Fluorescent probes for tracing beta-galactosidase, lysosomal ph and reactive oxygen species in aging process and synthetic methods and applications thereof

Abstract

The application belongs to the technical field of enzyme activity and active oxygen content detection, and particularly relates to a fluorescent probe for tracing beta-galactosidase, lysosome pH and active oxygen in the aging process. The application discloses a fluorescent probe capable of efficiently and selectively recognizing beta-galactosidase, lysosome pH and active oxygen, and having high sensitivity. Based on the spectral and imaging research at the cell and biological levels, it is shown that the fluorescent probe can track the aging process, has good stability in terms of sensitivity, anti-interference ability and imaging, and has good market application value for early diagnosis of aging.

Description

Technical Field

[0001] This invention belongs to the field of enzyme activity and reactive oxygen species (ROS) detection technology, and relates to a method and strategy that can simultaneously detect endogenous β-galactosidase, lysosomal pH, and ROS. More specifically, it relates to a method for synthesizing a multi-response fluorescent probe and its application in detecting aging processes. Background Technology

[0002] Aging is a complex, multi-stage process triggered by a range of factors, including DNA genetic and epigenetic alterations, decreased mitochondrial function, stem cell depletion, telomere shortening, and cellular damage caused by reactive oxygen species (ROS) produced by incomplete aerobic metabolism. Numerous reports indicate that ROS, despite being intracellular signaling and growth stimulants, are well-known contributors to aging. A key factor in the aging process is cellular senescence. Senescent cells are rare in the tissues of young organisms but become increasingly common with age, particularly in adipose tissue, muscle, and skin. Cellular senescence was long described as a result of replicative depletion, primarily aimed at preventing the proliferation of damaged or stressed cells and triggering tissue repair. However, the chronic accumulation of senescent cells in tissues can impair bodily functions, further exacerbating aging and inducing various diseases.

[0003] Early diagnosis of senescent cells can offer possibilities for therapeutic interventions in various diseases induced by tissue aging. After decades of effort, the precise tracking of aging through biomarkers, coupled with further intervention, is considered a promising approach to improving age-related diseases. Among these efforts, numerous β-galactosidase-targeting fluorescent probes have been developed to detect senescent cells. Endogenous β-galactosidases, abundant in human cells, originate from the GLB1 gene expressed in lysosomes and are associated not only with aging but also with certain cancers such as ovarian cancer.

[0004] Therefore, how to distinguish aging-related β-galactosidase (SA-β-gal) from β-galactosidase from other sources, while adding other recognition units to identify other biomarkers in the aging process, and thus providing a highly sensitive and selective fluorescent probe for tracking aging, is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, in order to solve this problem, the present invention discloses a multidimensional fluorescent probe that can be used simultaneously to detect endogenous β-galactosidase, lysosomal pH and reactive oxygen species in cells to trace aging.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The primary technical objective of this invention is to provide a fluorescent probe for tracing β-galactosidase, lysosomal pH, and reactive oxygen species during aging. The structural formula of the fluorescent probe is as follows:

[0008]

[0009] It should be noted that the fluorescent probe disclosed in this invention uses β-galactosidase, lysosomal pH, and reactive oxygen species (ROS) as biomarkers. Firstly, hemicyanine is used as the fluorescent signal reporter group, and the glycosidic bond is the site of action for β-galactosidase. Secondly, the pH-sensitive 4-hydroxybenzyl group is linked to the fluorophore via an ether bond, undergoing a spontaneous 1,6-elimination reaction under neutral or alkaline conditions, thereby distinguishing aging-related β-galactosidase from other sources. Simultaneously, the oxidation of the carbon-carbon double bond in the hemicyanine fluorophore by ROS releases an oxanthracene fluorophore, achieving a blue shift in the fluorescence spectrum. The change in the fluorescence ratio between the two fluorophores is used to identify ROS.

[0010] The second technical objective of this invention is to provide a method for synthesizing the fluorescent probe, specifically including the following steps:

[0011] (1) Add heptamethyl cyanine dye Cy-Cl, resorcinol, triethylamine and anhydrous NN dimethylformamide to a flask equipped with a DeanStark trap and a condenser; react the mixture at 110 °C for 0.5 h to obtain a blue solution, remove the solvent by vacuum distillation, and purify the crude product by silica gel column chromatography (dichloromethane / methanol 60:1) to obtain a dark blue solid HCy-OH;

[0012] (2) 4-hydroxybenzaldehyde, tetraacetyl-α-D-bromogalactose and cesium carbonate were dissolved in anhydrous acetonitrile and reacted at room temperature for 16 h under nitrogen protection. After the reaction was completed, the solvent was removed by filtration and reduced pressure, then extracted with dichloromethane, dried with anhydrous sodium sulfate, and the solvent was removed again by evaporation. The crude product was then purified by silica gel column chromatography (dichloromethane / methanol 100:1) to obtain compound 1.

[0013] (3) Dissolve compound 1 obtained in step (2) in methanol and add sodium borohydride to the stirred solution at 0°C; monitor the reaction by TLC, and after 5 minutes, dilute the solution with ethyl acetate and wash with saturated ammonium chloride solution and saturated brine; dry the organic layer on anhydrous sodium sulfate, remove the solvent under reduced pressure, and purify the crude residue by silica gel column chromatography (dichloromethane / methanol 90:1) to obtain compound 2;

[0014] (4) Compound 2 obtained in step (3) was dissolved in dichloromethane, and phosphorus tribromide was added to the stirred solution at 0°C. The reaction solution was stirred at 0°C for 30 minutes. After the reaction was completed, the solution was diluted with dichloromethane and washed with cold water and saturated brine. The organic layer was dried on anhydrous sodium sulfate and the solvent was removed under reduced pressure. The product was then purified by silica gel column chromatography (dichloromethane / methanol 100:1) to obtain compound 3.

