A dual-color aggregation-induced emission probe with hydrogen peroxide responsiveness, a preparation method thereof, and applications thereof

Abstract

The present invention relates to a dual-color aggregation-induced emission probe responsive to hydrogen peroxide, a preparation method thereof, and an application thereof, belonging to the technical field of fluorescent probes. The structural formula is as follows: #imgabs0# R is an alkoxy group, an alkylamino group or an aryl group, X ‑ is Br ‑ or PF6 ‑ . The probe has the characteristic of dual-color emission, and its fluorescence emission presents two colors, red and green, before and after detecting hydrogen peroxide, which provides convenience for its biological applications. In the monitoring of the simulated gravity effect, the change of endogenous hydrogen peroxide can be simply presented by the ratio of the green signal to the red signal, reducing the error of artificially observing the fluorescence color and reducing the background noise, which provides strong help for clarifying the oxidative stress phenomenon occurring under simulated microgravity.

Description

Technical Field

[0001] The present invention relates to a dual-color aggregation-induced emission probe with hydrogen peroxide responsiveness, a preparation method thereof, and an application thereof, belonging to the technical field of fluorescent probes. Background Art

[0002] Microgravity, as one of the complex space environments, has an important impact on the physical health of astronauts during long-term flights. The weightlessness effect will cause oxidative stress in human cells. As one of the important markers of oxidative stress, hydrogen peroxide (H2O2) also changes with the space flight time. Therefore, real-time monitoring of endogenous hydrogen peroxide generated in human cells under microgravity is very important for clarifying the oxidative stress damage mechanism and protection methods of astronauts' bodies. The existing main technologies for detecting endogenous hydrogen peroxide in cells are: (1) Xylenol Orange method: Hydrogen peroxide oxidizes ferrous ions to produce ferric ions, and xylenol orange selectively binds to ferric ions to form a colored (purple) product, which can be measured by colorimetry at 580 nm. Thus, the determination of hydrogen peroxide concentration is achieved. (2) Titanium sulfate colorimetric method: Hydrogen peroxide reacts with titanium sulfate to form a yellow titanium peroxide complex, which has a characteristic absorption at 415 nm. (3) Probe method: For example, in the presence of horseradish peroxidase (HRP), a specific probe reacts with hydrogen peroxide to generate a colored product with a maximum light absorption at 570 nm, and methods such as generating red fluorescence can also be used.

[0003] For detecting endogenous traditional endogenous hydrogen peroxide generated by cells under simulated microgravity conditions, the first two methods are not suitable. The reason is that the microgravity effect will recover when the simulation stops, and the detection methods used must be simple to operate and capable of rapid detection. Fluorescent probes have the characteristics of rapid detection, high sensitivity, non-invasiveness, low cost, etc., and are very suitable for detecting endogenous hydrogen peroxide generated by cells under simulated microgravity.

[0004] However, fluorescent probes face the problem of aggregation-caused quenching (ACQ). The condition that aggregation-induced quenching probes must be used at low concentrations will lead to bleaching phenomena under continuous illumination of a fluorescence microscope; at the same time, the disadvantage of requiring rinsing with phosphate buffer (PBS) for imaging also has an adverse impact on real-time monitoring of the simulated microgravity effect. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a dual-color aggregation-induced emission probe with hydrogen peroxide responsiveness, a preparation method thereof, and an application thereof.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows:

[0007] A dual-color aggregation-induced emission probe with hydrogen peroxide responsiveness, the structural formula of the probe is as follows:

[0008]

[0009] Wherein, R is an alkoxy group, an alkylamino group or an aryl group, and X - is Br - or PF6 - .

[0010] Preferably, the alkoxy group is a methoxy group; the alkylamino group is a dimethylamino group or a diethylamino group; the aryl group is a diphenylamino group.

[0011] Preferably, the structural formula of the probe is:

[0012]

[0013] A preparation method of the two-color aggregation-induced emission probe with hydrogen peroxide response according to the present invention, the method steps include:

[0014] First, a bromo-substituted cyanostilbene derivative and 4-pyridineboronic acid are reacted through a Suzuki coupling reaction under the action of a catalyst to generate an intermediate product, and the intermediate product is purified to obtain a precursor of a hydrogen peroxide-responsive aggregation-induced emission probe containing pyridine;

[0015] The precursor reacts with 4-methylphenylboronic acid pinacol ester through a nucleophilic substitution reaction to obtain a crude product, and through ultrasonic treatment, filtration, and drying, an X - is Br - two-color aggregation-induced emission probe with hydrogen peroxide response;

[0016] The X - is Br - two-color aggregation-induced emission probe with hydrogen peroxide response is replaced with hexafluorophosphate ions to obtain an X - is PF6 - two-color aggregation-induced emission probe with hydrogen peroxide response;

[0017] Wherein, the structural formula of the bromo-substituted cyanostilbene derivative is R is an alkoxy group, an alkylamino group or an aryl group.

