Photo-activated fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nano material synthesis and biological imaging analysis, in particular to a light-activated fluorescent probe as well as a preparation method and application thereof. The preparation method of the light-activated fluorescent probe comprises the following steps: adding o-phenylenediamine and lewis acid into low-carbon alcohol according to a molar ratio of 5: 1, and obtaining the light-activated fluorescent probe through a solvothermal method. The obtained light-activated fluorescent probe has no fluorescence, and is activated to emit fluorescence after being irradiated by exciting light, so that the light-activated fluorescent probe can regulate and control a fluorescence signal generated by the light-activated fluorescent probe by controlling parameters of the exciting light. According to the scheme, the light-activated probe which has the characteristic of no fluorescence and can generate fluorescence only under the irradiation of exciting light is prepared, so that the light-activated probe has the advantages of low background interference, high controllability, wide application scene, more stable performance and the like.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterial synthesis and bioimaging analysis technology, specifically to a photoactivated fluorescent probe and its preparation method and application. Background Technology

[0002] Carbon dots, also known as carbon quantum dots or carbon nanodots, are a class of zero-dimensional carbon nanomaterials with remarkable fluorescence properties. They are composed of ultrafine, dispersed, quasi-spherical carbon nanoparticles with a size of less than 10 nm. Carbon dots possess numerous advantages, including excellent optical properties, good water solubility, low toxicity, environmental friendliness, wide availability of raw materials, low cost, and good biocompatibility.

[0003] Due to their tunable optical properties, good biocompatibility, and low toxicity, carbon dots show great promise for applications in bioimaging. Therefore, carbon dots are generally used as probes in bioimaging. The photostability of probes is crucial in their optical properties. Some existing probes are self-fluorescent, which means that when using these probes for bioimaging, the spontaneous signals from the biological sample itself may superimpose with the probe's fluorescence, easily leading to blurred images and inaccurate target localization. Furthermore, existing probes quench with prolonged irradiation, resulting in insufficient photostability under continuous illumination conditions, which also limits their application in bioimaging.

[0004] Furthermore, lysosomes and mitochondria are important organelles within cells. Lysosomes are the centers of acidic digestion and recycling in eukaryotic cells, while mitochondria provide energy for the cell. Monitoring the dynamic relationship between these two plays a crucial role in biomedical research and drug development. However, current methods for simultaneous visualization of both require labeling mitochondria separately using lysosomal and mitochondrial staining. This multi-probe co-application technique faces challenges such as probe uptake efficiency, asynchronous biolocalization, high cytotoxicity, and photobleaching. Summary of the Invention

[0005] To address the technical problem that existing probes, due to their inherent fluorescence, easily lead to blurred images or inaccurate target localization when applied in the field of bioimaging, this invention provides a photoactivated fluorescent probe, its preparation method, and its application.

[0006] This invention is achieved using the following technical solution: a method for preparing a photoactivated fluorescent probe, comprising: adding o-phenylenediamine and Lewis acid in a molar ratio of 5:1 to a low-carbon alcohol, and obtaining the photoactivated fluorescent probe by a solvothermal method. The obtained photoactivated fluorescent probe is itself non-fluorescent, but it is activated and emits fluorescence after being irradiated with excitation light, thereby enabling the fluorescence signal generated by the photoactivated fluorescent probe to be modulated by controlling the parameters of the excitation light.

[0007] As a further improvement of the present invention, the excitation light is ultraviolet light or visible light.

[0008] As a further improvement of the present invention, the parameters of the excitation light include the irradiation time and the excitation light power; the photoactivated fluorescent probe generates fluorescence after the excitation light is turned on; the imaging time of the photoactivated fluorescent probe in the cell decreases as the excitation light power increases.

[0009] As a further improvement of the present invention, under 365nm ultraviolet light irradiation, the photoactivated fluorescent probe emits blue fluorescence in ethanol and red fluorescence in aqueous solution.

[0010] As a further improvement of the present invention, the preparation method includes: adding o-phenylenediamine and Lewis acid to a low-carbon alcohol at a molar ratio of 5:1 and dissolving them by ultrasonication; after ultrasonic dissolution, transferring the solution to a reaction vessel for heating reaction. After the heating reaction is completed, the supernatant in the reaction vessel is taken out, and the supernatant is purified, rotary evaporated, and dried sequentially to obtain the photoactivated fluorescent probe.

[0011] As a further improvement of the present invention, the amount of low-carbon alcohol used is 1 / 2 to 2 / 3 of the volume of the reaction vessel.

[0012] As a further improvement of this invention, the general chemical formula of the lower alcohol is C0. n H 2n+1 OH, where 1 < n ≤ 4.

[0013] As a further improvement of the present invention, the heating reaction temperature is 180℃~220℃, and the heating reaction time is 8h~12h.

[0014] As a further improvement of the present invention, the reaction vessel is a polytetrafluoroethylene-lined reaction vessel.

[0015] As a further improvement of the present invention, the supernatant is purified by column chromatography, and the eluent for purification by column chromatography is petroleum ether and dichloromethane, with a volume ratio of petroleum ether to dichloromethane of 100:1.

[0016] The present invention provides a photoactivated fluorescent probe, which is prepared by the photoactivated fluorescent probe preparation method described above.

[0017] As a further improvement of the present invention, the fluorescence excitation wavelength range of the photoactivated fluorescent probe is 360nm~900nm.

[0018] As a further improvement of the present invention, the diameter of the photoactivated fluorescent probe is in the range of 1.5 nm to 5.5 nm.

[0019] As a further improvement of the present invention, the maximum absorption wavelength of the photoactivated fluorescent probe in aqueous solution is 484 nm.