[0015] (5) The compound HCy-OH and cesium carbonate obtained in step (1) were dissolved in anhydrous dichloromethane in a round-bottom flask and stirred at room temperature for 15 min under nitrogen atmosphere; then the compound 3 obtained in step (4) was added and the reaction mixture was stirred at room temperature for 12 h; after the reaction was completed, the solvent was removed under reduced pressure and the product was purified by silica gel column chromatography (dichloromethane / methanol 20:1) to obtain blue solid compound 4;

[0016] (6) Dissolve compound 4 obtained in step (5) in anhydrous methanol, and add a methanol solution of sodium methoxide dropwise to the solution. Stir the solution at room temperature for 3 hours. After the reaction is complete, purify the product by silica gel column chromatography (dichloromethane / methanol 10:1) to obtain compound SA-HCy-1 in blue solid form, which is the fluorescent probe.

[0017] The synthetic route of the fluorescent probe described above is shown below:

[0018]

[0019] This invention uses β-galactosidase, lysosomal pH, and reactive oxygen species (ROS) as biomarkers for detection. First, hemicyanine is used as a fluorescent reporter group, with the glycosidic bond serving as the site of action for β-galactosidase. Second, a pH-sensitive 4-hydroxybenzyl group is linked to the fluorophore via an ether bond, undergoing a spontaneous 1,6-elimination reaction under weakly alkaline conditions. Only through a continuous reaction process can the bond be removed, generating a hemicyanine fluorophore as a near-infrared fluorescent indicator, thereby distinguishing aging-related β-galactosidase from other sources. Simultaneously, the oxidation of the carbon-carbon double bonds in the hemicyanine structure by ROS alters the near-infrared fluorophore structure, transforming it into an oxanthracene fluorophore with a shorter fluorescence emission wavelength. The change in the fluorescence ratio between the two fluorophores is used to identify ROS.

[0020] Preferably, the molar ratio of Cy-Cl, resorcinol, and triethylamine in step (1) is 1:(7.0-7.5):(9.0-10.0).

[0021] Preferably, the molar ratio of 4-hydroxybenzaldehyde, tetraacetyl-α-D-bromogalactose and cesium carbonate in step (2) is 1:(1.2-1.5):(3.5-4.5).

[0022] Preferably, the molar ratio of compound 1 and sodium borohydride in step (3) is 1:(1.5 to 2.5).

[0023] Preferably, the molar ratio of compound 2 and phosphorus tribromide in step (4) is 1:(1 to 1.5).

[0024] Preferably, the molar ratio of HCy-OH, compound 3 and cesium carbonate in step (5) is 1:(3-3.5):(4-5.5).

[0025] Preferably, the molar ratio of compound 4 and sodium methoxide in step (6) is 1:(6-10).

[0026] For the synthesis of fluorescent probes, the inventors obtained various raw material ratios through inventive experiments. Among them, the ratio of sodium borohydride and triethylamine is particularly important. Triethylamine affects the acid-base regulation of the reaction and is related to whether the reaction can proceed smoothly. The content of sodium borohydride directly affects the degree of reaction and is related to the steps of complete reaction and excess treatment.

[0027] In addition, the inventors characterized the fluorescent probe using methods such as proton nuclear magnetic resonance spectroscopy and mass spectrometry to verify the successful synthesis of the fluorescent probe.

[0028] A third technical objective of this invention is to provide the application of the above-mentioned fluorescent probe in products for detecting senescent cells and tissues, such as kits.

[0029] The aforementioned β-galactosidase and reactive oxygen species are overexpressed in aging biological samples, and aging-related β-galactosidases can be distinguished from β-galactosidases from other sources by utilizing lysosomal pH changes. The fluorescent probe SA-HCy-1 synthesized in this invention can successfully enter living cells or organisms for detection.

[0030] Specifically, the optimal reaction concentration of the synthesized fluorescent probe is 10 μmol·L⁻¹. -1 .

[0031] Furthermore, the optimal conditions for the reaction between the fluorescent probe and β-galactosidase are co-incubation at 37°C in PBS buffer solution at pH 8.0 for 20 minutes.

[0032] The optimal conditions for the fluorescent probe to react with reactive oxygen species after co-incubation with β-galactosidase for 20 minutes are 60 minutes at 37°C in PBS buffer solution at pH 8.0.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] This invention discloses a fluorescent probe that can efficiently and selectively identify β-galactosidase, lysosomal pH, and reactive oxygen species, and has high sensitivity. Furthermore, based on spectroscopic and imaging studies at the cellular and biological levels, this fluorescent probe can track the aging process and exhibits good stability in terms of sensitivity, anti-interference ability, and imaging. This has good market application value for the early diagnosis of aging. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0036] Figure 1 This is the proton NMR spectrum of the fluorescent probe of this invention.

[0037] Figure 2 This is the mass spectrum of the fluorescent probe of the present invention.

[0038] Figure 3 The images show the absorption and fluorescence emission spectra of the fluorescent probe of this invention after reacting with β-galactosidase and reactive oxygen species, where (a) is the ultraviolet-visible absorption spectrum, (b) is the near-infrared fluorescence spectrum, and (c) is the visible fluorescence spectrum.

