A carbon monoxide near-infrared fluorescent probe and its preparation method and application

Abstract

The present invention discloses a carbon monoxide near-infrared fluorescent probe, its preparation method, and its application, belonging to the technical field of fluorescent probes. The carbon monoxide near-infrared fluorescent probe provided by the present invention has good photostability, a short response time to CO, high selectivity, a low detection limit, and high sensitivity. Furthermore, the fluorescent probe is mitochondrial-targeting and can be used for the detection and imaging of CO in mitochondria. Furthermore, the fluorescent probe can also be used for the detection and imaging of CO in living cells and in human subjects, and in particular, can directly detect CO fluctuations in ocular blood samples from mouse models of lipopolysaccharide-induced inflammation. Therefore, the fluorescent probe has promising application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a carbon monoxide near-infrared fluorescent probe and a preparation method and application thereof. Background Art

[0002] Carbon monoxide (CO) is a colorless, odorless gas with a strong affinity for hemoglobin. It can rapidly deplete oxygen levels in the body, posing a significant threat to human health. CO has been shown to be a gaseous signaling molecule with various physiological functions, and abnormal CO levels are closely associated with serious diseases such as heart disease, Alzheimer's disease, cancer, and hypertension. In addition, oxidative stress induced by acute inflammation can increase CO levels because it acts as a scavenger of oxygen free radicals to protect cells. Therefore, the detection of CO is a potential method for monitoring CO-related diseases and early warning of acute inflammation.

[0003] Organic molecular fluorescent probes based on organic fluorescent dyes are widely used as practical technologies for bioimaging due to their significant selectivity, high sensitivity, non-invasive fluorescence imaging advantages, and precise spatiotemporal resolution. Recently, many small molecule probes for detecting CO in biological systems have been reported. For example, Wei et al. (J. Tang, P. Zhang, Z. Li, Y. Zhang, H. Chen, X. Li, et al., A simple ratiometric fluorescent probe for two-photonimaging of carbon monoxide in living cells and zebrafish, Bioorganic Chemistry, 135 (2023) 106489.) used coumarin to develop a new fluorescent probe RTFP, which showed significant selectivity and sensitivity in endogenous CO imaging. Lin et al. (Y. Zhang, Y. Tang, X. Kong, W. Lin, An endoplasmic reticulum targetable turn-on fluorescence probe for imaging application of carbon monoxide in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 247 (2021) 119150.) designed a Na-CM-ER probe based on classic naphthalene diimide dyes, which can quickly detect exogenous and endogenous CO in ER-targeted manner through the reduction reaction of nitro groups with amines. However, most of the currently available CO probes emit fluorescence less than 650 nm, which limits their ability to penetrate tissues and makes them susceptible to a large amount of interference from intrinsic biofluorescence. These limitations greatly limit the application of optical imaging in biological tissues. In contrast, near-infrared fluorescent probes are known for minimizing photodamage in biological samples, promoting deep tissue penetration, and minimal interference from biomolecular autofluorescence, making them very suitable for in vivo imaging.

[0004] However, existing near-infrared fluorescent probes have problems such as poor selectivity, low sensitivity and long response time. Summary of the Invention

[0005] The present invention aims to provide a carbon monoxide near-infrared fluorescent probe and its preparation method and application, so as to solve the problems of poor selectivity, low sensitivity and long response time of existing near-infrared fluorescent probes.

[0006] In a first aspect, the present invention provides a carbon monoxide near-infrared fluorescent probe having a structure shown in the following formula (1):

[0007]

[0008] In the carbon monoxide near-infrared fluorescent probe provided by the present invention, Nile blue is used as a fluorescent support and allyl carbonate group is used as a recognition unit. 2+ ) When Pd comes into contact with CO, 2+ First reduced to Pd by CO 0 , then mediates the Tsuji-Trost reaction, resulting in the breakage of the allyl formate and the release of the Nile blue fluorophore, thereby generating near-infrared fluorescence, and the intensity of the infrared fluorescence is linearly correlated with the concentration of CO. Therefore, the carbon monoxide near-infrared fluorescent probe in the present invention can be used for the quantitative detection of CO.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned carbon monoxide near-infrared fluorescent probe, comprising the following steps: mixing Nile blue with triethylamine and allyl chloroformate and conducting a chemical reaction, and separating and purifying the resulting product to obtain a carbon monoxide near-infrared fluorescent probe.

