PH response light-emitting type small organic molecule fluorescent probe, preparation method thereof and application of pH response light-emitting type small organic molecule fluorescent probe in lysosome imaging
By preparing a pH-responsive luminescent organic small molecule fluorescent probe SJ-INH, the problems of small Stokes shift, multiple synthesis steps, and high cytotoxicity in the existing technology were solved, and efficient and safe lysosomal imaging was achieved.
Patent Information
- Application Number
- CN202511743571.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-25
- Publication Date
- 2026-02-27
AI Technical Summary
Existing small molecule fluorescent probes have limitations in lysosomal imaging due to their small Stokes shift, multiple synthesis steps, and high cytotoxicity.
A pH-responsive luminescent organic small molecule fluorescent probe was developed. Using readily available 2-arylindole and ethyl azodicarbonate compounds as substrates and copper chloride as a catalyst, an indole-3-hydrazone fluorescent probe, named SJ-INH, was prepared in a one-step reaction. It exhibits a large Stokes shift, low cytotoxicity, and high quantum yield.
It achieves large Stokes shift, low cytotoxicity and high quantum yield, simplifies the synthesis steps and improves the accuracy and safety of lysosomal imaging.
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Figure CN121574087A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the preparation of fluorescent materials and the field of lysosome imaging technology, and in particular to a pH-responsive luminescent organic small-molecule fluorescent probe, a preparation method thereof and application thereof in lysosome imaging, and in particular to a preparation method of a series of visible light pH-responsive lysosome probes with biocompatibility and imaging application. BACKGROUND
[0002] Lysosome dysfunction can seriously affect the balance in the body and is directly related to a variety of diseases. Typical lysosomal storage diseases (LSDs) are caused by mutations in genes encoding lysosomal hydrolases, activators or transporters, resulting in the accumulation of substrates in lysosomes. Such diseases often manifest as mental deterioration, skeletal deformities and organ enlargement, are mostly autosomal recessive, and currently most lack effective treatment methods, but can be screened early through lysosome activity detection. Recent studies have shown that lysosome dysfunction is also related to a variety of diseases. For example, Gaucher disease, as the most common lysosomal storage disease, is caused by mutations in the GBA gene, resulting in loss of glucocerebrosidase activity, causing substrates to accumulate in lysosomes, forming "Gaucher cells", and then triggering multiple system damage, such as hepatosplenomegaly, cytopenia and bone damage.
[0003] Therefore, lysosome targeting and bioimaging technology have important value in related basic research, disease diagnosis and drug development. At present, lysosome imaging technology includes X-ray, magnetic resonance, ultrasound, acoustic radiation force pulse imaging and tomography. Although these technologies have different imaging principles, they can all achieve visualization detection of lysosomes. However, they generally have limitations such as limited precision, low image quality, complex data processing, radiation interference and hardware dependence.
[0004] In contrast, fluorescence spectroscopy has become a research hotspot in the field of lysosome imaging due to its high spatiotemporal resolution, high sensitivity, simple operation and ability to perform non-invasive in situ detection. However, small-molecule fluorescent probes still have some obvious shortcomings that limit their application in lysosome imaging, such as small Stokes shift, leading to poor anti-background interference ability, and background interference during lysosome imaging; most probe molecules have long synthesis steps, low efficiency and high cost, which is not conducive to industrial synthesis application. SUMMARY
[0005] The present application aims at providing a pH-responsive luminescent organic small-molecule fluorescent probe, a preparation method thereof and application thereof in lysosome imaging, and aims at solving the problems of small-molecule fluorescent probes in the prior art, such as small Stokes shift, many synthesis steps and high cytotoxicity.