[0020] As a further improvement of the present invention, the maximum absorption wavelength of the photoactivated fluorescent probe in ethanol solution is 475 nm.

[0021] The present invention also includes an application of the photoactivated fluorescent probe as described above, which is used as an anti-counterfeiting label based on the difference in fluorescence color in water and ethanol. The anti-counterfeiting label appears blue when applied with ethanol under excitation light and red when applied with water.

[0022] The present invention also includes an application of the photoactivated fluorescent probe as described above, which serves as a dual-targeting imaging reagent for lysosomes and mitochondria in tumor cells.

[0023] The technical solution provided by this invention has the following beneficial effects: (1) The photoactivated fluorescent probe provided by this invention is prepared by a solvothermal method using o-phenylenediamine and Lewis acid as the main raw materials. The entire preparation process is simple, the raw materials are inexpensive, and it is easy to repeat. Compared with conventional probes, the photoactivated fluorescent probe, which can be activated by light, has a longer photoexcited state duration, solving the problem of low photostability of current probes. In addition, the prepared photoactivated probe is completely non-fluorescent without excitation light and only emits light under excitation light. This allows the photoactivated probe in this scheme to achieve "on-demand emission" when used as a luminescent material for bioimaging, based on its non-fluorescence and fluorescence only under excitation light. It can also accurately distinguish the "signal region" where the probe is present and the "background region" where the probe is absent in bioimaging, avoiding the situation in the prior art where the probe's own fluorescence affects the imaging results or the accuracy of target localization. Thus, the photoactivated fluorescent probe in this scheme can achieve accurate and high-resolution bioimaging and targeted detection in the biological field.

[0024] (2) The photoactivated fluorescent probe provided by this invention emits blue fluorescence in ethanol solution and red fluorescence in aqueous solution. This gives the photoactivated fluorescent probe of this scheme good reversibility in switching between blue and red light, which makes the photoactivated fluorescent probe of this scheme have the advantages of cyclic reuse and intelligent optical response. In solvent detection or microenvironment sensing, the photoactivated fluorescent probe can achieve repeated switching of fluorescence color through solvent replacement, without the need to repeatedly prepare the photoactivated fluorescent probe, greatly reducing the detection cost, and making the photoactivated fluorescent probe suitable for some scenarios that require continuous detection.

[0025] (3) The photoactivated fluorescent probe provided by the present invention can be used as an imaging reagent to distinguish between tumor cells and normal cells, and can be used for tumor targeted therapy and surgical navigation. Attached Figure Description

[0026] Figure 1This is a simplified schematic diagram illustrating the preparation of the photoactivated fluorescent probe provided by the present invention.

[0027] Figure 2 This is a high-resolution mass spectrum of the photoactivated fluorescent probe provided by the present invention.

[0028] Figure 3 This is a high-resolution mass spectrum of the photoactivated fluorescent probe provided by the present invention.

[0029] Figure 4 The FTIR spectrum of the photoactivated fluorescent probe provided by this invention.

[0030] Figure 5 The FTIR spectrum of the photoactivated fluorescent probe provided in this invention.

[0031] Figure 6 The UV-Vis absorption spectra of the photoactivated fluorescent probe aqueous solution before and after activation provided by the present invention.

[0032] Figure 7 The fluorescence emission spectrum of the photoactivated fluorescent probe after activation by aqueous solution provided by the present invention.

[0033] Figure 8 The time-fluorescence spectrum of the photoactivated fluorescent probe aqueous solution provided by the present invention under 370 nm excitation light.

[0034] Figure 9 The time-fluorescence spectrum of the photoactivated fluorescent probe aqueous solution provided by the present invention under 480 nm excitation light.

[0035] Figure 10 The UV-Vis absorption spectra of the photoactivated fluorescent probe provided by this invention before and after activation with ethanol solution.

[0036] Figure 11 The fluorescence emission spectrum of the photoactivated fluorescent probe provided by this invention after activation with ethanol solution.

[0037] Figure 12 The time-fluorescence spectrum of the photoactivated fluorescent probe ethanol solution provided by the present invention under 370 nm excitation light.

[0038] Figure 13 The time-fluorescence spectrum of the photoactivated fluorescent probe ethanol solution provided by the present invention under 470 nm excitation light.

[0039] Figure 14 TEM characterization of the photoactivated fluorescent probe provided by the present invention.

[0040] Figure 15 TEM characterization of the photoactivated fluorescent probe provided by this invention.

[0041] Figure 16 This is a confocal imaging image of tumor cells after activation of the photoactivated fluorescent probe provided by the present invention.

[0042] Figure 17 This is a colocalization imaging diagram of the photoactivated fluorescent probe provided by the present invention in tumor cells.

[0043] Figure 18 This is a confocal image of human immortalized keratinocytes after activation by the photoactivated fluorescent probe provided by the present invention.

[0044] Figure 19 This is a confocal imaging image of mouse fibroblasts after activation of the photoactivated fluorescent probe provided by the present invention. Detailed Implementation

[0045] The present invention will now be further described in conjunction with specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0046] In the description of this invention, it should be noted that directional terms such as "center," "lateral," "longitudinal," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation and positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These are used only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. They should not be construed as limiting the specific scope of protection of this invention. The terms "first," "second," etc., in the specification and claims of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. The terms "comprising" and "having," and any variations thereof, in the specification and claims of this invention, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products, or devices.