[0010] In some embodiments, the molar ratio of Nile blue, triethylamine, and allyl chloroformate is (0.3-0.7):(1.5-2):(0.5-0.8).

[0011] In some embodiments, the chemical reaction step specifically comprises: stirring the reaction at a temperature of 20-25° C. under an inert atmosphere for 10-14 hours.

[0012] In a third aspect, the present invention provides use of the above-mentioned carbon monoxide near-infrared fluorescent probe in the detection and / or imaging of CO in living cells.

[0013] In some embodiments, CO includes exogenous CO and endogenous CO.

[0014] In some embodiments, endogenous CO is produced by introducing heme into living cells, or is induced by introducing lipopolysaccharide into living cells.

[0015] In a fourth aspect, the present invention provides use of the above-mentioned carbon monoxide near-infrared fluorescent probe in the detection and / or imaging of CO in a subject.

[0016] In some embodiments, CO includes exogenous CO and endogenous CO; and endogenous CO is induced by injecting lipopolysaccharide into the subject.

[0017] In some embodiments, the subject comprises a mouse.

[0018] The beneficial effects of the present invention are as follows: unlike the prior art, the carbon monoxide near-infrared fluorescent probe provided by the present invention has good photostability, a short response time to CO (less than 5 minutes), high selectivity, a low detection limit (47 nM), and high sensitivity. In addition, the fluorescent probe is targeted to mitochondria and can be used for the detection and imaging of CO in mitochondria. In addition, the fluorescent probe can also be used for the detection and imaging of CO (including exogenous CO and endogenous CO) in living cells and subjects (mice), and in particular can directly detect CO fluctuations in ocular blood samples of lipopolysaccharide-induced mouse inflammation models. Therefore, the fluorescent probe has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The fluorescent probe Nile-CO prepared in Example 1 of the present invention 1 HNMR spectrum;

[0020] Figure 2 The fluorescent probe Nile-CO prepared in Example 1 of the present invention 13 CNMR spectrum;

[0021] Figure 3 HRMS spectrum of the fluorescent probe Nile-CO prepared in Example 1 of the present invention;

[0022] Figure 4A This is the absorbance result diagram of the present invention after 10 μM fluorescent probe Nile-CO reacts with 100 μM CO and 10 μM PdCl2;

[0023] Figure 4B The fluorescence spectrum of the present invention after 10 μM fluorescent probe Nile-CO reacts with 100 μM CO and 10 μM PdCl2 (λ ex =580nm);

[0024] Figure 5A This is a graph showing the change in fluorescence intensity over time after the reaction of 10 μM fluorescent probe Nile-CO with 50 μM CO and 10 μM PdCl2 in the present invention;

[0025] Figure 5B The fluorescence spectrum of the present invention after the reaction of 10 μM fluorescent probe Nile-CO with 100 μM analyte and 10 μM PdCl2 was obtained 30 minutes after the addition of the analyte, and the results were repeated three times. ex =580nm, and 1-21 represent Li + 、Na + , K + Mg 2+ 、Fe3+ 、Fe 2+ 、Zn 2+ , Ca 2+ 、Cu 2+ 、SO4 2- 、CO3 2- 、SO3 2- 、NO2 - 、NO、ONOO - , H2O2, Cys, Hcy, GSH, H2S, CO;

[0026] Figure 5C The fluorescence spectrum results of the fluorescent probe Nile-CO detecting CO in solution systems with different pH values ​​(2-11) in the presence of palladium ions in the present invention;

[0027] Figure 5D This is a graph showing the optical stability of the fluorescent probe Nile-CO at an excitation wavelength of 580 nm in the present invention;

[0028] Figure 6 The linear relationship between the fluorescence intensity change of the fluorescent probe Nile-CO at 664m and the CO concentration is shown in the figure. Each data point is obtained 30 minutes after adding the analyte and repeated three times. ex =580nm;

[0029] Figure 7A This is a graph showing the LC-MS analysis results of a 100 mM fluorescent probe Nile-CO solution (containing 100 μM PdCl2) in the present invention;

[0030] Figure 7B This is the LC-MS analysis result of adding 500 μM CO to a 100 mM fluorescent probe Nile-CO solution (containing 100 μM PdCl2) in the present invention;

[0031] Figure 8 Schematic diagram of the detection principle of CO by the fluorescent probe Nile-CO in the present invention;