[0018] The reaction equation is:

[0019]

[0020] Preferably, the catalyst is a palladium-based catalyst. More preferably, the catalyst is tetrakis(triphenylphosphine)palladium.

[0021] An application of the two-color aggregation-induced emission probe with hydrogen peroxide response according to the present invention in detecting hydrogen peroxide.

[0022] Application of the two-color aggregation-induced emission probe with hydrogen peroxide response in the present invention in monitoring the change of endogenous hydrogen peroxide produced by cells induced by simulated microgravity for non-disease diagnosis and treatment purposes.

[0023] Preferably, when in use, first stain the cells with the probe, then place them in a simulated microgravity environment, and finally take them out for imaging.

[0024] Application of the two-color aggregation-induced emission probe with hydrogen peroxide response in the present invention in the preparation of products for detecting endogenous hydrogen peroxide produced by cells induced by simulated microgravity.

[0025] Application of the two-color aggregation-induced emission probe with hydrogen peroxide response in the present invention in the preparation of products for detecting hydrogen peroxide in mitochondria.

[0026] Beneficial effects

[0027] The present invention provides a two-color aggregation-induced emission probe with hydrogen peroxide response. The probe has the characteristic of two-color emission, and its fluorescence emission presents two colors, red and green, before and after detecting hydrogen peroxide respectively, which provides convenience for its biological applications. In biological applications, especially in the monitoring of simulated gravity effects, the change of endogenous hydrogen peroxide can be simply presented by the ratio of the green signal to the red signal, reducing the error of artificially observing the fluorescence color and reducing the background noise, which provides strong help for clarifying the oxidative stress phenomenon occurring under simulated microgravity.

[0028] The present invention provides a two-color aggregation-induced emission probe with hydrogen peroxide response. Different donor groups (alkoxy, alkylamino or aryl) are connected to the cyanostilbene end of the probe to form a Donor-π-Acceptor structure with pyridinium salt (acceptor), which can bring a red-shifted emission for the probe. When hydrogen peroxide is detected, the 4-bromomethylphenylboronic acid pinacol ester group is cut off as the reaction site of hydrogen peroxide, so that the pyridinium salt becomes pyridine and no longer serves as an electron acceptor in the structure of the present invention. Therefore, the change of the overall structure leads to a blue shift of the molecular emission of the present invention, thus achieving the effect of the emission changing from red to green before and after detecting hydrogen peroxide.

[0029] The two-color aggregation-induced emission probe provided by the present invention has good biocompatibility, and at the same time has high targeting to mitochondria in living cells, and has the ability to in-situ detect hydrogen peroxide produced in cell mitochondria. At the same time, due to the advantages of aggregation-induced emission, the probe in the present invention has excellent photostability, which lays a foundation for long-term staining methods.

[0030] The dual-color aggregation-induced emission probe provided by the present invention is used to detect the endogenous hydrogen peroxide generated by cells induced by simulated microgravity, providing a new method for exploring issues in space life science.

[0031] The aggregation-induced emission probe provided by the present invention is simple to synthesize and has low raw material costs. It can also perform biofluorescence imaging under high-concentration conditions, having great advantages in organisms compared to probes with traditional aggregation-induced quenching phenomena.

[0032] Compared with the prior art, the fluorescence detection method in the present invention has the characteristics of rapid detection, high sensitivity, non-invasiveness, and low cost, providing strong assistance for detecting substances to be detected in cells in complex environments (simulated microgravity or other space environments).

[0033] Compared with traditional fluorescence, the aggregation-induced emission probe described in the present invention has extremely strong photostability and can be stained in cells for a long time without being bleached, enabling cells to be rapidly imaged after the completion of simulated microgravity, avoiding the recovery of microgravity. Similarly, the aggregation-induced emission probe has the characteristic of being wash-free, which can also help to quickly detect the microgravity effect of cells.

[0034] Compared with monochromatic fluorescence probes, the dual-color emission fluorescence probe described in the present invention exhibits two different colors in the emission before and after detecting the analyte, and the ratio thereof can be output as a detection signal. This can reduce the error during photographing and can amplify the tiny color change in the form of a color ratio, achieving the purpose of detecting tiny effects under simulated microgravity.