[0039] Figure 4 This is a fluorescence intensity diagram of the fluorescence emission peak at 713 nm after the fluorescent probe of the present invention reacts with β-galactosidase in buffer solutions of different pH values.

[0040] Figure 5 The bar charts show the reaction ability of the fluorescent probe of the present invention with different substances and β-galactosidase added to the buffer solution (a) and the fluorescent probe treated with β-galactosidase in the buffer solution and then with different substances (b) and reactive oxygen species.

[0041] Figure 6 (a) is the fluorescence emission spectrum of the fluorescent probe of the present invention after adding different concentrations of β-galactosidase; (b) is the relationship between the fluorescence intensity of the fluorescent probe of the present invention at 713 nm and the concentration of β-galactosidase after adding different concentrations of β-galactosidase; (c) is the linear relationship between the fluorescence intensity at 713 nm and the enzyme concentration when 0-25 μM / mL of β-galactosidase is added to the fluorescent probe of the present invention.

[0042] Figure 7 (a) and (b) show the fluorescent probe of the present invention after being added to 100 μmol·ml. -1After treatment with β-galactosidase, add 0-250 μmol·L -1 Fluorescence emission spectra in the near-infrared and visible regions after reactive oxygen species (ROS) are shown. (c) shows the ratio of fluorescence intensity at 468 nm to 713 nm, and (d) shows the linear relationship between the ratio of fluorescence intensity at 468 nm to 713 nm and enzyme concentration when ROS concentration is between 0 and 25 μM.

[0043] Figure 8 The images show confocal fluorescence images of RAW264.7 cells treated with doxorubicin for different numbers of days and RAW264.7 cells treated with doxorubicin for 3 days followed by azithromycin or vitamin E.

[0044] Figure 9 This is a confocal fluorescence image of the fluorescent probe of the present invention in B16 cells treated with doxorubicin for 2 days.

[0045] Figure 10 This is a confocal fluorescence image showing the regulation of reactive oxygen species (ROS) content in normally cultured RAW264.7 cells and senescent RAW264.7 cells by the fluorescent probe of this invention.

[0046] Figure 11 This is a confocal fluorescence image of the fluorescent probe of the present invention in naturally passaged senescent cells.

[0047] Figure 12 The probe of this invention was used to detect fluorescence images of β-galactosidase and reactive oxygen species in a mouse skin photoaging model. Detailed Implementation

[0048] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0049] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0050] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0051] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0052] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

[0053] This invention discloses a fluorescent probe for tracing β-galactosidase, lysosomal pH, and reactive oxygen species during aging, as well as its synthesis method and application.

[0054] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0055] Example 1

[0056] Synthesis of fluorescent probes

[0057] 1. Synthesis Steps

[0058] (1) Add 0.64 g Cy-Cl, 0.83 g resorcinol, 1.4 mL triethylamine and 15 mL anhydrous N,N dimethylformamide to a flask equipped with a DeanStark trap and condenser. React the mixture at 110 °C for 0.5 h to obtain a blue solution. Remove the solvent by vacuum distillation. Purify the crude product by silica gel column chromatography (dichloromethane / methanol 60:1) to obtain a dark blue solid HCy-OH.

[0059] (2) 0.16 g of 4-hydroxybenzaldehyde, 0.82 g of tetraacetyl-α-D-bromogalactose, and 2.1 g of cesium carbonate were dispersed in 20 mL of anhydrous acetonitrile. The mixture was stirred at room temperature for 16 h under nitrogen protection. After the reaction was completed, the mixture was filtered, the solvent was removed under reduced pressure, and then extracted with dichloromethane. After drying with anhydrous sodium sulfate, the solvent was removed again by evaporation, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol 100:1) to obtain the desired product 1.

[0060] (3) Dissolve 0.45 g of compound 1 obtained in step (2) in 5 mL of methanol, and add 0.076 g of sodium borohydride to the stirred solution at 0 °C. Monitor the reaction by TLC. After 5 minutes, dilute the solution with 20 mL of ethyl acetate, and wash with saturated ammonium chloride solution and saturated brine. Dry the organic layer with anhydrous sodium sulfate, remove the solvent under reduced pressure, and purify the crude residue by silica gel column chromatography (dichloromethane / methanol 90:1) to obtain the desired compound 2.

[0061] (4) Dissolve 0.23 g of compound 2 obtained in step (3) in 15 mL of dichloromethane, and add 57 μL of phosphorus tribromide to the stirred solution at 0 °C. Stir the reaction solution at 0 °C for 30 minutes. After the reaction is complete, dilute the solution with 20 mL of dichloromethane and wash with cold water and saturated saline. Dry the organic layer on anhydrous sodium sulfate and remove the solvent under reduced pressure. Then purify the product by silica gel column chromatography (dichloromethane / methanol 100:1) to obtain the desired product 3.

[0062] (5) Disperse 53 mg of compound HCy-OH and 0.16 g of cesium carbonate obtained in step (1) in 15 mL of anhydrous dichloromethane in a round-bottom flask and stir at room temperature for 15 min under a nitrogen atmosphere. Then add 0.15 g of compound 3 obtained in step (4) and stir the reaction mixture further at room temperature for 12 h. After the reaction is complete, remove the solvent under reduced pressure and purify the product by silica gel column chromatography (dichloromethane / methanol 20:1) to obtain blue solid compound 4.

[0063] (6) Dissolve 61 mg of compound 4 obtained in step (5) in 7 mL of anhydrous methanol, and add 3 mL of methanol solution containing 34 mg of sodium methoxide dropwise to the solution, and stir the solution at room temperature for 3 h. After the reaction is complete, purify the product by silica gel column chromatography (dichloromethane / methanol 10:1) to obtain compound SA-HCy-1 in the form of a blue solid.