[0032] Figure 9 This is the MTT result of HeLa cells treated with 0-40 μM fluorescent probe Nile-CO in the present invention (all experiments were repeated 3 times);

[0033] Figure 10 The cell imaging images of HeLa cells in the present invention were first incubated with 0, 5, 10, and 30 μM CO for 30 min, and then incubated with 5 μM probe solution (Nile-CO + PdCl2, 1:1) for 30 min;

[0034] Figure 11The cell imaging images of HeLa cells in the present invention were first incubated with 30 μM CO for 30 min, and then incubated with 0, 2, 5, and 10 μM probe solutions (Nile-CO + PdCl2, 1:1) for 30 min;

[0035] Figure 12 These are cell imaging images of HeLa cells in the present invention after incubation with 100 μM heme for 2, 4, and 8 hours, and then with 5 μM probe solution (Nile-CO + PdCl2, 1:1) for 30 minutes;

[0036] Figure 13 These are cell imaging images of HeLa cells in the present invention after being incubated with 1 μg / mL LPS for 1, 4, and 16 hours, and then incubated with 5 μM probe solution (Nile-CO+PdCl2, 1:1) for 30 minutes;

[0037] Figure 14 This is the result of cell co-localization using 5 μM probe solution (Nile-CO+PdCl2, 1:1) and 5 μM mitochondrial green fluorescent probe in the present invention;

[0038] Figure 15 The images of mice were taken within 0-60 min after the probe (100 μM Nile-CO+PdCl2) and CORM-3 (500 μM) were injected into the same cavity of the mouse in the present invention;

[0039] Figure 16 These are images of mice injected intraperitoneally with 4 mg / kg LPS for 1 hour and 16 hours, followed by a tail vein injection of 5 mg / kg probe (Nile-CO + PdCl2, 1:1) according to the present invention.

[0040] Figure 17 This is the result of administering 0, 2, and 4 mg / kg LPS to the tail vein of mice for 1 hour and 16 hours, respectively, and then adding 300 μM fluorescent probe Nile-CO, and observing the fluorescence intensity of mouse eye blood samples between 650-750 nm. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] For experimental methods in the examples where specific conditions are not specified, generally conventional conditions and conditions described in the manual or conditions recommended by the manufacturer were followed. The general equipment, materials, reagents, etc. used were all commercially available unless otherwise specified.

[0043] Example 1

[0044] This embodiment provides a carbon monoxide near-infrared fluorescent probe Nile-CO, which has a structure shown in the following formula (1):

[0045]

[0046] The synthesis reaction formula of the carbon monoxide near-infrared fluorescent probe Nile-CO is as follows:

[0047]

[0048] Specifically, compound 1 (200 mg, 0.57 mmol), triethylamine (172 mg, 1.7 mmol), and allyl chloroformate (81.75 mg, 0.68 mmol) were dissolved in dichloromethane to obtain a mixture, and the mixture was stirred and reacted at room temperature for 12 h under an N2 atmosphere. The resulting solution was quenched with water and extracted three times with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and concentrated using a rotary evaporator. The crude mixture was further purified by column chromatography (dichloromethane / methanol (100: 1 to 10: 1)) to obtain a red powder probe (65 mg, yield 26.26%).

[0049] The red powder probe was subjected to structural characterization test. 1 HNMR, 13 CNMR and HRMS spectra are shown in Figure 1-3 shown.

[0050] from Figure 1-3 It can be seen that the intermediate compound has the following structural information: 1 HNMR(400MHz,Chloroform-d)δ8.54(d,J=7.9Hz,1H),8.41(d,J=7.9Hz,1H),7.67-7.38(m,3H),6.74-6.46(m,2H),6.30(d,J=2.8Hz,1H),6.02(ddt, J=16.5,11.0,5.8Hz,1H),5.37(d,J=17.2Hz,1H),5.23(d,J=10.4Hz,1H), 4.73(d,J=5.9Hz,2H), 3.36(q,J=7.1Hz,4H), 0.79(q,J=10.2,8.3Hz,6H). 13CNMR (101MHz, Chloroform-d)δ162.74,160.40,149.62,148.53,145.66,139.16,131.60,130.74,129.83,128.77,124.53,122.73,117.49,108.76,99.08,95.17,65.94,44.05,28.68,21.67,11.60. Chemical formula: C 24 H 24 N3O3, exact mass: 402.1812, found mass: 402.1804.