[0047] This embodiment provides a method for preparing a photoactivated fluorescent probe, comprising: adding o-phenylenediamine and Lewis acid in a molar ratio of 5:1 to a low-carbon alcohol, and obtaining the photoactivated fluorescent probe by a solvothermal method. This one-step synthesis method effectively simplifies the synthesis steps of the photoactivated fluorescent probe, and the obtained photoactivated fluorescent probe itself is non-fluorescent, only emitting fluorescence after being activated by excitation light irradiation. Therefore, the fluorescence signal generated by the photoactivated fluorescent probe can be modulated by controlling the parameters of the excitation light. This method, by preparing a photoactivated fluorescent probe that is non-fluorescent itself and only fluoresces under excitation light irradiation, gives the photoactivated fluorescent probe advantages such as low background interference, strong controllability, wide application scenarios, and more stable performance. Regarding the advantage of low background interference, the photoactivated fluorescent probe of this solution is completely non-fluorescent in the absence of excitation light, emitting light only under excitation light. This allows it to achieve "on-demand luminescence" when used as a luminescent material for bioimaging, based on its non-fluorescent nature and fluorescence emission only under excitation light. This enables precise differentiation between the "signal region" where the probe is present and the "background region" where the probe is absent in bioimaging, avoiding the situation in existing technologies where the probe's own fluorescence affects imaging results or target localization accuracy. Therefore, the photoactivated fluorescent probe of this solution can achieve accurate and high-resolution bioimaging and targeted detection in the biological field. Furthermore, the photoactivated fluorescent probe provided by this solution can also be applied to the field of sensing and detection. Based on the non-fluorescent nature of the photoactivated fluorescent probe, the detection limit can be reduced to a very low level, enabling precise quantification of trace substances (such as heavy metal ions, pathogens, biomarkers, etc.). This solves the technical problem of existing probes having fluorescent signals that mask weak detection signals, resulting in higher detection limits. Regarding the advantage of stability, existing probes generally possess intrinsic fluorescence, and their continuous luminescence depends on the continuous transition of their own electrons. Therefore, existing probes are prone to rapid degradation and fluorescence quenching due to photo-oxidation and electron quenching. In contrast, the photoactivated fluorescent probe of this scheme only generates fluorescence through electron transitions under excitation light. Without excitation light, it remains in a stable ground state, avoiding unnecessary electronic reactions and significantly reducing non-specific losses, thus improving its stability. Regarding the advantage of controllability, the fluorescence generation of the photoactivated fluorescent probe in this scheme depends entirely on the excitation light. This allows for focusing the excitation light (such as with a laser) to ensure that the probe emits fluorescence only in a designated micro-area, enabling pinpoint monitoring such as micro-area imaging and single-particle tracking. Furthermore, the photoactivated fluorescent probe provided by this scheme only generates fluorescence under excitation light, allowing for dynamic real-time monitoring (such as monitoring dynamic changes in intracellular reactive oxygen species and the actual progress of chemical reactions), avoiding the signal superposition caused by the continuous luminescence of existing probes, which affects the detection results.As can be seen from the above description, the probe of this solution can be used not only in the field of biological imaging, but also in the field of sensing and detection, thus increasing its application scenarios.

[0048] The excitation light can be ultraviolet or visible light; ultraviolet light can be generated by irradiation with a 365nm ultraviolet lamp. Parameters of the excitation light include irradiation time and excitation power. The photoactivated fluorescent probe produces fluorescence after irradiation with the excitation light. The imaging time of the photoactivated probe within cells decreases as the excitation light power increases. This allows for the preferential selection of channels with lower excitation power in cell experiments, thereby extending the cell imaging time.

[0049] Under 365nm ultraviolet light irradiation, the photoactivated fluorescent probe emits blue fluorescence in ethanol solution and red fluorescence in aqueous solution. This gives the photoactivated fluorescent probe of this scheme excellent reversibility in switching between blue and red light, a characteristic that allows for cyclic reuse and intelligent optical response. In solvent detection or microenvironment sensing, the photoactivated fluorescent probe can achieve repeated switching of fluorescence color through solvent replacement, eliminating the need for repeated preparation of the probe and significantly reducing detection costs. This makes the photoactivated fluorescent probe suitable for scenarios requiring continuous detection. Furthermore, the photoactivated fluorescent probe can be fabricated into anti-counterfeiting labels, displaying a blue pattern in ethanol and a red pattern in water, thereby enhancing its anti-counterfeiting level. Moreover, the photoactivated fluorescent probe of this scheme can also be used to detect the purity of ethanol or to screen the moisture content of ethanol solvents in chemical production, pharmaceuticals, and video processing. Specifically, it emits pure blue fluorescence in pure ethanol; if trace amounts of water are present, the fluorescence of the photoactivated fluorescent probe will gradually change from blue to red as the moisture content increases. Since red and blue light and red light are characteristic wavelengths in the visible light region, this identification process can be performed directly with the naked eye, simplifying the detection or screening process.

[0050] Specifically, the preparation method of the photoactivated fluorescent probe is as follows: o-phenylenediamine and Lewis acid are added to a low-carbon alcohol at a molar ratio of 5:1 and dissolved by ultrasonication. After ultrasonic dissolution, the solution is transferred to a reaction vessel for heating. After the heating reaction is completed, the supernatant in the reaction vessel is removed and purified, rotary evaporated, and dried sequentially to obtain the photoactivated fluorescent probe. The amount of low-carbon alcohol used is 1 / 2 to 2 / 3 of the reaction vessel volume. The entire synthesis process is simple, the raw materials are inexpensive, and it is easy to repeat. Compared with conventional fluorescent probes, the photoactivated fluorescent probe has a longer photoexcited state duration, which solves the problem of low photostability of current fluorescent probes. Lewis acids passivate surface defects and fix radiative transition channels, resulting in quantum yields far exceeding those of acid-free or protonic acid catalytic systems. Furthermore, the abundant nitrogen / coordinating groups on the surface of Lewis acids can form differentiated hydrogen bonds and coordination interactions with solvents of different polarities (such as ethanol or water), giving them a natural solvent-phase responsive characteristic: producing blue light in the alcohol phase and red light in the aqueous phase.