[0064] 2. Test Analysis

[0065] Figure 1 The 1H NMR spectrum of probe SA-HCy-1 shows the following peak values: 1HNMR(500MHz,DMSO-d6)δ(ppm)8.60(d,J=15.3Hz,1H),7.76(d,J=7.5Hz,1H),7.69(d,J=8.1Hz,1H),7.54(t,J=8.3Hz,2H), 7.50(s,1H),7.50-7.40(m,3H),7.19(s,1H),7.07(d,J=8.4Hz,3H),6.56(d,J=15.0Hz,1H),5.22(s,2H),5.16(d,J=5.0Hz, 1H), 4.88(d, J = 5.6 Hz, 1H), 4.84(d, J = 7.6 Hz, 1H), 4.66(d, J = 5.4 Hz, 1H), 4.53(d, J = 4.6 Hz, 1H), 4.44(d, J = 7.3 Hz, 2H), 3.70(s, 2H), 3.56(s, 2H), 3.48(s, 1H), 3.40(s, 1H), 2.72(s, 2H), 2.69(s, 2H), 1.83(s, 2H), 1.76(s, 6H), 1.37(t, J = 7.2 Hz, 3H). These correspond to the probe group, confirming the successful synthesis of the probe.

[0066] Figure 2 This is the mass spectrum of probe SA-HCy-1, with specific information as follows: C 40 H 44 NO8 + 666.3074 (Theoretical value: 666.3061). Corresponding to the theoretical value, this further confirms the correctness of the probe structure.

[0067] Example 2

[0068] (1) Add 0.64 g Cy-Cl, 0.77 g resorcinol, 1.2 mL triethylamine and 13 mL anhydrous N,N dimethylformamide to a flask equipped with a DeanStark trap and condenser. React the mixture at 110 °C for 0.5 h to obtain a blue solution. Remove the solvent by vacuum distillation. Purify the crude product by silica gel column chromatography (dichloromethane / methanol 60:1) to obtain a dark blue solid HCy-OH.

[0069] (2) 0.16 g of 4-hydroxybenzaldehyde, 0.75 g of tetraacetyl-α-D-bromogalactose, and 1.9 g of cesium carbonate were dispersed in 15 mL of anhydrous acetonitrile. The mixture was stirred at room temperature for 16 h under nitrogen protection. After the reaction was complete, the mixture was filtered, the solvent was removed under reduced pressure, and then extracted with dichloromethane. After drying with anhydrous sodium sulfate, the solvent was removed again by evaporation. The crude product was then purified by silica gel column chromatography (dichloromethane / methanol 100:1) to obtain the desired product 1.

[0070] (3) Dissolve 0.45 g of compound 1 obtained in step (2) in 5 mL of methanol, and add 0.038 g of sodium borohydride to the stirred solution at 0 °C. Monitor the reaction by TLC. After 5 minutes, dilute the solution with 15 mL of ethyl acetate, and wash with saturated ammonium chloride solution and saturated brine. Dry the organic layer with anhydrous sodium sulfate, remove the solvent under reduced pressure, and purify the crude residue by silica gel column chromatography (dichloromethane / methanol 90:1) to obtain the desired compound 2.

[0071] (4) Dissolve 0.23 g of compound 2 obtained in step (3) in 15 mL of dichloromethane, and add 47 μL of phosphorus tribromide to the stirred solution at 0 °C. Stir the reaction solution at 0 °C for 30 minutes. After the reaction is complete, dilute the solution with 15 mL of dichloromethane and wash with cold water and saturated saline. Dry the organic layer on anhydrous sodium sulfate and remove the solvent under reduced pressure. Then purify the product by silica gel column chromatography (dichloromethane / methanol 100:1) to obtain the desired product 3.

[0072] (5) Disperse 53 mg of compound HCy-OH and 0.18 g of cesium carbonate obtained in step (1) in 15 mL of anhydrous dichloromethane in a round-bottom flask and stir at room temperature for 15 min under a nitrogen atmosphere. Then add 0.18 g of compound 3 obtained in step (4) and stir the reaction mixture further at room temperature for 12 h. After the reaction is complete, remove the solvent under reduced pressure and purify the product by silica gel column chromatography (dichloromethane / methanol 20:1) to obtain blue solid compound 4.

[0073] (6) Dissolve 61 mg of compound 4 obtained in step (5) in 7 mL of anhydrous methanol, and add 3 mL of methanol solution containing 20 mg of sodium methoxide dropwise to the solution, and stir the solution at room temperature for 3 h. After the reaction is complete, purify the product by silica gel column chromatography (dichloromethane / methanol 10:1) to obtain compound SA-HCy-1 in the form of a blue solid.

[0074] To further verify the superiority of the technology of the present invention, the inventors conducted the following effect verification experiment on the fluorescent probe synthesized in Example 1 above, and the specific operation is as follows:

[0075] Experiment 1:

[0076] Study on the recognition performance of fluorescent probes for aging-related β-galactosidase and reactive oxygen species in buffer solution.

[0077] The synthesized fluorescent probe was prepared into a 5.0 mmol·L⁻¹ solution. -1 Take 4 μL of the solution and add it to a container containing 2 mL of PBS (10 mmol·L⁻¹). -1 In centrifuge tubes (pH = 8.0), first use 100 μmol / mL... -1β-galactosidase was incubated at 37°C for 20 minutes, followed by the addition of 100 μmol·L⁻¹ to the solution. -1 Changes in absorption and fluorescence emission spectra were detected after 60 minutes of different ROS treatments.