[0051] Example 2

[0052] This example studies the absorption and fluorescence spectra of the fluorescent probe Nile-CO interacting with CO.

[0053] Specifically, the absorption spectrum (Hitachi F-4500 UV-visible spectrophotometer) and fluorescence spectrum (Hitachi F-2500 spectrophotometer) of the fluorescent probe Nile-CO before and after the reaction with CO were measured in phosphate buffer (PBS, 10 mM, pH 7.4) containing 1% DMSO at 24°C. The results are as follows: Figure 4A and 4B shown.

[0054] from Figure 4A As can be seen in the figure, the primary absorption band of the fluorescent probe Nile-CO (10 μM) is located at 534 nm. No significant changes were observed in the absorption spectrum after the addition of 10 μM PdCl2. However, when 100 μM CO (using CORM-3 as the CO source) and 10 μM PdCl2 were added simultaneously, the absorption band at 534 nm disappeared significantly, undergoing a red shift and the emergence of a new, significantly enhanced absorption band at 628 nm.

[0055] from Figure 4B It can be seen that due to the quenching effect of allyl formate, the fluorescent probe Nile-CO has no obvious near-infrared emission peak in the near infrared (664nm) after adding 10μM PdCl2; after adding 100μM CO and 10μM PdCl2 at the same time, a clear near-infrared emission peak appeared at 664nm. This is because Pd 2+ First reduced to Pd by CO 0 , which then mediates the Tsuji-Trost reaction, leading to the cleavage of the allyl formate group and the release of the Nile blue fluorophore, thereby generating near-infrared fluorescence.

[0056] Example 3

[0057] This example studies the response time, selectivity, pH adaptability, and photostability of the fluorescent probe Nile-CO to CO.

[0058] Specifically, after adding 50 μM CO to a 10 μM fluorescent probe Nile-CO solution (containing 10 μM PdCl2), the fluorescence intensity (wavelength of 664 nm) at different times was measured. The results are as follows: Figure 5A shown.

[0059] from Figure 5A As can be seen from the figure, the fluorescence intensity increases slowly within 1 minute, then increases rapidly within 5 minutes and reaches saturation fluorescence intensity. The results show that the fluorescent probe Nile-CO has a fast response capability for the detection of CO.

[0060] Next, 21 common analytes (Li + 、Na + , K + Mg 2+ 、Fe 3+ 、Fe 2+ 、Zn 2+ , Ca 2+ 、Cu 2+ 、SO4 2- 、CO3 2- 、SO3 2- 、NO2 - 、NO、ONOO - , H2O2, Cys, Hcy, GSH, H2S, CO) were introduced into a solution containing 10 μM fluorescent probe Nile-CO (containing 10 μM PdCl2), and the selectivity of the fluorescent probe Nile-CO was evaluated based on the measured fluorescence intensity (wavelength of 664 nm). The results are shown in Figure 2. Figure 5B shown.

[0061] from Figure 5B As can be seen from the figure, only the addition of CO can cause a significant increase in fluorescence intensity. After the addition of other analytes, the fluorescence intensity is extremely low. The results show that the fluorescent probe Nile-CO has good selectivity for CO.

[0062] Furthermore, the effect of different pH values ​​(2-11) on the fluorescence spectrum of the fluorescent probe Nile-CO in detecting CO was studied. After adding 100 μM CO to a 10 μM fluorescent probe Nile-CO solution (with different pH values ​​and containing 10 μM PdCl2), the fluorescence intensity (wavelength of 664 nm) was measured. The results are as follows: Figure 5C shown.

[0063] from Figure 5CAs can be seen in the figure, the fluorescence intensity of the fluorescent probe Nile-CO itself does not increase significantly in solutions with various pH values. However, after the addition of CO, the probe's fluorescence intensity is significantly enhanced within the pH range of 6-9, and reaches its highest intensity at pH 7-8. This is very beneficial for CO detection and bioimaging applications.

[0064] Finally, after adding 100 μM CO to a 10 μM fluorescent probe Nile-CO solution (containing 10 μM PdCl2), the fluorescence intensity (wavelength 664 nm) was measured at different time points under an excitation wavelength of 580 nm to evaluate the optical stability of the fluorescent probe Nile-CO. The results are shown in Figure 2. Figure 5D shown.