[0051] The amount of lower alcohol used is 1 / 2 to 2 / 3 of the reactor volume. The reactor can be a polytetrafluoroethylene-lined reactor. The general chemical formula of the lower alcohol can be C0. n H 2n+1 OH, where 1 < n ≤ 4. The heating temperature is 180℃~220℃, and the heating time is 8h~12h. The supernatant is purified by column chromatography, with petroleum ether and dichloromethane as the eluent in a volume ratio of 100:1. Lewis acids can be any one of GdCl3•6H2O, EuCl3•6H2O, ErCl3•6H2O, ZrCl3•6H2O, AlCl3•6H2O, and FeCl3•6H2O. o-Phenylenediamine is a known good material for condensation, polymerization, and carbonization into probes. It can be used as a carbon source, and gadolinium chloride hexahydrate as a catalyst can expand the effective aromatic sp. 2 Due to the conjugated structure, this scheme can prepare a photoactivated fluorescent probe with high fluorescence quantum yield by using o-phenylenediamine as a nitrogen-containing aromatic carbon source and combining it with gadolinium chloride hexahydrate.

[0052] The following provides a specific preparation method for a photoactivated fluorescent probe using o-phenylenediamine and gadolinium chloride hexahydrate as the main raw materials. Please refer to [link / reference needed]. Figure 1 The process involves adding 0.54 g of o-phenylenediamine and 0.37 g of gadolinium chloride hexahydrate to 30 mL of ethanol at a molar ratio of 5:1, dissolving them by sonication, and then transferring the mixture to a 50 mL polytetrafluoroethylene-lined reactor. The reactor is heated at 200 °C for 12 h. After the reaction, the supernatant is removed and purified by column chromatography. The purified supernatant is then subjected to rotary evaporation at 38 °C and drying at 60 °C to obtain the photoactivated fluorescent probe. The photoactivated fluorescent probe prepared by this method can directly absorb excitation light and react to produce fluorescent substances. Furthermore, the photoactivated fluorescent probe can gradually release fluorescent substances, maintaining a low concentration suitable for bioimaging. Therefore, the photoactivated fluorescent probe of this scheme is an excitation light-dependent photoactivated fluorescent probe, which changes from a dark state to a bright state after irradiation with a 365 nm ultraviolet lamp or visible light.

[0053] This solution also provides a photoactivated fluorescent probe, which is prepared by the method described above. The excitation wavelength range of the prepared photoactivated fluorescent probe is 340 nm to 660 nm, and the optimal excitation wavelength of the photoactivated fluorescent probe in aqueous solution is 340 nm; the optimal excitation wavelength of the photoactivated fluorescent probe in ethanol solution is 360 nm. The diameter of the prepared photoactivated fluorescent probe is distributed in the range of 1.5 nm to 5.0 nm, with an average diameter of about 3.45 nm.

[0054] Building upon the above description, this solution also provides an application of a photoactivated fluorescent probe, which can be used as an anti-counterfeiting label based on the difference in its fluorescence color in water and ethanol. The anti-counterfeiting label appears blue when applied with ethanol under excitation light and red when applied with water. By using the photoactivated fluorescent probe of this solution as an anti-counterfeiting label, the anti-counterfeiting level of the label can be improved.

[0055] This solution provides a photoactivated fluorescent probe that can also serve as a dual-targeting imaging reagent for lysosomes and mitochondria in tumor cells. The mechanism is as follows: the photoactivated fluorescent probe enters the cell through endocytosis, and the natural endpoint of the endocytic pathway is the lysosome, thus it can be used as a targeted imaging reagent for lysosomes. Furthermore, based on the high negative potential of the mitochondrial inner membrane, the positively charged photoactivated fluorescent probe can be driven into the mitochondrial matrix through electrostatic attraction, thereby achieving targeted imaging of mitochondria. Therefore, the photoactivated fluorescent probe of this solution has dual targeting capabilities for lysosomes and mitochondria. This solution enables real-time imaging observation and functional regulation of lysosomes and mitochondria, overcoming the problems of photobleaching of current dye probes and cytotoxicity of multi-probe combinations, making it more suitable for simultaneous real-time imaging of lysosomes and mitochondria in living cells. Moreover, mitochondria generally serve as the cell's energy factory, producing reactive oxygen species (ROS) or regulating apoptosis; lysosomes, as the cell's degradation center, maintain cellular homeostasis and clear damaged mitochondria. Abnormalities in both often affect aging, metabolism, and even increase the risk of cancer. Therefore, using a single probe to target both lysosomes and mitochondria effectively allows for simultaneous monitoring of both systems, avoiding spectral crosstalk, toxicity confounding, and inaccurate localization caused by multiple probes. The photoactivated fluorescent probe provided in this solution possesses dual-targeting capability for both lysosomes and mitochondria, enabling it to function as a "dual-lens microscope" for observing both energy metabolism and degradation / clearance systems. This provides a more comprehensive, precise, and efficient approach than single-target probes in terms of spatiotemporal resolution, physiological accuracy, disease mechanisms, and drug screening. Furthermore, tumor cells exhibit high metabolic activity, strong negative charge on their cell membranes, high fluidity, and often overexpress specific receptors. In contrast, normal cells have intact cell membrane structures, normal membrane potential and fluidity, endocytosis at a basal physiological level, inactive macropinocytosis, and lack highly expressed receptors specific to most nanomaterials. Moreover, normal cells are largely part of the normal vascular system, making it difficult for nanomaterials to accumulate. These differences result in a stronger uptake capacity of the photoactivated fluorescent probe in tumor cells compared to normal cells under the same conditions. Therefore, based on the difference in uptake efficiency of photoactivated fluorescent probes in normal cells and tumor cells, photoactivated fluorescent probes can be used as imaging reagents to distinguish tumor cells from normal cells, and can be used for tumor targeted therapy and surgical navigation.