[0035] In summary, the discovery of the aggregation-induced emission (AIE) fluorescent molecules used in the present invention effectively overcomes the characteristics of high detection limits, strong detection timeliness, and long monitoring time required under simulated microgravity conditions, becoming a new method for quickly and timely detecting the effects of weightlessness environments. In addition, the fluorescence probe with dual-color emission can output the results through the signal ratio of two fluorescence channels, playing a role in correcting background interference and reducing information misguidance. At the same time, the characteristic of amplifying tiny changes through the ratio of fluorescence signals is also very suitable for detecting microgravity effects. Developing a dual-color aggregation-induced emission probe with hydrogen peroxide response is of great significance for exploring the oxidative stress of human cells under simulated microgravity and even in future real space environments. As the beginning of the combination of AIE and space life science, the strategy of solving problems in space life science effects based on aggregation-induced emission technology has good application prospects. Description of the Drawings

[0036] Figure 1 1H NMR spectrum of the dual-color aggregation-induced emission probe precursor ACP described in Example 1.

[0037] Figure 2 1H NMR spectrum of the precursor ACP of the dual-color aggregation-induced emission probe described in Example 1.

[0038] Figure 3 High-resolution mass spectrum (ESI) of the precursor ACP of the dual-color aggregation-induced emission probe described in Example 1.

[0039] Figure 4 1H NMR spectrum of the dual-color aggregation-induced emission probe ASCPB described in Example 1.

[0040] Figure 5 13C NMR spectrum of the dual-color aggregation-induced emission probe ASCPB described in Example 1.

[0041] Figure 6 ESI spectrum of the dual-color aggregation-induced emission probe ASCPB described in Example 1.

[0042] Figure 7 (A) shows the normalized UV absorbance of ASCPB described in Example 1; (B) shows the fluorescence emission spectra of the ASCPB in dimethyl sulfoxide (DMSO) and DMSO / toluene mixtures with different toluene percentages (f T ) ; (C) shows the normalized ratio diagrams of the ASCPB in DMSO and DMSO / toluene mixtures with different toluene percentages (f T ).

[0043] Figure 8 Mechanism diagram of the response of ASCPB described in Example 1 to hydrogen peroxide.

[0044] Figure 9 ESI spectrum of the product obtained by the reaction of equimolar amounts of ASCPB and hydrogen peroxide in Example 1.

[0045] Figure 10 (A) shows the normalized UV absorbance of ACP described in Example 1; (B) shows the fluorescence emission spectra of the ACP in DMSO and DMSO / water mixtures with different water percentages (f W ) ; (C) shows the normalized ratio diagrams of the ACP in DMSO and DMSO / water mixtures with different water percentages (f W ).

[0046] Figure 11(A) shows the UV absorption spectra of the ASCPB (20 μM) described in Example 1 in the presence of hydrogen peroxide at different concentrations (1 - 18 μM); (B) shows the PL spectra of the ASCPB (20 μM) in the presence of hydrogen peroxide at different concentrations (0 - 180 μM); (C) shows the normalized fluorescence emission ratio diagram of the reaction of the ASCPB with hydrogen peroxide at different concentrations (I / I0 is a function of the hydrogen peroxide concentration, I is the fluorescence intensity of the ASCPB treated with different concentrations of hydrogen peroxide at 525 nm, and I0 is the fluorescence intensity of the ASCPB without hydrogen peroxide treatment at 525 nm); (D) shows the selectivity bar chart of the ASCPB for various ROS and RNS (I is the fluorescence intensity of the ASCPB (20 μM) after treatment with different ROS / RNS, and I0 is the fluorescence intensity of the ASCPB without ROS / RNS treatment at 525 nm).

[0047] Figure 12 It is the toxicity assay of different concentrations of ASCPB and ACP on U87 - MG cells in Example 1.

[0048] Figure 13 It is the laser confocal imaging diagram of the co - staining of U87 - MG cells with the ASCPB and MTG described in Example 1 for 48 h. The ASCPB is in the red channel (a), the MTG is in the green channel (b), (c) is the merged image of (a) and (b), and (d) is the signal distribution diagram of the red channel of the ASCPB and the green channel of the MTG. The Pearson correlation coefficient is 0.95 (experimental conditions: the concentration of the ASCPB is 1 μM, the concentration of the MTG is 100 nM; for the red channel of the ASCPB: λ ex = 485.7 nm, λ em = 620 - 720 nm; for the green channel of the MTG: λ ex = 485.7 nm, λ em = 500 - 550 nm, and the scale bar is 20 μM).

[0049] Figure 14 It is the functional relationship between the normalized signal intensities of the ASCPB and MTG described in Example 1 and the number of scans of the laser confocal microscope (experimental conditions: the concentration of the ASCPB is 1 μM, the concentration of the MTG is 100 nM; for the red channel of the ASCPB: λ ex = 485.7 nm, λ em = 620 - 720 nm; for the green channel of the MTG: λ ex = 485.7 nm, λ em = 500 - 550 nm).

