (TPE) with classic aggregation-induced emission (AIE) characteristics and coumarin with good photon yield and stability through double bonds to obtain a TPEC-OH intermediate. Subsequently, the classic response group 2 of H2S , 4-Dinitrobenzenesulfonyl (DNBS) was used as a quencher to synthesize the fluorescent probe TPEC-DNBS with weak fluorescence emission. The presence of H2S can cleave the DNBS fragment to produce the TPEC-OH precursor. The precursor fragment has a classic D-π-A molecular structure, and its spectrum changes significantly with the changes in external environmental parameters (polarity and viscosity, etc.). In addition, due to the AIE characteristics of the TPE molecule, it exhibits weak green fluorescence at low polarity and strong red fluorescence at high polarity, which is exactly the opposite of the characteristics of strong fluorescence at low polarity and weak fluorescence at high polarity of traditional polarity probes. Therefore, the fluorescent probe is a multifunctional fluorescent probe that responds to H2S, polarity and viscosity. By constructing an ischemia-reperfusion cell model, it was further proved that the fluorescent probe has low cytotoxicity and high targeting ability to lipid droplets and mitochondria. The fluorescent probe can track the changes in cell polarity and viscosity under physiological conditions, and reveals that the cell microenvironment (viscosity and polarity) before and after defective reperfusion injury has obvious changes. When treated with H2S, the fluorescent probe can track the process of cell polarity and viscosity recovery during treatment in real time.

[0098] The basic principles, main features and advantages of the present invention are shown and described above. Without departing from the spirit and scope of the present invention, the present invention may also be subject to various changes and improvements, which all fall within the scope of the present invention to be protected.

Claims

1. A fluorescent probe for tracking microenvironment changes before and after treatment of ischemia-reperfusion injury, characterized in that: The fluorescent probe can produce a cascade response to H2S, polarity and viscosity, and the structural formula of the fluorescent probe is:

2. A method for preparing a fluorescent probe for tracking microenvironment changes before and after treatment of ischemia-reperfusion injury according to claim 1, characterized in that The specific steps are: Step S1: adding 7-hydroxycoumarin-4-acetic acid and compound TPE-CHO to methanol, then adding piperidine and reflux reaction, monitoring the reaction process by thin layer chromatography, and after the reaction is completed, separating the obtained mixed solution by column chromatography to obtain a yellow solid compound TPEC-OH; Step S2: Add compound TPEC-OH and triethylamine to dichloromethane and stir, then add 2 , 4-dinitrobenzenesulfonyl chloride, the resulting solution was stirred at room temperature, and the reaction process was monitored by thin layer chromatography. After the reaction was completed, the resulting mixed solution was separated by column chromatography to obtain orange solid TBEC-DNBS; The corresponding synthetic route in the preparation process is:

3. Use of the fluorescent probe for tracking microenvironment changes before and after ischemia-reperfusion injury treatment as claimed in claim 1 in the preparation of a selective detection preparation for endogenous or exogenous hydrogen sulfide in LO2 living cells.

4. Use of the fluorescent probe for tracking microenvironment changes before and after ischemia-reperfusion injury treatment as claimed in claim 1 in the preparation of a detection preparation for tracking cell microenvironment changes in an ischemia-reperfusion injury cell model.

5. The use according to claim 4, characterized in that: The cell microenvironment change is specifically a change in cell polarity and / or viscosity.

6. The use according to claim 3 or 4, characterized in that The specific process is as follows: the fluorescent probe used to track the changes in the microenvironment before and after the treatment of ischemia-reperfusion injury has its own maximum absorption peak at 385nm in a PBS solution containing 20% ​​THF by volume. After the addition of H2S, the maximum absorption wavelength does not change, and the absorption intensity is slightly enhanced. Under 385nm excitation, the fluorescent probe exhibits extremely weak fluorescence emission. After the addition of H2S, it emits obvious yellow-green fluorescence at 563nm, and the peak intensity is significantly improved relative to the fluorescent probe. The Stocks shift between its excitation and emission peaks reaches 178nm. The TPEC-OH generated after the fluorescent probe reacts with H2S has an obvious solvation effect. In solvents of different polarities, its emission wavelength is significantly red-shifted with the increase of solvent polarity, and the emission intensity is enhanced with the increase of polarity. When the viscosity of the solvent is increased, the fluorescence wavelength of TPEC-OH remains almost unchanged, and the fluorescence intensity is significantly enhanced with the increase of viscosity.

7. Use of the fluorescent probe for tracking microenvironment changes before and after ischemia-reperfusion injury treatment as claimed in claim 1 in the preparation of cell or in vivo cell imaging preparations.

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