[0056] The maximum absorption wavelength of the photoactivated fluorescent probe in aqueous solution is 484 nm, and the maximum absorption wavelength of the photoactivated probe in ethanol solution is 475 nm.

[0057] Performance testing To verify the performance of the photoactivated fluorescent probe provided in this embodiment, the technicians conducted the following verification experiment.

[0058] Preparation of the photoactivated fluorescent probe: 0.54 g o-phenylenediamine and 0.37 g gadolinium chloride hexahydrate were added to 30 mL of ethanol and dissolved by sonication. The mixture was then transferred to a 50 mL polytetrafluoroethylene-lined reactor and heated at 200 °C for 12 h. After the reaction was completed, the supernatant was collected from the reactor and purified by column chromatography. The purified supernatant was then subjected to rotary evaporation at 38 °C and drying at 60 °C to obtain the photoactivated fluorescent probe.

[0059] (I) Mass Spectrometry Analysis The photoactivated fluorescent probe prepared above was analyzed using a high-resolution mass spectrometer to obtain... Figure 2 The results were obtained. The photoactivated fluorescent probe prepared above was then irradiated with a 365nm ultraviolet lamp and analyzed using a high-resolution mass spectrometer. Figure 3 The result.

[0060] Through the Figure 2 and Figure 3 Analysis shows that, in positive ion mode, the mass of the photoactivated fluorescent probe prepared by this method is 165, and the mass of the photoactivated fluorescent probe after activation by 365nm ultraviolet lamp is 323. This indicates that the structure of the photoactivated fluorescent probe is different before and after 365nm ultraviolet lamp irradiation, which further indicates that the photoactivated fluorescent probe absorbs excitation light under 365nm ultraviolet lamp irradiation, causing the probe to react and produce fluorescent substances.

[0061] (ii) FTIR spectral analysis (i.e., Fourier transform infrared spectroscopy) The photoactivated fluorescent probe prepared above was analyzed using an FTIR spectrometer to obtain... Figure 4 The results were obtained. The photoactivated fluorescent probe prepared above was then irradiated with a 365 nm ultraviolet lamp and analyzed using an FTIR spectrometer. Figure 5 The result.

[0062] Through the Figure 4 Analysis shows that the photoactivated fluorescent probe without excitation light exposure is at 3436 cm⁻¹ -1 The nearby absorption bands may be attributed to NH bonds on its surface, at 3085 cm⁻¹. -1 The nearby absorption band is likely attributed to CH bonds, at 1573 cm⁻¹ -1 The nearby absorption bands are likely attributed to C=O / C=N bonds, at 1300 cm⁻¹. -1 ~1000cm -1 The nearby absorption bands may be attributed to CO bonds. (This is based on...) Figure 5 Analysis shows that the photoactivated probe, after being irradiated with excitation light, is at 3000 cm⁻¹ -1The nearby absorption bands may be attributed to NH and CH bonds on its surface, at 1300 cm⁻¹. -1 ~1000cm -1 The nearby absorption bands may be attributed to CO bonds. (This is based on...) Figure 4 and Figure 5 Comprehensive analysis reveals that the functional groups on the surface of the photoactivated probe in this scheme change before and after excitation light irradiation. Specifically, at 1500 cm⁻¹... -1 The initial absorption peak remained almost unchanged, mainly because the carbon core remained the same; the subsequent change in the absorption band was due to the alteration of the probe surface structure after activation by excitation light. This demonstrates that the photoactivated fluorescent probe of this scheme absorbs excitation light under 365nm ultraviolet light irradiation, causing the photoactivated fluorescent probe to react and produce fluorescent substances.

[0063] (III) UV-Vis spectroscopic analysis of photoactivated fluorescent probes in aqueous solution The photoactivated fluorescent probe prepared above was placed in an aqueous solution, and the solution was irradiated with excitation light. The absorbance of the photoactivated fluorescent probe solution before and after irradiation with excitation light was obtained by ultraviolet-visible spectrophotometry, and the specific results are as follows: Figure 6 As shown. Figure 6 This is a spectrum showing the absorption wavelength and corresponding absorbance of an aqueous solution of a photoactivated fluorescent probe before and after excitation light irradiation. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Figure 6 Analysis revealed that the photoactivated fluorescent probe aqueous solution produced almost no fluorescence before excitation light irradiation, but fluorescence was generated after excitation light irradiation, with the maximum absorption wavelength of the photoactivated fluorescent probe aqueous solution being 484 nm. Furthermore, from... Figure 6 As shown in the illustrated photographs, the aqueous solution of the photoactivated fluorescent probe is transparent and colorless to the naked eye before light irradiation and shows no fluorescence upon exposure to ultraviolet light. After irradiation with a 365nm ultraviolet lamp, the solution appears orange-red to the naked eye and exhibits deep red fluorescence upon further ultraviolet light irradiation. This demonstrates that the aqueous solution of the photoactivated fluorescent probe provided in this scheme does not produce fluorescence without excitation light irradiation, but produces red fluorescence under 365nm ultraviolet light irradiation.

[0064] (iv) Fluorescence emission spectroscopy analysis of photoactivated fluorescent probes placed in aqueous solution The photoactivated fluorescent probe prepared above was placed in an aqueous solution, and its fluorescence intensity at different excitation wavelengths was measured using a fluorescence spectrophotometer. The results are as follows: Figure 7 As shown. Figure 7 This is the fluorescence emission spectrum of the photoactivated fluorescent probe in aqueous solution. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Figure 7Analysis shows that as the fluorescence excitation wavelength increases from 340 nm to 660 nm, the emission peak shifts from 440 nm to 680 nm. The optimal excitation wavelength corresponding to the emission peak at 440 nm is 340 nm, and the optimal excitation wavelength corresponding to the emission peak at 680 nm is 660 nm. This proves that the photoactivated fluorescent probe provided in this scheme has excitation light dependence.