[0050] Figure 15For the U87-MG cells in Example 1, they were stained with ASCPB and MTG respectively, and the laser confocal fluorescence images were obtained under continuous excitation. The scans were 0, 30, and 60 times respectively (experimental conditions: the concentration of ASCPB was 1 μM, and the concentration of MTG was 100 nM; for the red channel of ASCPB: λ ex = 485.7 nm, λ em = 620 - 720 nm; for the green channel of MTG: λ ex = 485.7 nm, λ em = 500 - 550 nm; the scale bar was 20 μM).

[0051] Figure 16 For the layer scan stereogram of ASCPB and MTG described in Example 1, the number of scans was from 0 to 293 times (experimental conditions: the concentration of ASCPB was 1 μM, and the concentration of MTG was 100 nM; for the red channel of ASCPB: λ ex = 485.7 nm, λ em = 620 - 720 nm; for the green channel of MTG: λ ex = 485.7 nm, λ em = 500 - 550 nm; the scale bar was 20 μM).

[0052] Figure 17 For the response of ASCPB to exogenous hydrogen peroxide in U87-MG cells: (A) shows the laser confocal fluorescence images of U87-MG cells stained with ASCPB in the green (a) and red (b) channels for 48 h, (c) is the merged image of (a) and (b), the laser confocal fluorescence images of U87-MG cells stained with ASCPB after incubation with hydrogen peroxide in the green (d) and red (e) channels for 48 h, and (f) is the merged image of (d) and (e); (B) shows the ratio of the fluorescence emission intensity of the green channel to the red channel in the control group and the hydrogen peroxide treatment group (experimental conditions: the concentration of ASCPB was 1 μM, and the concentration of hydrogen peroxide was 5 mM; for the red channel: λ ex = 485.7 nm, λ em = 620 - 720 nm; for the green channel: λ ex = 405 nm, λ em = 500 - 550 nm; the scale bar was 30 μM).

[0053] Figure 18Response of ASCPB to exogenous hydrogen peroxide in AC16 cells: (A) Laser confocal fluorescence images of ASCPB-stained AC16 cells at 48 h in the green (a) and red (b) channels. (c) is the merged image of (a) and (b). Laser confocal fluorescence images of ASCPB-stained AC16 cells after incubation with hydrogen peroxide for 48 h in the green (d) and red (e) channels. (f) is the merged image of (d) and (e); (B) Ratio of fluorescence emission intensity in the green channel to that in the red channel in the control group and the hydrogen peroxide treatment group (experimental conditions: concentration of ASCPB is 1 μM, concentration of hydrogen peroxide is 5 mM; red channel: λ ex = 485.7 nm, λ em = 620 - 720 nm; green channel: λ ex = 405 nm, λ em = 500 - 550 n; scale bar is 50 μM).

[0054] Figure 19 Long-term dual-channel imaging of endogenous hydrogen peroxide in U87-MG cells by ASCPB under simulated microgravity: (A) Ratio of fluorescence emission intensity in the green channel to that in the red channel of ASCPB-stained U87-MG cells after 6, 24, and 48 h of simulated microgravity stimulation and unstimulated control; (B) Confocal laser fluorescence images of ASCPB-stained U87-MG cells in the control group (a-c) and after 48 h of simulated microgravity stimulation (d-f) in the green (a and d) and red (b and e) channels. (c) is the merged image of (a) and (b), and (f) is the merged image of (d) and (e) (experimental conditions: concentration of ASCPB is 1 μM; red channel: λ ex = 485.7 nm, λ em = 620 - 720 nm; green channel: λ ex = 405 nm, λ em = 500 - 550 nm; scale bar is 50 μM).

[0055] Figure 20 Confocal laser fluorescence images of ASCPB-stained U87-MG cells in the control group (a-c) and after 6 h of simulated microgravity stimulation (d-f) in the green (a and d) and red (b and e) channels. (c) is the merged image of (a) and (b), and (f) is the merged image of (d) and (e) (experimental conditions: concentration of ASCPB is 1 μM; red channel: λ ex = 485.7 nm, λ em = 620 - 720 nm; green channel: λ ex = 405 nm, λ em = 500 - 550 nm; scale bar is 50 μM).

[0056] Figure 21 Confocal laser fluorescence images of U87-MG cells stained with ASCPB in the control group (a-c) and after 24 h of simulated microgravity stimulation (d-f), in the green (a and d) channel and the red (b and e) channel. (c) is the merged image of (a) and (b), and (f) is the merged image of (d) and (e) (experimental conditions: the concentration of ASCPB is 1 μM; red channel: λ ex = 485.7 nm, λ em = 620 - 720 nm; green channel: λ ex = 405 nm, λ em = 500 - 550 nm; scale bar is 50 μM).