[0078] Figure 3 The absorption and fluorescence emission spectra of the fluorescent probe before and after the reaction with aging-related β-galactosidase and reactive oxygen species are shown. The black curve represents the fluorescent probe (10 μmol·L⁻¹). -1 The red curve represents the fluorescent probe (10 μmol·L⁻¹). -1 ) and β-galactosidase (100 μmol·ml) -1 The mixture of 10 μmol·L⁻¹, with the green curve representing the fluorescent probe (10 μmol·L⁻¹). -1 ) and reactive oxygen species (100 μmol·L -1 The mixture of 10 μmol·L⁻¹, the blue curve represents the fluorescent probe (10 μmol·L⁻¹). -1 ), β-galactosidase (100 μmol·ml) -1 ) and reactive oxygen species (100 μmol·L -1 A mixture of )

[0079] Depend on Figure 3 It was found that SA-HCy-1 exhibited a maximum absorption peak at 600 nm and two smaller peaks at 650 nm and 566 nm in aqueous solution. This was observed when reacting with β-galactosidase (100 μmol / mL). -1 After incubation for 20 minutes, the absorption peak of SA-HCy-1 at 600 nm weakened, while a new peak near 680 nm increased. Subsequently, different reactive oxygen species (100 μmol·L⁻¹) were added to the solution. -1 The presence of β-galactosidase leads to an absorption peak at 360 nm. The fluorescence spectrum shows that the fluorescent probe exhibits weak fluorescence, with a maximum peak at 674 nm, indicating its potential for high sensitivity. With the addition of β-galactosidase, the fluorescent probe shows a strong and rapid onset of fluorescence at 713 nm. The coexistence of β-galactosidase and reactive oxygen species (ROS) induces a more significant spectral response. Initially, the fluorescent probe showed a weak emission signal at 468 nm in the presence of β-galactosidase; however, the subsequent addition of ROS significantly enhanced the fluorescence emission at 468 nm, while the fluorescence emission at 713 nm slightly decreased. The fluorescent probe showed no significant response to UV absorption or fluorescence emission in the presence of different ROS.

[0080] This demonstrates that the probe can determine the content of β-galactosidase not only based on fluorescence emission at 713 nm, but also based on the ratio of fluorescence at 468 nm to fluorescence at 713 nm to determine the content of reactive oxygen species (ROS). Therefore, this carefully designed probe is considered a dual-response probe for β-galactosidase and ROS, which improves the diagnostic accuracy of cellular senescence.

[0081] Experiment 2

[0082] The fluorescent probe was treated with β-galactosidase in buffer solutions of different pH values ​​to further explore whether the probe could distinguish between aging-related β-galactosidase and β-galactosidase from other sources based on pH value.

[0083] The synthesized fluorescent probe was prepared into a 5.0 mmol·L⁻¹ solution. -1 Take 4 μL of the solution and add it to 2 mL of PBS (10 mmol·L⁻¹) with different pH values ​​(4.0-9.0). -1 In a centrifuge tube, use 100 μmol·ml -1 The changes in fluorescence emission spectrum of β-galactosidase were detected after incubation at 37°C for 20 minutes.

[0084] Figure 4 The UV-Vis absorption and fluorescence spectra of the fluorescent probe interacting with β-galactosidase in buffer solutions at different pH values ​​were obtained.

[0085] Depend on Figure 4 It can be seen that the structure of the fluorescent probe remains quite stable when the pH value is adjusted within the range of 4.0-9.0, while the fluorescent probe responds well to β-galactosidase within the pH range of 6.0-9.0.

[0086] This demonstrates that glycosidic bonds can selectively and rapidly react with β-galactosidase, but the 1,6-elimination reaction of the 4-hydroxybenzyl group is pH-dependent and can be accelerated in an alkaline environment. This further proves that the probe can distinguish aging-related β-galactosidase from other sources by pH. Simultaneously, it also exhibits good reactivity to β-galactosidase at the physiological pH of 7.4, indicating that this fluorescent probe can be used for imaging aging-related β-galactosidase in cells and living tissues.

[0087] Experiment 3

[0088] The fluorescent probe was tested by adding different substances and β-galactosidase to a buffer solution, or by adding β-galactosidase to a buffer solution before adding different substances and reactive oxygen species.

[0089] The synthesized fluorescent probe was prepared into a 5.0 mmol·L⁻¹ solution. -1Take 4 μL of the solution from 40 centrifuge tubes and add it to a solution containing 2 mL of PBS (10 mmol·L⁻¹). -1 In centrifuge tubes (pH = 8.0), starting with tube number 2, add 100 μmol·L⁻¹ of the solution sequentially. -1 Salts, amino acids, or 100 μL / ml -1 β-galactosidase was measured by detecting changes in fluorescence emission spectra after incubation at 37°C for 20 minutes.

[0090] The synthesized fluorescent probe was prepared into a 5.0 mmol·L⁻¹ solution. -1 Take 39 centrifuge tubes and add 4 μL of each tube to a solution containing 2 mL of PBS (10 mmol·L⁻¹). -1 Add 100 μmol·ml to a centrifuge tube (pH = 8.0). -1 β-galactosidase was then added sequentially, starting with tube 2, at a rate of 100 μmol·L⁻¹. -1 Salts, amino acids, or 100 μmol·L -1 The reactive oxygen species were measured, and the changes in fluorescence emission spectra were detected after incubation at 37°C for 60 minutes.