[0065] from Figure 5D As can be seen in the figure, the fluorescence signal of the fluorescent probe Nile-CO itself does not change significantly over time. However, after the addition of CO, the fluorescence signal of the fluorescent probe Nile-CO does not decrease significantly. After 1 hour of excitation, it maintains 90% of its initial fluorescence intensity, indicating that the fluorescent probe Nile-CO has good optical stability.

[0066] Example 4

[0067] This example studies the quantitative detection of CO by the fluorescent probe Nile-CO.

[0068] Specifically, 10 μM PdCl2 and different concentrations (0-35 μM) of CO were added to a 10 μM fluorescent probe Nile-CO solution, and the fluorescence intensity (emission wavelength was 664 nm) was measured. A linear equation was established with the fluorescence intensity as the ordinate and the CO concentration as the abscissa. The results are shown in Figure 2. Figure 6 shown.

[0069] from Figure 6 As can be seen from the figure, there is a good linear relationship between the fluorescence intensity at an emission wavelength of 664 nm and the CO concentration (0-30 μM), and the detection limit is 47 nM; therefore, CO can be quantitatively detected using the fluorescent probe Nile-CO in the present invention.

[0070] Example 5

[0071] This example studies the detection mechanism of CO by the fluorescent probe Nile-CO.

[0072] Specifically, LC-MS was used to analyze the reaction process of the fluorescent probe Nile-CO with CO and PdCl2. Mobile phase A was an aqueous solution containing 0.05% trifluoroacetic acid, mobile phase B was acetonitrile, the elution conditions were mobile phase A / mobile phase B = 50 / 50, and the column temperature was 40°C. First, 100mM fluorescent probe Nile-CO solution (containing 100μM PdCl2) was subjected to LC-MS analysis test. The results are as follows: Figure 7A As shown, 500 μM CO was added to a 100 mM fluorescent probe Nile-CO (containing 100 μM PdCl2) solution, and LC-MS analysis was performed. The results were as follows. Figure 7B shown.

[0073] from Figure 7A It can be seen that the chromatographic peak of the fluorescent probe Nile-CO appears at 5.31min, with a molecular weight of 402.38. Figure 7B It can be seen that after adding 500μM CO, a new chromatographic peak appeared at 5.326min, with a molecular weight of 318.25, which is consistent with the molecular weight of Nile blue. Figure 8 shown.

[0074] from Figure 8 It can be seen that in the detection process of CO by fluorescent probe Nile-CO, Pd 2+ First reduced to Pd by CO 0 , which then mediates the Tsuji-Trost reaction, leading to the cleavage of the allyl formate group and the release of the Nile blue fluorophore, thereby generating near-infrared fluorescence.

[0075] Application Test Example 1

[0076] This application test case studies the application of the fluorescent probe Nile-CO in the detection and imaging of exogenous CO in living cells.

[0077] First, HeLa cells (purchased from Procell Life Science & Technology) were used for cell culture and cytotoxicity experiments. Specifically, the cells were cultured in MEM containing 10% fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 U / mL); the cytotoxic effect of the fluorescent probe Nile-CO was determined using the MTT assay. HeLa cells (1×10 4Cells were seeded in 96-well flat-bottom plates containing 100 μL of culture medium and incubated at 37°C, 5% CO2 for 24 h. The cells were then incubated in fresh culture medium containing different concentrations of the fluorescent probe Nile-CO (0.0, 2.5, 5.0, 10.0, 20.0, and 40.0 μM) for 24 h. MTT solution (5.0 mg / mL, PBS) was then added to each well (10 μL / well, 0.5 mg / mL), and the residual MTT solution was removed after 4 h. Finally, 150 μL of DMSO was added to each well to dissolve the formazan crystals. After shaking for 10 min, the absorbance of the wells was measured by enzyme-linked immunosorbent assay at 570 nm, and the cytotoxic effect of the fluorescent probe Nile-CO was evaluated using the formula A / A0×100% (where A and A0 are the absorbance values ​​of the experimental group and the control group, respectively). The results are shown in Figure 2. Figure 9 shown.

[0078] from Figure 9 As can be seen in the figure, after HeLa cells were cultured in a solution containing 0-40 μM fluorescent probe Nile-CO for 24 hours, the cell survival rate exceeded 85%. The results show that the probe has low toxicity and can be used for in vivo cell and mouse imaging.