[0057] Figure 22 Detection of endogenous hydrogen peroxide in cells under simulated microgravity using a commercial hydrogen peroxide detection kit: (A) Photograph of the experiment when using a T-12.5 cell culture flask in an SM-31 biaxial gyrator for simulation; (B) Determination of the endogenous hydrogen peroxide level in U87-MG cells after 6, 24, and 48 h of simulated microgravity stimulation by a commercial hydrogen peroxide kit.

[0058] Figure 23 Long-term dual-channel imaging of endogenous hydrogen peroxide in AC16 cells by ASCPB under simulated microgravity: (A) Ratio of fluorescence emission intensity in the green channel to that in the red channel of AC16 cells stained with ASCPB after 6, 24, and 48 h of simulated microgravity stimulation and in the unstimulated control group; (B) Confocal laser fluorescence images of AC16 cells stained with ASCPB in the control group (a-c) and after 48 h of simulated microgravity stimulation (d-f), in the green (a and d) channel and the red (b and e) channel. (c) is the merged image of (a) and (b), and (f) is the merged image of (d) and (e) (experimental conditions: the concentration of ASCPB is 1 μM; red channel: λ ex = 485.7 nm, λ em = 620 - 720 nm; green channel: λ ex = 405 nm, λ em = 500 - 550 nm; scale bar is 50 μM).

[0059] Figure 24 Confocal laser fluorescence images of AC16 cells stained with ASCPB in the control group (a-c) and after 6 h of simulated microgravity stimulation (d-f), in the green (a and d) channel and the red (b and e) channel. (c) is the merged image of (a) and (b), and (f) is the merged image of (d) and (e) (experimental conditions: the concentration of ASCPB is 1 μM; red channel: λ ex= 485.7 nm, λ em = 620 - 720 nm; Green channel: λ ex = 405 nm, λ em = 500 - 550 nm; Scale bar is 50 μM).

[0060] Figure 25 Confocal laser fluorescence images of AC16 cells stained with ASCPB in the control group (a - c) and after 24 h of simulated microgravity stimulation (d - f) in the green (a and d) and red (b and e) channels. (c) is the merged image of (a) and (b), and (f) is the merged image of (d) and (e) (Experimental conditions: The concentration of ASCPB is 1 μM; Red channel: λ ex = 485.7 nm, λ em = 620 - 720 nm; Green channel: λ ex = 405 nm, λ em = 500 - 550 nm; Scale bar is 50 μM). Detailed implementation manners

[0061] The present invention will be further described in detail below in conjunction with specific embodiments.

[0062] Example 1

[0063] In a 250 ml two - necked round - bottom flask, add respectively (1.500 g, 4.584 mmol), 4 - pyridineboronic acid (1.038 g, 8.444 mmol), tetrakis(triphenylphosphine)palladium (0.78 g, 0.6750 mmol) and anhydrous potassium carbonate (6.219 g, 45.00 mmol). Then, under a nitrogen atmosphere, deoxygenated tetrahydrofuran (150 mL) and water (22.5 mL) are injected into the flask. Stir the reaction mixture and reflux it under nitrogen overnight. After removing most of the volatiles under reduced pressure, add water and extract three times with dichloromethane. Collect the organic phase and dry it with anhydrous sodium sulfate. After the solvent has evaporated completely, purify the crude product by silica gel column chromatography with a DCM / MeOH mixture (50 / 1, v / v) to obtain the dual - color hydrogen peroxide - responsive aggregation - induced emission probe precursor (ACP) with a yield of 48%.

[0064] The reaction equation is:[[]]

[0065]

[0066] The ACP (100.0 mg, 0.3073 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl bromide (88.00 mg, 0.2963 mmol) were added to a 50 mL three-necked round-bottom flask and dissolved in 10 mL of acetonitrile. The reaction was stirred and heated overnight under nitrogen. After the reaction was completed, sticky solid blocks could be found on the inner wall of the flask. After cooling to room temperature, the reaction mixture at the bottom was slowly sucked out with a dropper, and the solid blocks were ultrasonically treated with petroleum ether. After suction filtration and washing 3 times with methyl tert-butyl ether, a dark red solid was obtained, which was a dual-color hydrogen peroxide-responsive aggregation-induced emission probe (ASCPB), with a yield of 16%.

[0067] The reaction equation is as follows:

[0068]

[0069] Chemical structure characterization of the ASCPB and ACP:

[0070] The chemical structure of the synthesized ACP was characterized by NMR. The 1H NMR spectrum is as Figure 1 shown; the 13C NMR spectrum is as Figure 2 shown, and the high-resolution mass spectrometry characterization is as Figure 3 shown.