[0091] Figure 5 (a) is a bar chart showing the fluorescence intensity of the fluorescent probe of the present invention at the maximum emission wavelength after the addition of different substances. Among them, β-galactosidase showed a significant fluorescence response, while other substances had little effect on the fluorescence intensity.

[0092] Figure 5 (b) The fluorescent probe of the present invention was added to 100 μmol·ml -1 A bar chart showing fluorescence intensity at the maximum emission wavelength after processing β-galactosidase and other substances. Reactive oxygen species (ROS) exhibit a significant fluorescence response, with ClO₂ being a prominent example. - The fluorescence response was the most significant, while other substances had little effect on the fluorescence intensity.

[0093] This demonstrates that the fluorescent probe SA-HCy-1 did not show a significant response to most of the interfering substances tested, indicating that it has good anti-interference capabilities.

[0094] Experiment 4

[0095] Determination of the limit of detection (LOD) of β-galactosidase and reactive oxygen species using the fluorescent probe SA-HCy-1.

[0096] The synthesized fluorescent probe was prepared into a 5.0 mmol·L⁻¹ solution. -1 Take 4 μL of the solution and add it to a solution containing 2 mL of PBS (10 mmol·L⁻¹). -1 In centrifuge tubes (pH = 8.0), use 0 to 500 μmol / mL. -1After incubating β-galactosidase at 37°C for 20 minutes, the change in fluorescence emission spectrum was detected. 100 μmol / mL of the fluorescent probe was added. -1 Treat with β-galactosidase for 20 minutes, then add 0-250 μmol·L⁻¹ -1 Changes in fluorescence emission spectra were detected after incubation of reactive oxygen species at 37°C for 60 minutes.

[0097] Figure 6 The fluorescent probe of this invention is added in the form of 0 to 500 μmol / ml -1 The fluorescence emission spectrum and fluorescence intensity at the maximum emission wavelength after β-galactosidase were obtained. Calculations showed that the limit of detection for β-galactosidase was 3.457 × 10⁻⁶ U / mL.

[0098] Figure 7 The fluorescent probe of this invention was added to 100 μmol / ml -1 After treatment with β-galactosidase, add 0-250 μmol·L -1 The fluorescence emission spectrum and the fluorescence intensity ratio of ratio fluorescence after reactive oxygen species were obtained. Calculations showed that the detection limit of the probe for ClO- was 184.0 nM.

[0099] This demonstrates that the probe exhibits high sensitivity for the detection of β-galactosidase and reactive oxygen species, indicating its potential application value in the efficient detection of these two substances.

[0100] Experiment 5

[0101] The specific applications of fluorescent probes in detecting aging-related β-galactosidase and reactive oxygen species in live cells and mouse imaging.

[0102] (1) Specific application of fluorescent probes in detecting β-galactosidase and reactive oxygen species in drug-induced senescent RAW264.7 cells.

[0103] RAW264.7 cells were treated with 0.1 μmol·L⁻¹ -1 Doxorubicin (DOX) was used to induce senescence in cells after treatment for different durations (day 0, day 1, day 2, and day 3). Cells treated for senescence for 3 days were then treated with 100 μmol·L⁻¹. -1 Anti-aging drug azithromycin or 100 μmol·L -1 Vitamin E (VE) treatment for 60 minutes to remove senescent cells, followed by treatment with 10 μmol·L⁻¹ -1 The probe SA-HCy-1 was incubated together for 30 min. X-gal staining was then performed, and the cyan channel (λ) was collected simultaneously. ex (377nm) and near-infrared fluorescence channel (λ) exFluorescence images (628 nm) were obtained and fluorescence intensity was recorded.

[0104] Figure 8 (a) 0.1 μmol·L⁻¹ was used for RAW264.7 cells. -1 Doxorubicin (DOX) was used to treat cells for 0, 1, 2, and 3 days to induce senescence. Cells treated for 3 days were then treated with an anti-senescence drug at 100 μmol·L⁻¹. -1 Azithromycin or 100 μmol·L -1 Vitamin E (VE) treatment of the cyan fluorescence channel (λ) ex (377nm) and near-infrared fluorescence channel (λ) ex Fluorescence image (628nm). Figure 8 (b) 0.1 μmol·L⁻¹ for RAW264.7 cells -1 Doxorubicin (DOX) was used to treat cells for 0, 1, 2, and 3 days to induce senescence. Cells treated for 3 days were then treated with 100 μmol·L⁻¹. -1 Azithromycin or 100 μmol·L -1 Image of X-gal staining after vitamin E (VE) treatment. Figure 8 (c)-(e) are respectively Figure 8 (a) Average fluorescence intensity of the cyan channel and the near-infrared fluorescence channel, and the ratio of the average fluorescence intensity of the cyan channel to the near-infrared fluorescence channel (Ic). Cyan / I NIR ) and the sum of the average fluorescence intensities of the cyan channel and the near-infrared fluorescence channel (I Cyan +I NIR ).

[0105] like Figure 8 It was found that after treatment with the fluorescent probe SA-HCy-1, the control group showed weak fluorescence in both the cyan and near-infrared channels under imaging. With increasing DOX treatment time, the fluorescence signals in both the cyan and near-infrared channels of RAW264.7 cells increased. The degree of cell senescence was verified by a commercial X-gal staining kit for cell senescence. Simultaneously, compared with senescent RAW264.7 cells (treated with DOX for 3 days), the fluorescence intensity in both the cyan and near-infrared channels of the same cells treated with azithromycin and vitamin E was weakened, and the total fluorescence intensity (IL) was significantly reduced. Cyan +I NIR ) and fluorescence intensity ratio (I Cyan / I NIR () also decreased significantly.