[0006] The present application aims at providing a pH-responsive luminescent organic small-molecule fluorescent probe, a preparation method thereof and application thereof in lysosome imaging, and aims at solving the problems of small-molecule fluorescent probes in the prior art, such as small Stokes shift, many synthesis steps and high cytotoxicity. The first object of the present application is to provide a pH-responsive luminescent organic small-molecule fluorescent probe, which comprises one or more of the structures shown in general formula A or solvates of the structures shown in general formula A, and the general formula A is as follows:
[0007] In the formula, R is independently selected from alkyl, halogen, ester group, nitro group, cyano group and trifluoromethyl group. R 1 is independently selected from alkyl, halogen, ester group, nitro group, cyano group and trifluoromethyl group. R 2 is independently selected from alkyl, halogen, ester group, nitro group, cyano group and trifluoromethyl group. Ar is independently selected from aromatic group and substituted aromatic group. Further, R 1 is independently selected from C1-C6 alkyl, halogen, C1-C6 ester group, nitro group, cyano group and trifluoromethyl group.
[0008] Further, R 2 is independently selected from C1-C6 alkyl, halogen, C1-C6 ester group, nitro group, cyano group and trifluoromethyl group.
[0009] Further, Ar is independently selected from C1-C12 aromatic group or C1-C12 substituted aromatic group.
[0010] Further, the organic small-molecule fluorescent probe is a visible light small-molecule fluorescent probe with an indole skeleton and a hydrazone functional group, and is an indole-3-hydrazone fluorescent probe.
[0011] Further, the maximum absorption wavelength of the small-molecule probe is between 300 nm, the maximum emission wavelength is between 500-510 nm, and the molecular weight is between 365-500 Da. Further, the structure of the small-molecule probe is any one of formulae 3a-4n, and further, when R 1 is H, R 2 is -CO2Et, and the 2nd position is phenyl (i.e. 3a), the organic small-molecule fluorescent probe is named SJ-INH.
[0012] Furthermore, the structure of the SJ-INH is as follows: .
[0013] Furthermore, the solvate of the structure represented by general formula A is a solvate formed by general formula A and solvents such as dichloromethane, ethanol, or dimethyl sulfoxide.
[0014] The second objective of this invention is to provide a method for preparing a pH-responsive luminescent organic small molecule fluorescent probe, the method comprising the following steps: Step 1: Compound (3), compound (1) (2-arylindole), and compound (2) (ethyl azodicarbonate) are reacted with copper chloride to obtain compound (3). Wherein, equation (1) is Equation (2) is Equation (3) is The compound shown in formula (3) is the organic small molecule fluorescent probe; The R 1 Independently selected from alkyl, halogen, ester, nitro, cyano, and trifluoromethyl groups; R 2 Ar is independently selected from ester groups; Ar is independently selected from aromatic groups or substituted aromatic groups.
[0015] Furthermore, both the compounds shown in formula (1) and formula (2) are commercially available.
[0016] Furthermore, SJ-INH was directly prepared by copper chloride catalysis using readily available compounds of formula (1) and formula (2).
[0017] Furthermore, the preparation method specifically includes the following steps: In a first organic solvent environment, under the catalysis of copper chloride, the compound shown in formula (1) and the compound shown in formula (2) were heated and stirred to obtain the compound shown in formula (3).
[0018] The first organic solvent is selected from one or more combinations of toluene, xylene, acetonitrile, ethyl acetate, dichloromethane, 1,2-dichloroethane, tetrahydrofuran, and diethyl ether.
[0019] A third objective of this invention is to provide an application of a pH-responsive luminescent organic small molecule fluorescent probe for lysosomal imaging, specifically, the application of the organic small molecule fluorescent probe in the preparation of reagents or kits for lysosomal imaging.
[0020] Furthermore, the visible light small molecule fluorescent probe exhibits a response behavior to pH values less than 7, specifically a fluorescence response behavior under acidic conditions.
[0021] Further, when R 1 is H, R 2 is -CO2Et, and Ar is phenyl, the organic small molecule fluorescent probe is named SJ-INH, and the visible light small molecule fluorescent probe SJ-INH has a response behavior to pH less than 7, specifically a fluorescence response behavior under acidic conditions.
[0022] Further, the fluorescence response behavior is manifested as fluorescence enhancement.
[0023] Further, the excitation wavelength of the visible light small molecule fluorescent probe is 380-420 nm, and the maximum emission wavelength is 505-550 nm.