[0071] The chemical structure of the synthesized ASCPB was characterized by NMR. The 1H NMR spectrum is as Figure 4 shown; the 13C NMR spectrum is as Figure 5 shown, and the high-resolution mass spectrometry characterization is as Figure 6 shown.

[0072] Photophysical property characterization of the ASCPB:

[0073] The photophysical properties of the ASCPB were studied in solvents with different polarities and in the solid state. The results are as Figure 7 shown. All data were collected at room temperature, and the excitation wavelength was 475 nm. The results showed that the ASCPB had similar ultraviolet absorbances, and its peak was around 475 nm. Using this parameter as the excitation wavelength, the fluorescence emission spectrum of the ASCPB in a mixture of the good solvent DMSO and the poor solvent toluene was measured. In the mixture with a toluene volume fraction (f T ) less than 90%, the fluorescence intensity was very low. When f T increased to 95%, a sharp increase in the PL intensity (about 500 times at the emission wavelength of 620 nm) was observed. This indicates that the ASCPB has typical AIE properties.

[0074] Study on the response of the ASCPB to hydrogen peroxide:

[0075] The reaction mechanism of the two-color aggregation-induced emission probe ASCPB with hydrogen peroxide response is as follows Figure 8 shown. In the ASCPB, the pyridinium salt group serves as a targeting group for mitochondria, facilitating in-situ monitoring of substances produced by cells; while the 4-methylphenylboronic acid ester group, as a targeting group responsive to hydrogen peroxide, is connected to the pyridine on ASCPB. In the presence of hydrogen peroxide, hydrogen peroxide can cleave the boronic acid pinacol ester, causing the 4-methylphenylboronic acid ester group on ASCPB to detach and generate ACP. At the same time, the cleavage of the D-π-A structure of ASCPB leads to an obvious low chromatic aberration shift of the emitter.

[0076] Figure 9 Figure Figure 8 is the ESI diagram of the product obtained from the reaction of equimolar equivalents of ASCPB with hydrogen peroxide. The ASCPB was incubated with an equal amount of hydrogen peroxide at 37 °C, and then the reaction mixture was detected by electrospray ionization mass spectrometry (ESI-MS). In the figure, ion peaks 432.2060 and 326.1640 (m / z) can be found, and in Figure 8 these two peaks belong to the reaction intermediate and ACP respectively. At the same time, the synthesized ACP was used as a model compound to measure its absorption spectrum ( Figure 10 A) and emission spectrum ( Figure 10 B), which were matched with the corresponding spectra of ASCPB after treatment with hydrogen peroxide in Figure 7 A and 7B respectively. These results confirmed the reaction mechanism of ASCPB with H2O2 hypothesized in Figure 8 . In addition, ACP also has AIE properties. As the fraction of the poor solvent water in the good solvent DMSO solution of ACP gradually increases, the fluorescence intensity gradually increases, and the intensity is the maximum when f W (water fraction) is 70% ( Figure 10 B and C). The AIE properties of ACP result in the change of the emission color of ASCPB after reaction with hydrogen peroxide and the formation of molecular aggregation.

[0077] Figure 11 Figure A is the ultraviolet-visible absorption spectrum of ASCPB in the presence of different concentrations of hydrogen peroxide. As the concentration of H2O2 increases, the absorption of ASCPB at 465 nm changes proportionally, an absorption band appears at 405 nm, and the absorption intensity gradually increases. At the same time, the fluorescence emission behavior of ASCPB also changes significantly. In the presence of hydrogen peroxide, a new emission peak appears at around 525 nm. And as the concentration of hydrogen peroxide gradually increases, the emission intensity also gradually increases ( Figure 11 B). When the concentration of hydrogen peroxide reaches 190 μM, the light intensity at 525 nm increases by about 40 times. In addition, the emission intensity shows a good linear relationship with the hydrogen peroxide concentration in the range of 0 - 180 μM ( Figure 11C), the detection limit was estimated to be 170 nM based on 3δ / slope. Meanwhile, the selectivity of ASCPB towards different reactive oxygen species (ROS) and reactive nitrogen species (RNS) was investigated. As can be seen from Figure 11 the data in D, at 525 nm, only hydrogen peroxide could significantly trigger fluorescence enhancement, while other ROS and RNS had poor responses to ASCPB. Therefore, ASCPB has high sensitivity and good selectivity and is suitable for the quantitative determination of hydrogen peroxide.

[0078] Bioimaging test of ASCPB:

[0079] The cell counting Kit-8 (CCK-8) method was used to evaluate the biocompatibility of ASCPB and ACP. Human brain astrocytoma cells (U87-MG) were used as model cells. As can be seen from Figure 12 the results, when the concentrations of both ASCPB and ACP were 5 μM, the cell viability still remained at a relatively high level, indicating that the entire probe system had good biocompatibility.