[0106] (2) Specific applications of fluorescent probes in detecting β-galactosidase and reactive oxygen species in drug-induced senescent B16 cells

[0107] B16 cells were treated with 0.1 μmol·L⁻¹ -1 Doxorubicin (DOX) was used to treat the organisms for different durations (day 0 and day 2) to induce senescence, followed by treatment with 10 μmol·L⁻¹. -1 The probe SA-HCy-1 was incubated together for 30 min. X-gal staining was then performed, and the cyan channel (λ) was collected simultaneously. ex (377nm) and near-infrared fluorescence channel (λ) ex Fluorescence images (628 nm) were obtained and fluorescence intensity was recorded.

[0108] Figure 9 (a) 0.1 μmol·L⁻¹ was used for B16 cells. -1 Doxorubicin (DOX) treatment for 0 and 2 days induced the senescent cyan fluorescence channel (λ). ex (377nm) and near-infrared fluorescence channel (λ) ex Fluorescence image (628nm). Figure 9 (b) 0.1 μmol·L⁻¹ for B16 cells -1 X-gal staining images of doxorubicin (DOX) after treatment for 0 days and 2 days to induce senescence.

[0109] like Figure 9 It can be seen that after treatment with the fluorescent probe SA-HCy-1, the control group showed weak fluorescence in both the cyan and near-infrared channels under imaging, while the fluorescence signals in both the cyan and near-infrared channels of the senescent B16 cells were enhanced, and the total fluorescence intensity (I) was significantly higher. Cyan +I NIR ) and fluorescence intensity ratio (I Cyan / I NIR The degree of cellular senescence also increased significantly. This can be verified using a commercially available X-gal staining kit for cellular senescence.

[0110] (3) Specific applications of fluorescent probes in regulating reactive oxygen species content in normal and senescent RAW264.7 cells.

[0111] Cells in the blank control group were cultured normally, while RAW264.7 cells in the senescent group were cultured with 0.1 μmol·L⁻¹. -1 Doxorubicin (DOX) treatment for 3 days induced senescence. Cells from the normal and senescent groups were then treated with lipopolysaccharide (LPS) (1.0 μg / mL). -1 ) and propylene glycol methyl ether acetate (PMA) (0.5 μg·mL -1Pretreatment for 60 min induces more reactive oxygen species (ROS), or pretreatment with N-acetylcysteine ​​(NAC) (100 mM) for 60 min clears ROS, followed by culture with probe SA-HCy-1 for 30 min for cell fluorescence imaging. The cyan channel (λ) is collected. ex (377nm) and near-infrared fluorescence channel (λ) ex The fluorescence image (628 nm) was obtained and the fluorescence intensity was recorded, and then X-gal staining was performed.

[0112] Figure 10 X-gal staining images of cellular senescence and live-cell fluorescence imaging at emission wavelengths of 447 nm and 593 nm. Figure 10 (a) (Ⅰ-Ⅲ) is the fluorescent probe SA-HCy-1 (10 μmol·L⁻¹). -1 Incubate for 30 min, then use lipopolysaccharide (LPS) (1.0 μg·mL⁻¹). -1 ) and propylene glycol methyl ether acetate (PMA) (0.5 μg·mL -1 Cell fluorescence imaging was performed after pretreatment for 60 min followed by culture with probe SA-HCy-1 for 30 min, and after pretreatment with N-acetylcysteine ​​(NAC) (100 mM) for 60 min followed by culture with probe SA-HCy-1 for 30 min. Figure 10 (b) X-gal staining images of normal cells and senescent cells. Figure 10 (c)-(e) are respectively Figure 10 (a) The average fluorescence intensity of the cyan fluorescence channel and the near-infrared fluorescence channel, and the ratio of the average fluorescence intensity of the cyan fluorescence channel and the near-infrared fluorescence channel to the sum of the average fluorescence intensities.

[0113] from Figure 10 It was found that treatment with doxorubicin increased the fluorescence intensity in both channels of senescent RAW264.7 cells. Lipopolysaccharide (LPS) and propylene glycol methyl ether acetate (PMA) represent typical stimulants that can trigger the production of reactive oxygen species in living cells. When senescent RAW264.7 cells were treated with a mixture of LPS and PMA, the fluorescence intensity in the cyan channel was observed to be greater than that in senescent cells not treated with LPS and PMA, while near-infrared fluorescence decreased, but the fluorescence intensity was lower than that in (I1). Cyan / I NIR The sum of fluorescence intensity (I) and fluorescence intensity Cyan +I NIR The fluorescence intensity of the near-infrared channel also increased significantly. Furthermore, when N-acetyl-L-cysteine ​​(NAC), a widely used reactive oxygen species scavenger, was introduced into senescent cells, the fluorescence signal in the near-infrared channel showed no significant change, while the fluorescence intensity in the cyan channel was impaired, and the ratio of fluorescence intensity (IL) was significantly reduced. Cyan / INIR The sum of fluorescence intensity (I) and fluorescence intensity Cyan +I NIR The fluorescence intensity also decreased significantly. However, when a mixture of lipopolysaccharide and diol methyl ether acetate was added to normally cultured cells or when treated with N-acetyl-L-cysteine ​​(NAC), there were no significant changes in the fluorescence of the near-infrared and cyan channels.

[0114] This indicates that the fluorescent probe SA-HCy-1 will only continue to respond to reactive oxygen species when the glycosidic bond is broken in the presence of senescence-related β-galactosidase. This avoids the fluorescence changes in non-senescent cells when oxidative stress occurs, making it easier to accurately detect the condition of senescent cells.