[0024] Further, when R 1 is H, R 2 is -CO2Et, and Ar is phenyl, the organic small molecule fluorescent probe is named SJ-INH, and the excitation wavelength of the visible light small molecule fluorescent probe SJ-INH is 397 nm, and the maximum emission wavelength is 509 nm.
[0025] Further, when R 1 is H, R 2 is -CO2Et, and Ar is phenyl, the organic small molecule fluorescent probe is named SJ-INH, and the fluorescence enhancement is manifested as that the fluorescence intensity of the visible light small molecule fluorescent probe SJ-INH changes more obviously at pH acidity equal to 5 compared with neutral or alkaline environment.
[0026] Further, the organic small molecule fluorescent probe is used for live cell lysosome imaging, that is, the application of the organic small molecule fluorescent probe in the preparation of a reagent or kit for live cell lysosome imaging. The organic small molecule fluorescent probe can be imaged on live cell lysosomes, and has low cytotoxicity.
[0027] Further, the cell is a tumor cell, especially a malignant tumor cell.
[0028] Further preferably, the cell is a HeLa cell.
[0029] Further, the live cell lysosome imaging is: co-incubate the probe SJ-INH, Lyso tracker red, and Hoechst 33342 with HeLa cells for 30 min, and then wash with PBS buffer solution. Biological imaging is performed using a confocal microscope.
[0030] Further, SJ-INH has the advantages of large stokes shift (112 nm), less synthesis steps (1 step), high quantum yield, and low cytotoxicity compared with commercially available commercial probes Lyso tracker series and other partial reporter probes.
[0031]
[0032] Compared with the prior art, the beneficial effects of the present application are embodied in the following aspects: 1) The present application relates to a pH-responsive luminescent organic small molecule fluorescent probe, a preparation method thereof, and application thereof in lysosome imaging. The organic small molecule fluorescent probe has the advantages of large stokes shift, less synthesis steps (1 step), high quantum yield, and low cytotoxicity.
[0033] 2) The present application relates to a pH-responsive luminescent organic small molecule fluorescent probe, a preparation method thereof, and application thereof in lysosome imaging. When R 1 is H, R 2 is -CO2Et, and Ar is phenyl, the organic small molecule fluorescent probe is named SJ-INH. SJ-INH has the advantages of large stokes shift (112 nm), less synthesis steps (1 step), high quantum yield, and low cytotoxicity compared with commercially available commercial probes Lyso tracker series and other partial reporter probes.
[0034] 3) The present application relates to a pH-responsive luminescent organic small molecule fluorescent probe, a preparation method thereof, and application thereof in lysosome imaging. Commercially available 2-arylindole and azodicarboxylic acid ethyl ester compounds are used as substrates, and copper chloride is used as a catalyst to construct the fluorescent probe material in one step, thereby avoiding the step of pre-functionalization at the 3-position required for traditional construction of indole-3-hydrazone materials.
[0035] The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, and to implement the content of the description, the following describes the preferred embodiments of the present application with reference to the detailed description and accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS
[0036] Figure 1 FIG. 1 is a nuclear magnetic resonance hydrogen spectrum (a) and a carbon spectrum (b) of SJ-INH synthesized in Example 1.
[0037] Figure 2The emission spectrum of SJ-INH synthesized in Example 1 in the buffer solution at different pH under the irradiation of 397 nm as excitation wavelength.
[0038] Figure 3 The fluorescence emission spectrum of SJ-INH synthesized in Example 1 under the irradiation of 397 nm as excitation wavelength in the buffer solution with pH between 5 and 8 under reversible cycle.
[0039] Figure 4 The fluorescence emission spectrum of SJ-INH synthesized in Example 1 under the irradiation of 397 nm as excitation wavelength in the buffer solution with pH of 5, 7 and 8 for 20 min.
[0040] Figure 5 The cytotoxicity experiment chart of SJ-INH synthesized in Example 1 configured as an ethanol solution with different concentrations, incubated in HeLa cells for 24 hours, and measured by CCK-8.
[0041] Figure 6 The lysosome co-localization experiment chart of SJ-INH synthesized in Example 1, Lyso tracker red and Hoechst 33342 in HeLa cells.