[0080] ASCPB has good selectivity for mitochondria. U87-MG cells were co-stained with 1 μM ASCPB and 100 nM commercial mitochondrial dye Mito-Tracker Green (MTG) for 48 h and imaged using a laser confocal microscope ( Figure 13 ). The figure shows that the red fluorescence of ASCPB has excellent correlation with the green fluorescence of MTG (correlation coefficient is 0.95), indicating that the pyridinium group on the probe endows ASCPB with good mitochondrial targeting ability. Mitochondria cause the aggregation of ASCPB, leading to the RIM effect, which makes the ASCPB probe show strong fluorescence.

[0081] Since the monitoring experiment of hydrogen peroxide under simulated microgravity usually needs to last for more than 48 h, the long-term photostability of the probe is a key parameter that needs to be evaluated. U87-MG cells were co-stained with working concentrations of ASCPB and MTG and continuously scanned by laser irradiation on a confocal laser scanning microscope. After 60 scans, the fluorescence signal of MTG decreased sharply from 100% to 40%, while the signal loss of ASCPB was only about 10% ( Figure 14 、 15 ). Meanwhile, fluorescence signal monitoring for a longer number of times (0 - 293 times) was further studied ( Figure 16 ). As the number of scans increased, the ASCPB signal maintained good quality, while the MTG signal decreased significantly and disappeared in the later stage. These results revealed the excellent photostability of ASCPB.

[0082] Response test of ASCPB to exogenous hydrogen peroxide in living cells:

[0083] U87-MG cells were first stained with 1 μM ASCPB for 30 min. Without rinsing with PBS buffer, they were then treated with 5 mM hydrogen peroxide for 48 h. The control group was the same as the treatment group except that hydrogen peroxide was not added. After the reaction was completed, imaging was performed using a laser confocal microscope. As Figure 17 shown in A, the fluorescence intensity of the green channel in the hydrogen peroxide treatment group was significantly higher than that in the control group, and the fluorescence intensity of the red channel in the treatment group was significantly lower than that in the control group. By processing and analyzing the fluorescence intensity ratio of the green channel to the red channel using software, it was found that the fluorescence intensity of the hydrogen peroxide treatment group was significantly higher than that of the control group ( Figure 17 B). These results are attributed to the conversion of ASCPB to ACP under the action of hydrogen peroxide. The hydrophobic ACP formed during the conversion aggregates, resulting in enhanced green fluorescence due to the RIM effect. In addition, Figure 18 for the response of ASCPB to exogenous hydrogen peroxide in AC16 cells, its confocal images and data processing results also showed obvious changes in the fluorescence signals between the red and green channels. The above results indicate that ASCPB is a good two-color probe for tracking hydrogen peroxide in living cells.

[0084] Test of the response of ASCPB to intracellular hydrogen peroxide induced by simulated microgravity:

[0085] The probe was used to study the production of endogenous hydrogen peroxide during oxidative stress in living cells induced by simulated microgravity. A simulated microgravity environment was established using a two-axis gyroscope. In this study, U87-MG cells in a culture dish were first stained with ASCPB (1 μM), and then the culture dish was filled with culture medium to prevent the formation of bubbles (the presence of bubbles in the culture medium during simulated microgravity would generate shear force on the cells, affecting cell adhesion). The sample was fixed on the two-axis gyroscope using a sterile mold. During the whole experiment, since the cells were pre-stained, the sample could be taken off the gyroscope at any time for rapid imaging and then re-fixed for continued rotation after imaging. In this way, by continuously monitoring the same sample at different time points, the differences between different samples could be reduced, making the experimental results more accurate. Imaging was performed at the 6 h, 24 h, and 48 h time points of the sample ( Figure 19 B, Figure 20 , Figure 21 ), and the software was used to process the ratios between the green (λ ex = 405 nm; λ em = 500 - 550 nm) and red (λ ex = 485.7 nm; λ em = 620 - 720 nm) channels of the microgravity group and the two channels of the control group, indicating that endogenous hydrogen peroxide could be produced at the three time points under simulated microgravity conditions (Figure 19 A). In addition, there were significant differences in the ratio of the two channels between the SMG group and the control group under simulated microgravity for 6 h. As the time extended to 24 h, this difference decreased slightly. As the simulated time extended to 48 h, this difference increased again and was significantly higher than that at 6 h. This change in difference reflected the change in endogenous hydrogen peroxide generated under simulated microgravity during the experiment. The short-term stress and long-term damage effects under microgravity might be the reasons for this difference. When U87-MG cells were initially stimulated by a short-term microgravity environment, they became non-adaptive and triggered an oxidative stress response. However, as time increased, the microgravity effect began to accumulate continuously in the cells, and the cells gradually adapted to oxidative stress to a certain extent, so they returned to a level close to that of the control group under simulated microgravity for 24 h. As the simulated microgravity time further increased, the gradually accumulated microgravity effect exceeded the adaptability of the cells, thus showing a long-term microgravity effect, and a large amount of hydrogen peroxide was generated.