[0065] (v) Changes in fluorescence intensity of photoactivated fluorescent probe aqueous solution under 370nm light irradiation over time The photoactivated fluorescent probe prepared above was placed in an aqueous solution and irradiated with 370 nm light for 50 minutes. During this process, the fluorescence intensity was measured every 5 minutes using a fluorescence spectrophotometer, and the results are as follows. Figure 8 As shown. By analyzing... Figure 8 Analysis revealed that the photoactivated fluorescent probe aqueous solution exhibited peaks at both 435 nm and 665 nm during 50 min of 370 nm light irradiation. Furthermore, through... Figure 8 Furthermore, it was found that the photoactivated fluorescent probe aqueous solution reached equilibrium (i.e., the fluorescence intensity tended to stabilize) after 50 minutes of irradiation with 370 nm light. Its fluorescence intensity at 435 nm was approximately 1.6 times that of its unactivated fluorescence intensity, and its fluorescence intensity at 665 nm was approximately 5 times that of its unactivated fluorescence intensity. This demonstrates that the photoactivated fluorescent probe of this scheme is excitation light dependent, and its fluorescence intensity increases with increasing irradiation time.

[0066] (vi) Changes in fluorescence intensity of photoactivated fluorescent probe aqueous solution under 480 nm light irradiation over time The photoactivated fluorescent probe prepared above was placed in an aqueous solution and irradiated with 480 nm light for 26 minutes. During this process, the fluorescence intensity was measured every 2 minutes using a fluorescence spectrophotometer, and the results are as follows. Figure 9 As shown. By analyzing... Figure 9 Analysis revealed that the aqueous solution of the photoactivated fluorescent probe exhibited a peak at 665 nm during 26 minutes of 480 nm light irradiation. Furthermore, through... Figure 9 Furthermore, it was found that the aqueous solution of the photoactivated fluorescent probe reached equilibrium after 26 minutes of irradiation with 480 nm light, and its fluorescence intensity at 665 nm was approximately 11 times that of its fluorescence intensity without 480 nm light activation. This demonstrates that the photoactivated fluorescent probe of this scheme is excitation light dependent, and its fluorescence intensity increases with increasing irradiation time.

[0067] Through the Figure 8 and Figure 9Analysis shows that the time required for the fluorescence of the photoactivated fluorescent probe aqueous solution to reach dynamic equilibrium differs after excitation by 370nm and 480nm light. This indicates that the fluorescence intensity increases with increasing excitation light irradiation time, proving that the fluorescence intensity of the photoactivated fluorescent probe provided by this scheme does not decrease with increasing irradiation time. This indirectly proves that the photoactivated fluorescent probe of this scheme has good photostability and is suitable for applications requiring long-duration bioimaging. Furthermore, the photoactivated fluorescent probe aqueous solution of this scheme requires 50 minutes to reach dynamic equilibrium at the short wavelength of 370nm, while it requires 26 minutes at the long wavelength of 480nm. This shows that the time required for the fluorescence of the photoactivated fluorescent probe aqueous solution to reach dynamic equilibrium is shorter at longer wavelengths and longer at shorter wavelengths. Therefore, in practical applications, the excitation wavelength can be controlled by adjusting the excitation light power, thereby controlling the imaging duration. Furthermore, when conducting cell experiments, channels with lower excitation power can be selected first, thereby prolonging the cell imaging time of the photoactivated fluorescent probe and increasing the duration of the cell experiment.

[0068] (vii) UV-Vis spectrophotometric analysis of photoactivated fluorescent probes placed in ethanol solution The photoactivated fluorescent probe prepared above was placed in an ethanol solution, and the ethanol solution was irradiated with excitation light. The absorbance of the photoactivated fluorescent probe ethanol solution before and after excitation light irradiation at different absorption wavelengths was obtained using a UV-Vis spectrophotometer. Specific results are as follows: Figure 10 As shown. Figure 10 The spectrum of the absorption wavelength and corresponding absorbance of the photoactivated fluorescent probe ethanol solution before and after excitation light irradiation is shown. Figure 10 Analysis revealed that the photoactivated fluorescent probe ethanol solution exhibited almost no peaks before excitation light irradiation, but peaks appeared after excitation light irradiation, with the maximum absorption wavelength of the photoactivated fluorescent probe ethanol solution being 475 nm. Furthermore, from... Figure 10 As shown in the illustrated photograph, the photoactivated fluorescent probe ethanol solution is transparent and colorless to the naked eye before light irradiation and shows no fluorescence upon UV irradiation. After irradiation with a 365nm UV lamp, the solution appears orange-red to the naked eye and exhibits blue fluorescence upon UV irradiation. This proves that the photoactivated fluorescent probe ethanol solution provided in this scheme does not produce fluorescence without excitation light irradiation, but produces blue fluorescence under 365nm UV irradiation.

[0069] (viii) Fluorescence emission spectroscopy analysis of photoactivated fluorescent probes placed in ethanol solution The photoactivated fluorescent probe prepared above was placed in an ethanol solution, and its fluorescence intensity at different excitation wavelengths was measured using a fluorescence spectrophotometer. The results are as follows: Figure 11 As shown. Figure 11 This is the fluorescence emission spectrum of the photoactivated fluorescent probe in ethanol solution. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Figure 11 Analysis shows that as the fluorescence excitation wavelength increases from 340 nm to 660 nm, the optimal excitation wavelength is 360 nm, with the corresponding emission peak around 450 nm. Furthermore, the fluorescence intensity at other excitation wavelengths is weaker than that at 360 nm. This demonstrates that the photoactivated fluorescent probe provided in this scheme exhibits excitation light dependence.