[0042] Figure 7 The cell imaging chart of SJ-INH probes with different concentrations in Example 1 in HeLa cells. DETAILED DESCRIPTION
[0043] The present application will be described in detail below in conjunction with specific embodiments, but is by no means limited to the present application. If the preparation means, materials, structures or composition ratio of the features are not explicitly stated in the technical solution, they are considered as common technical features disclosed in the prior art.
[0044] The present application relates to a pH-responsive luminescent organic small molecule fluorescent probe and a preparation method and application thereof, the organic small molecule fluorescent probe in the present application is an indole-3-hydrazone fluorescent probe, 2-arylindole and azo dimethyl acid ethyl ester compounds are used as substrates, copper chloride is used as a catalyst, and the fluorescent probe material can be obtained in one step, avoiding the step of pre-functionalization at the 3rd position required by traditional construction of indole-3-hydrazone materials, the fluorescent material has the structure shown in general formula A, wherein when R 1 is H, R 2When Ar is -CO2Et and Ar is phenyl, this material is named SJ-INH. Its absorption and emission wavelengths are in the visible light region of 300-400 / 500-510 nm, exhibiting strong visible light absorption and emission characteristics, significant acid-responsive behavior, and excellent biocompatibility. Furthermore, this probe possesses advantages such as large Stokes shift, good photostability, high quantum yield under acidic conditions, and low cytotoxicity, and can be used for lysosomal imaging, demonstrating certain commercial value.
[0045] General Formula A:
[0046] In the formula: R 1 Independently selected from alkyl, halogen, ester, nitro, cyano, and trifluoromethyl groups; R 2 Independently selected from ester groups; Ar is independently selected from aromatic groups and / or substituted aromatic groups.
[0047] SJ-INH: .
[0048] The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.
[0049] In this embodiment, the hydrogen nuclear magnetic resonance spectrum of the compound ( 1 H NMR) and carbon nuclear magnetic resonance (CNMR) 13 The C NMR was determined by a Bruker AVANCE III HD 400; all reagents used were commercially available.
[0050] Synthesize indole-3-hydrazone fluorescent probes:
[0051] This invention provides a method for preparing a pH-responsive luminescent organic small molecule fluorescent probe (indole-3-hydrazone fluorescent probe), comprising the following steps: 0.2 mmol of 2-arylindole was added to a 10 mL reaction tube, along with an azo compound (an ethyl azodicarbonate compound, with a molar ratio of azo compound to 2-arylindole of 1–3:1, preferably 2:1), copper chloride (with a molar ratio of copper chloride to 2-arylindole of approximately 0.05–0.3:1, preferably 0.1:1), and toluene (toluene volume being 1 / 10–1 / 2, preferably 1 / 5, of the reaction tube volume). The reaction was heated for 6–12 h at 100–150 °C and stirred at 500–1000 rpm. After cooling to room temperature, the mixture was filtered through a diatomaceous earth liner, the organic phase was concentrated, and column chromatography was used to obtain the indole-3-hydrazone product, i.e., the indole-3-hydrazone fluorescent probe.
[0052] Example 1: Synthesis of fluorescent probe SJ-INH
[0053] This embodiment provides a method for preparing a pH-responsive luminescent organic small molecule fluorescent probe (indole-3-hydrazone fluorescent probe) SJ-INH, including the following steps: In a 100 mL round-bottom flask, 1.1 g (5.5 mmol) of 2-phenylindole, 2 mL (11 mmol) of DEAD (diethyl azodicarbonate), 85 mg (0.55 mmol) of copper chloride, and 50 mL of toluene were added. The reaction mixture was heated to 120 °C for 8 h with a stirring rate of 800 rpm. After cooling to room temperature, the mixture was filtered through a diatomaceous earth liner, the organic phase was concentrated, and column chromatography was used to obtain product SJ-INH, a red solid of 1.63 g, in 81% yield. Figure 1 The image shows the proton and carbon NMR spectra of the product SJ-INH.