[0115] (4) Specific applications of fluorescent probes in detecting β-galactosidase and reactive oxygen species in naturally passaged senescent cells.

[0116] RAW264.7 cells were cultured from passage 2 (P2) to passage 11 (P11) to obtain replicative senescent cells at different senescence stages, and then treated with 10 μmol·L⁻¹. -1 Incubate probe SA-HCy-1 together for 30 min, and collect cyan channel (λ) simultaneously. ex (377nm) and near-infrared fluorescence channel (λ) ex The fluorescence image (628 nm) was obtained and the fluorescence intensity was recorded, and then X-gal staining was performed.

[0117] Figure 11 (a) Cell fluorescence images of RAW264.7 cells from passage 2 (P2) to passage 11 (P11). Figure 11 (b) X-gal staining images of RAW264.7 cells from passage 2 (P2) to passage 11 (P11). Figure 11 (c)-(f) represent the average fluorescence intensity of two channels in RAW264.7 cells from passage 2 (P2) to passage 13 (P11), respectively, and the ratio of the average fluorescence intensity of the cyan channel to the near-infrared fluorescence channel (Ic). Cyan / I NIR ) and the sum of the average fluorescence intensities of the cyan channel and the near-infrared fluorescence channel (I Cyan +I NIR ).

[0118] like Figure 11 It can be seen that the fluorescence of second-generation RAW264.7 cells treated with the fluorescent probe SA-HCy-1 in the cyan and near-infrared channels is negligible. With increasing passage number (P2-P11), the fluorescence intensity of RAW264.7 cells in both channels gradually increases, while the sum of the fluorescence intensities (IL) decreases. Cyan +I NIRThe ratio of fluorescence intensity to fluorescence intensity (I) Cyan / I NIR All three channels showed an increase in fluorescence intensity. Specifically, the near-infrared channel showed an earlier increase, with significant fluorescence enhancement observed in the fifth generation, while the cyan channel fluorescence increased significantly after the seventh generation. This increase is determined by the specific structure of the probes that sequentially recognize β-galactosidase and reactive oxygen species. Furthermore, X-Gal staining of senescent cells could be observed after the tenth generation.

[0119] This indicates that the levels of these two biomarkers accumulate continuously during the aging process. Therefore, SA-HCy-1 is considered a powerful tool for dynamically observing early cellular senescence. Furthermore, the fluorescent probe SA-HCy-1 can not only effectively assess the degree of cellular senescence, but is also a promising tool for evaluating anti-aging drugs.

[0120] In summary, logical and sensing strategies for intracellular aging-related β-galactosidases and reactive oxygen species are considered feasible and plausible, implying potential applications in organisms.

[0121] (5) Specific applications of fluorescent probes in detecting β-galactosidase and reactive oxygen species in a mouse skin photoaging model.

[0122] Pretreatment was performed on 1.5 x 1.5 cm areas on both sides of the mouse back for 15 minutes using either pure substrate (sesame oil) (100 μL) or a 3% isoflavone preparation made from diluted sesame oil (100 μL). The mice were then placed in a 5 x 10 cm rectangular experimental chamber and irradiated with a long-wave ultraviolet lamp with a peak wavelength of 365 nm. Irradiation was performed five times a week (Monday to Friday) from the start of irradiation. In weeks 1-3, irradiation was performed for 20, 40, and 60 minutes each time; in weeks 4 and 5, irradiation was performed for 80 minutes each time, for a total of 5 weeks. Fluorescence imaging was performed every Sunday during the irradiation period. Before fluorescence imaging, 1 mmol·L⁻¹ of sesame oil was added to the irradiated area. -1 The probe SA-HCy-1 (10 μL) was applied to the pretreatment area for 30 minutes. Then, the cyan fluorescence channel (λ) was collected. ex / em =420 / 480nm) and near-infrared fluorescence channel (λ ex / em Fluorescence images (600 / 710 nm) were obtained and the fluorescence intensity was recorded.

[0123] Figure 12 (a) Photoaging of mouse skin in the cyan fluorescence channel (λ) ex / em =420 / 480nm) and near-infrared fluorescence channel (λ ex / em Fluorescence images (600 / 710 nm) Figure 12 (b)-(e) are Figure 12 (a) Average fluorescence intensity of the cyan channel and the near-infrared fluorescence channel, and the ratio of the average fluorescence intensity of the cyan channel to the near-infrared fluorescence channel (Ic).Cyan / I NIR ) and the sum of the average fluorescence intensities of the cyan channel and the near-infrared fluorescence channel (I Cyan +I NIR ).

[0124] like Figure 12 It was observed that with increasing irradiation time, the fluorescence intensity of the near-infrared fluorescence channel in the left dorsal region of the mouse increased, and the fluorescence of the cyan fluorescence channel was strongly enhanced. Simultaneously, both the ratio and the sum of fluorescence intensities increased. Compared to the left side, the mice coated with the isoflavone preparation did not show a significant increase in near-infrared fluorescence, while the fluorescence of the cyan channel showed negligible enhancement.

[0125] This demonstrates that the fluorescent probe SA-HCy-1 can effectively assess the degree of cellular senescence and is also a promising tool for evaluating drugs that can improve skin tissue inflammation induced by ultraviolet radiation.

[0126] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fluorescent probe, characterized in that, The structural formula of the fluorescent probe is: 。 2. The use of the fluorescent probe as described in claim 1 in the preparation of a reagent for the simultaneous detection of β-galactosidase and reactive oxygen species.

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