[0086] A commercial hydrogen peroxide detection kit (Beyotime, hydrogen peroxide detection kit) was used to verify the detection results of ASCPB ( Figure 22 ). As can be seen from the figure, the results had a similar trend to those of the ASCPB determination, verifying the reliability of ASCPB in imaging endogenous hydrogen peroxide in living cells under simulated microgravity conditions.

[0087] Human cardiomyocyte AC16 was used to further evaluate the effectiveness and universality of the probe system ( Figure 24 - 25 ). This experiment revealed that ASCPB could also detect endogenous hydrogen peroxide in AC16 cells under simulated microgravity. At the same time, compared with U87-MG cells, the accumulation of hydrogen peroxide in AC16 cells was more obvious at 48 h, which might be attributed to the fact that AC16 had more abundant mitochondria than U87-MG. This experiment further demonstrated that ASCPB was a powerful probe for monitoring hydrogen peroxide in living cells under simulated microgravity.

[0088] Example 2

[0089] Replace the in the first-step reaction of Example 1 with The rest was the same as in Example 1, and a two-color aggregation-induced emission probe with hydrogen peroxide responsiveness was obtained.

[0090] The probe had a hydrogen peroxide detection effect similar to that of the probe described in Example 1.

[0091] Example 3

[0092] Replace the in the first-step reaction of Example 1 with The rest is the same as in Example 1, and a two-color aggregation-induced emission probe with hydrogen peroxide responsiveness is obtained.

[0093] The probe has a hydrogen peroxide detection effect similar to that of the probe described in Example 1.

[0094] Example 4

[0095] Replace the in the first-step reaction in Example 1 with The rest is the same as in Example 1, and a two-color aggregation-induced emission probe with hydrogen peroxide responsiveness is obtained.

[0096] The probe has a hydrogen peroxide detection effect similar to that of the probe described in Example 1.

[0097] In summary, the invention includes but is not limited to the above embodiments. Any equivalent replacement or partial improvement made under the spirit and principle of the present invention shall be regarded as being within the protection scope of the present invention.

Claims

1. A dual-color aggregation-induced emission probe with hydrogen peroxide response, characterized in that: The structural formula of the probe is as follows: 。 2. A preparation method of a dual-color aggregation-induced emission probe with hydrogen peroxide response as described in claim 1, characterized in that: The method steps include: First, a bromine-substituted cyanostilbene derivative and 4-pyridylboronic acid are subjected to a Suzuki coupling reaction under the action of a catalyst to generate an intermediate product, and the intermediate product is purified to obtain a precursor of a hydrogen peroxide-responsive aggregation-induced emission probe containing pyridine; The precursor undergoes a nucleophilic substitution reaction with 4-methylphenylboronic acid pinacol ester to obtain a crude product, and through ultrasonic treatment, filtration, and drying, a dual-color aggregation-induced emission probe with hydrogen peroxide responsiveness is obtained; Among them, the structural formula of the bromine-substituted cyanostilbene derivative is .

3. The preparation method of a two-color aggregation-induced emission probe with hydrogen peroxide response as described in claim 2, characterized in that: The catalyst is a palladium-based catalyst.

4. Use of a dual-color aggregation-induced emission probe with hydrogen peroxide responsiveness as described in claim 1 for detecting hydrogen peroxide for non-disease diagnosis and treatment purposes.

5. Use of a dual-color aggregation-induced emission probe with hydrogen peroxide responsiveness as described in claim 1 for monitoring changes in endogenous hydrogen peroxide generated by cells induced by simulated microgravity for non-disease diagnosis and treatment purposes.

6. Use of a two-color aggregation-induced emission probe with hydrogen peroxide responsiveness in monitoring changes in endogenous hydrogen peroxide produced by cells induced by simulated microgravity for non-disease diagnosis and treatment purposes, characterized in that: During use, first stain the cells with the probe, then place them in a simulated microgravity environment, and finally take them out for imaging.

7. Use of a dual-color aggregation-induced emission probe with hydrogen peroxide responsiveness as described in claim 1 for preparing a product for detecting endogenous hydrogen peroxide generated by cells induced by simulated microgravity.

8. Use of a dual-color aggregation-induced emission probe with hydrogen peroxide responsiveness as described in claim 1 for preparing a product for detecting hydrogen peroxide in mitochondria.

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