[0054] 1 H NMR (400 MHz, Chloroform- d ) δ 8.38 – 8.33 (m, 2H), 7.59 (d, J =7.2 Hz, 1H), 7.54 – 7.47 (m, 5H), 7.23 – 7.19 (m, 1H), 4.31 (q, J = 7.2 Hz,4H), 1.25 (t, J = 7.2 Hz, 6H). 13 C NMR (101 MHz, Chloroform- d) δ 169.1, 167.4,156.6, 150.6, 134.5, 131.7, 131.4, 130.1, 128.5, 127.7, 126.0, 121.9, 121.7,64.1, 14.1. Example 2: Fluorescence pH Response Behavior of Probe SJ-INH The probe SJ-INH prepared in Example 1 was dissolved in anhydrous ethanol to prepare a 50 mM stock solution, which was then diluted to 50 μM with PBS buffers of different pH values for fluorescence spectroscopy analysis (λ). ex = 397 nm, slit width = 5 nm), such as Figure 2 As shown, the fluorescence of the system is weak under neutral and alkaline conditions, while the fluorescence intensity is significantly enhanced under acidic conditions. The fluorescence is activated under acidic conditions, and the fluorescence intensity is nearly 24 times stronger at pH 5.0 compared to pH 8.0 under alkaline conditions.
[0055] Example 3: Fluorescent pH Reversible Cyclic Experiment of Probe SJ-INH The probe SJ-INH prepared in Example 1 was dissolved in anhydrous ethanol to prepare a 50 mM stock solution, which was then diluted with PBS buffer at pH 5 and 8 for fluorescence spectroscopy analysis (λ). ex = 397 nm, slit width = 5 nm). The specific procedure is as follows: Prepare 20 mL of 50 μM probe SJ-INH in PBS buffer at pH 5. After thorough shaking, take 2 mL for measurement. Then, return the sample to the system, adjust the pH to 8 with NaOH solid, take 2 mL of the sample again for measurement, and after completion, return the sample to the system and adjust the pH to 5 with concentrated hydrochloric acid. Repeat the above operation 5 times until the corresponding results are obtained. Figure 3 As shown, the probe SJ-INH exhibits good pH reversibility and can be used for multiple detections.
[0056] Example 4: Photostability Study of the SJ-INH Probe Using λ ex A laser with a wavelength of 397 nm and a slit width of 5 nm irradiated the probe SJ-INH in a 50 μM PBS buffer solution at pH 5 for 20 minutes. Figure 4 As shown, the probe maintained good fluorescence intensity within 20 minutes, indicating that the probe has good anti-bleaching ability and photostability.
[0057] Example 5: Cytotoxicity assay of probe SJ-INH on HeLa cells The probe SJ-INH prepared in Example 1 was dissolved in anhydrous ethanol to prepare a 50 mM stock solution. This stock solution was then diluted with PBS buffer (pH 7.4) to concentrations of 0, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 μM. These solutions were then co-incubated with HeLa cells in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin for 24 hours. Toxicity was tested using the CCK-8 assay. Figure 5 As shown, the probe SJ-INH did not produce significant cytotoxicity to HeLa cells, indicating that the probe SJ-INH has good biocompatibility.
[0058] Example 6: Co-localization experiment of probe SJ-INH and commercial probe Lyso tracker red on lysosomes: The probe SJ-INH prepared in Example 1 was dissolved in anhydrous ethanol to prepare a 50 mM stock solution, which was then diluted with PBS buffer (pH 7.4) to a concentration of 12.5 μM. This SJ-INH solution, along with Lyso tracker red and Hoechst 33342 (prepared according to the manufacturer's instructions), was incubated with HeLa cells in DMEM medium (Dürbeco modified Eagle medium) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin for 30 min, followed by washing with PBS buffer. Bioimaging was performed using a confocal microscope (LSM 800). Hoechst 33342 staining represented cell nuclei (blue channel), Lyso tracker red staining represented lysosomes (red channel), and SJ-INH staining represented lysosomes (green channel). Results are as follows: Figure 6 As shown, this indicates that the probe SJ-INH can be used for lysosomal targeting.