[0070] (ix) Changes in fluorescence intensity of photoactivated fluorescent probe ethanol solution under 370 nm light irradiation over time The photoactivated fluorescent probe prepared above was placed in an ethanol solution and irradiated with 370 nm light for 80 minutes. During this process, the fluorescence intensity was measured every 8 minutes using a fluorescence spectrophotometer, and the results are as follows. Figure 12 As shown. By analyzing... Figure 12 Analysis revealed that the photoactivated fluorescent probe ethanol solution exhibited a peak at 438 nm during 80 minutes of 370 nm light irradiation. Furthermore, through analysis of… Figure 12 The analysis also revealed that the photoactivated fluorescent probe ethanol solution reached equilibrium (i.e., the fluorescence intensity tended to stabilize) after 80 minutes of irradiation with 370 nm light, and its fluorescence intensity at 438 nm was approximately 20 times that of its fluorescence intensity without 370 nm light activation. This demonstrates that the photoactivated fluorescent probe of this scheme is excitation light dependent, and its fluorescence intensity increases with increasing irradiation time.

[0071] (x) Changes in fluorescence intensity of photoactivated fluorescent probe ethanol solution under 470 nm light irradiation over time The photoactivated fluorescent probe prepared above was placed in an ethanol solution and irradiated with 470 nm light for 32 minutes. During this process, the fluorescence intensity was measured every 2 minutes using a fluorescence spectrophotometer, and the results are as follows. Figure 13 As shown. By analyzing... Figure 13 Analysis revealed that the photoactivated fluorescent probe ethanol solution exhibited a peak at 655 nm during 32 minutes of 470 nm light irradiation. Furthermore, through... Figure 13 Furthermore, it was found that the fluorescence intensity of the photoactivated fluorescent probe ethanol solution reached equilibrium after 32 minutes of irradiation with 470 nm light, and its fluorescence intensity at 655 nm was approximately seven times that of its fluorescence intensity without 470 nm irradiation. This demonstrates that the photoactivated fluorescent probe of this scheme is excitation light dependent, and its fluorescence intensity increases with increasing irradiation time.

[0072] Through the Figure 12 and Figure 13 Analysis shows that the time required for the fluorescence of the photoactivated fluorescent probe aqueous solution to reach dynamic equilibrium differs between 370nm and 470nm light excitation. This indicates that the fluorescence intensity increases with increasing excitation light irradiation time, proving that the fluorescence intensity of the photoactivated fluorescent probe provided by this scheme does not decrease with increasing irradiation time. This indirectly proves that the photoactivated fluorescent probe of this scheme has good photostability and is suitable for applications requiring long-duration bioimaging. Furthermore, the ethanol solution of the photoactivated fluorescent probe of this scheme requires 80 minutes to reach dynamic equilibrium at the short wavelength of 370nm, while it requires 32 minutes at the long wavelength of 470nm. This shows that the ethanol solution of the photoactivated fluorescent probe of this scheme requires less time to reach dynamic equilibrium at longer wavelengths and more time at shorter wavelengths. Therefore, in practical applications, the excitation wavelength can be controlled by adjusting the excitation light power, thereby controlling the imaging duration.

[0073] (xi) TEM characterization of photoactivated fluorescent probes before and after excitation light activation The photoactivated fluorescent probe prepared above was observed under a transmission electron microscope and photographed. Figure 14 The photoactivated fluorescent probe prepared above was irradiated with a 365nm ultraviolet lamp and then observed and photographed under a transmission electron microscope. Figure 15 .

[0074] in Figure 14 This is a photograph taken by transmission electron microscopy of a photoactivated fluorescent probe without excitation light. Figure 14 It can be seen that the shape of the photoactivated fluorescent probe is approximately spherical. The size distribution results of the photoactivated fluorescent probe show that the diameter of the photoactivated fluorescent probe is distributed in the range of 1.5-5.5 nm, with an average diameter of about 3.45 nm, and it exhibits a uniform dispersion in the solvent.

[0075] Figure 15 This is a photograph taken using a transmission electron microscope after a photoactivated fluorescent probe has been irradiated with a 365nm ultraviolet lamp. Figure 15 The two images are taken with a transmission electron microscope at different nanometer scales; the left image is taken at 100 nm, and the right image is taken at 2 nm. Through analysis of... Figure 14 and 15 Analysis shows that the morphology of the photoactivated fluorescent probe changes and aggregates after activation compared to before activation, and obvious lattice fringes are observed on a 2nm scale, with a lattice spacing of 0.21nm.

[0076] (xii) Intracellular colocalization imaging An analyzer employing light-activated fluorescent probes with the ability to specifically target lysosomes and mitochondria (i.e., an analyzer for co-localization imaging in cells) can be used to simultaneously label live cell lysosomes and mitochondria, enabling real-time imaging of lysosomes and mitochondria.

[0077] In the co-localization experiment, human cervical cancer cells (HeLa) were first co-incubated with a light-activated fluorescent probe (10 mg / mL) for 30 min, followed by incubation with 0.5 μM lysosomal staining reagent for 5 min. Finally, the cells were washed twice with PBS solution (pH 7.4) and imaged using a laser confocal microscope. The results are shown below. Figure 16 As shown, through Figure 16 Analysis showed that the co-localization coefficient between the photoactivated fluorescent probe channel and the mitochondrial sigmata (Mito Tracker) channel was 70%. This demonstrates that the photoactivated fluorescent probe of this protocol can locate mitochondrial organelles. Furthermore, human liver cancer cells (G2) were co-incubated with the photoactivated fluorescent probe (10 mg / mL) for 30 min, followed by incubation with 0.5 μM lysosomal sigmata reagent for 5 min. Finally, the cells were washed twice with PBS solution (pH 7.4) and imaged using a laser confocal microscope to obtain... Figure 17 Through the Figure 17 Analysis revealed that the co-localization coefficient between the photoactivated fluorescent probe channel and the lysosomal tracker channel was 80%. Furthermore, through... Figure 17 It can be clearly observed that the photoactivated fluorescent probe can also be localized to lysosomes. Therefore, in tumor cells, the photoactivated fluorescent probe provided by this protocol can serve as a dual-targeting imaging probe targeting both lysosomes and mitochondria.

[0078] Mouse fibroblasts (L929) were co-incubated with a photoactivated fluorescent probe (10 mg / mL) for 30 min, and the images were obtained using a laser confocal microscope. Figure 18 Furthermore, human immortalized keratinocytes (HACAT) were co-incubated with a photoactivated fluorescent probe (10 mg / mL) for 30 min, and the images were obtained using a laser confocal microscope. Figure 19 Through analysis of 18 and Figure 19 Analysis shows that when photoactivated fluorescent probes were added to normal cells in both images, their fluorescence was very weak. This proves that normal cells have low uptake efficiency of photoactivated fluorescent probes. Therefore, photoactivated fluorescent probes can effectively distinguish between tumor cells and normal cells.

[0079] In summary, the photoactivated fluorescent probe prepared in this application fluoresces upon irradiation with 365nm or visible light, making it suitable for bioimaging. It can target lysosomes and mitochondria, enabling live-cell imaging and effectively distinguishing tumor cells from normal cells, significantly enhancing its application in the biological field without causing radiation damage to biological samples. Furthermore, the photoactivated fluorescent probe prepared in this application exhibits high photostability and good biocompatibility, overcoming the photobleaching properties of current dye probes and the cytotoxicity associated with multi-probe combinations.

[0080] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made without departing from the spirit and scope of the invention, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection claimed by this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a photoactivated fluorescent probe, characterized in that, It includes: o-phenylenediamine and Lewis acid were added to a low alcohol in a molar ratio of 5:1, and a photoactivated fluorescent probe was obtained by solvothermal method. The obtained photoactivated fluorescent probe itself is non-fluorescent, but it is activated and emits fluorescence after being irradiated with excitation light. This allows the photoactivated fluorescent probe to regulate the fluorescence signal it generates by controlling the parameters of the excitation light.

2. The method for preparing the photoactivated fluorescent probe as described in claim 1, characterized in that, The excitation light is ultraviolet light or visible light; And / or, the parameters of the excitation light include the irradiation time and the excitation light power; the photoactivated fluorescent probe generates fluorescence after the excitation light is turned on; the imaging time of the photoactivated fluorescent probe in the cell decreases as the excitation light power increases.

3. The method for preparing the photoactivated fluorescent probe as described in claim 1, characterized in that, Under 365nm ultraviolet light irradiation, the photoactivated fluorescent probe emits blue fluorescence in ethanol and red fluorescence in aqueous solution.

4. The method for preparing the photoactivated fluorescent probe as described in claim 1, characterized in that, The preparation method includes: adding o-phenylenediamine and Lewis acid to a low-carbon alcohol at a molar ratio of 5:1 and dissolving them by ultrasonication; after ultrasonic dissolution, transferring the solution to a reaction vessel for heating reaction; after the heating reaction is completed, taking out the supernatant from the reaction vessel and purifying, rotary evaporating and drying the supernatant in sequence to obtain the photoactivated fluorescent probe.

5. The method for preparing the photoactivated fluorescent probe as described in claim 4, characterized in that, The amount of the low-carbon alcohol used is 1 / 2 to 2 / 3 of the volume of the reaction vessel; And / or, the general chemical formula of the lower alcohol is C n H 2n+1 OH, where 1 < n ≤ 4.

6. The method for preparing the photoactivated fluorescent probe as described in claim 4, characterized in that, The heating reaction temperature is 180℃~220℃, and the heating reaction time is 8h~12h; And / or, the reactor is a polytetrafluoroethylene-lined reactor; And / or, the supernatant is purified by column chromatography, and the eluent for purification by column chromatography is petroleum ether and dichloromethane, wherein the volume ratio of petroleum ether to dichloromethane is 100:

1.

7. A photoactivated fluorescent probe, characterized in that, It is prepared using the method for preparing photoactivated fluorescent probes as described in any one of claims 1-6.

8. The photoactivated fluorescent probe as described in claim 7, characterized in that, The fluorescence excitation wavelength range of the photoactivated fluorescent probe is 360 nm to 900 nm; And / or, the diameter of the photoactivated fluorescent probe is in the range of 1.5 nm to 5.5 nm; And / or, the maximum absorption wavelength of the photoactivated fluorescent probe in aqueous solution is 484 nm; And / or, the maximum absorption wavelength of the photoactivated fluorescent probe in ethanol solution is 475 nm.

9. An application of the photoactivated fluorescent probe as described in claim 7 or 8, characterized in that, The photoactivated fluorescent probe is used as an anti-counterfeiting label based on the difference in its fluorescence color in water and ethanol. The anti-counterfeiting label appears blue when applied with ethanol under excitation light and red when applied with water.

10. An application of the photoactivated fluorescent probe as described in claim 7 or 8, characterized in that, The photoactivated fluorescent probe serves as a dual-targeting imaging reagent for lysosomes and mitochondria in tumor cells.

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