[0059] Example 7: Imaging experiment of lysosomes with different concentrations of probe SJ-INH Different concentrations (0 μM, 12.5 μM, 25 μM, 50 μM) of probes SJ-INH, Lyso tracker red, and Hoechst 33342 (prepared according to the manufacturer's instructions) prepared in Example 1 were co-incubated with HeLa cells in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin for 30 min, followed by washing with PBS buffer. Bioimaging was performed using a confocal microscope (LSM 800). Hoechst 33342 staining represented cell nuclei (blue channel), Lyso tracker red staining represented lysosomes (red channel), and SJ-INH staining represented lysosomes (green channel). Results are as follows: Figure 7 As shown, cell imaging exhibits the strongest fluorescence intensity at a concentration of 12.5 μM.
[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A pH-responsive luminescent organic small molecule fluorescent probe, characterized in that, The small molecule fluorescent probe comprises one or more of the structure shown in general formula A and solvates of the structure shown in general formula A, wherein general formula A is as follows: In the formula: R1 is independently selected from alkyl, halogen, ester, nitro, cyano, and trifluoromethyl groups; R2 is independently selected from ester groups; Ar is independently selected from aromatic groups and substituted aromatic groups.
2. The pH-responsive luminescent organic small molecule fluorescent probe according to claim 1, characterized in that, The maximum absorption wavelength of the small molecule probe is between 300 nm and the maximum emission wavelength is between 500-510 nm; the molecular weight is between 365-600 Da.
3. The pH-responsive luminescent organic small molecule fluorescent probe according to claim 1, characterized in that, The structure of the small molecule fluorescent probe is any one of the following formulas 3a-4n: 。 4. A method for preparing a pH-responsive luminescent organic small molecule fluorescent probe as described in any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Compound (3) is obtained by reacting the compound (1) and the compound (2) with copper chloride catalysis to obtain compound (3). Wherein, equation (1) is Equation (2) is Equation (3) is The compound shown in formula (3) is the organic small molecule fluorescent probe; The R 1 Independently selected from alkyl, halogen, ester, nitro, cyano, and trifluoromethyl groups; R 2 Ar is independently selected from ester groups; Ar is independently selected from aromatic groups or substituted aromatic groups.
5. The method for preparing the pH-responsive luminescent organic small molecule fluorescent probe according to claim 4, characterized in that, The preparation method specifically includes the following steps: In a first organic solvent environment, under the catalysis of copper chloride, the compound shown in formula (1) and the compound shown in formula (2) were heated and stirred to obtain the compound shown in formula (3); The first organic solvent is selected from one or more combinations of toluene, xylene, acetonitrile, ethyl acetate, dichloromethane, 1,2-dichloroethane, tetrahydrofuran, and diethyl ether.
6. The application of a pH-responsive luminescent organic small molecule fluorescent probe as described in any one of claims 1-3, characterized in that, The organic small molecule fluorescent probe was used for lysosomal imaging. The small molecule fluorescent probe is a visible light small molecule fluorescent probe.
7. The application of the pH-responsive luminescent organic small molecule fluorescent probe according to claim 6, characterized in that, The visible light small molecule fluorescent probe exhibits responsive behavior to pH values less than 7.
8. The application of the pH-responsive luminescent organic small molecule fluorescent probe according to claim 6, characterized in that, When R 1 For H, R 2 When -CO2Et is used and Ar is a phenyl group, the visible light small molecule probe is named SJ-INH. The visible light small molecule fluorescent probe SJ-INH has a responsive behavior to pH less than 7.
9. The application of the pH-responsive luminescent organic small molecule fluorescent probe according to claim 6, characterized in that, When R 1 When H is present, R2 is -CO2Et, and Ar is phenyl, the visible light small molecule probe is named SJ-INH. The excitation wavelength of SJ-INH is 397 nm, and the maximum emission wavelength is 509 nm.
10. The application of the pH-responsive luminescent organic small molecule fluorescent probe according to claim 6, characterized in that, When R 1 For H, R 2 When -CO2Et is used and Ar is phenyl, the visible light small molecule probe is named SJ-INH. Under acidic conditions, the fluorescence intensity of SJ-INH changes more significantly compared to neutral or alkaline environments.