[0079] Next, live cell imaging experiments were performed on HeLa cells using the fluorescent probe Nile-CO dissolved in DMSO. A 20.0 mM stock solution was prepared and cells were plated at 1.8 × 10 4 Cells were seeded at a density of 10 cells / mL in 15 mm glass-bottomed cell culture dishes for confocal microscopy imaging. In the cell imaging experiments, the probe (red channel) was excited at 590 nm and emitted in the range of 603-688 nm; Hoechst 33342 (blue channel) was excited at 405 nm and emitted in the range of 425-474 nm; and Mito-Tracker Green (green channel) was excited at 499 nm and emitted in the range of 517-591 nm.

[0080] The cultured cells were divided into four groups for exogenous CO imaging experiments. First, each group of cells was incubated with 0, 5, 10, and 30 μM CORM-3 (CO donor) solutions for 30 minutes. Subsequently, each group of cells was incubated with 5 μM probe solution (Nile-CO + PdCl2, 1:1) for 30 minutes and stained with Hoechst 33342. The cells were washed 2-3 times with PBS and imaged using a confocal microscope. The results are shown in Figure 2. Figure 10 shown.

[0081] from Figure 10It can be seen that when HeLa cells were incubated with 5 μM probe solution (Nile-CO + PdCl2, 1:1) in the absence of CORM-3, a weak fluorescence signal was observed. However, after the cells were pretreated with different concentrations of CORM-3 (5, 10, and 30 μM) and then incubated with 5 μM probe solution (Nile-CO + PdCl2, 1:1), the cell fluorescence intensity continued to increase with the increase of CORM-3 concentration.

[0082] The above cultured cells were divided into four groups for the probe imaging ability test. First, each group of cells was incubated with 30μM CORM-3 solution for 30min. Then, each group of cells was incubated with 0, 2, 5 or 10μM probe solution (Nile-CO + PdCl2, 1:1) and stained with Hoechst 33342. The cells were washed 2-3 times with PBS and imaged using a confocal microscope. The results are shown in Figure 2. Figure 11 shown.

[0083] from Figure 11 It can be seen that as the concentration of the fluorescent probe Nile-CO solution increases, the fluorescence intensity of HeLa cells gradually increases.

[0084] The above results indicate that the ability of the fluorescent probe Nile-CO to detect and image exogenous CO in living cells is dose-dependent with the concentration of CO.

[0085] Application Test Example 2

[0086] This application test case studies the application of the fluorescent probe Nile-CO in the detection and imaging of endogenous CO in living cells.

[0087] It is reported that heme produces endogenous CO in living cells, so heme was introduced into HeLa cells to evaluate the ability of the fluorescent probe Nile-CO to detect endogenous CO. Specifically, the HeLa cells cultured in Application Test Example 1 were incubated with heme (100 μM) at 37 ° C for 2, 4 and 8 h, and then the treated cells were incubated with 5 μM probe solution (Nile-CO + PdCl2, 1: 1) at 37 ° C for 30 min, and HeLa cells were directly incubated with 5 μM probe solution (Nile-CO + PdCl2, 1: 1) at 37 ° C for 30 min as a control group. The above cells were then stained with Hoechst 33342, washed 2-3 times with PBS, and imaged using a confocal microscope. The results are as follows Figure 12 shown.

[0088] from Figure 12As can be seen, after the cells were pretreated with 100 μM hemoglobin and then incubated with 5 μM probe solution, the fluorescence intensity in the cells gradually increased with the extension of hemoglobin incubation time. The results showed that the fluorescent probe Nile-CO can detect and image endogenous CO in living cells.

[0089] In order to further verify the effectiveness of the fluorescent probe Nile-CO in detecting endogenous CO, an in vivo CO fluorescence tracking experiment was performed using lipopolysaccharide (LPS, which is a well-known inflammatory inducer that can stimulate the production of CO). Specifically, the HeLa cells cultured in Application Test Example 1 were incubated with LPS (1 μg / ml) at 37°C for 1, 4, and 16 hours, respectively, and then the above-treated cells were incubated with 5 μM probe solution (Nile-CO+PdCl2, 1:1) at 37°C for 30 minutes, and HeLa cells were directly incubated with 5 μM probe solution (Nile-CO+PdCl2, 1:1) at 37°C for 30 minutes as a control group. The above cells were then stained with Hoechst33342, washed 2-3 times with PBS, and imaged using a confocal microscope. The results are shown in Figure 2. Figure 13 shown.

[0090] from Figure 13 As can be seen from the figure, as the incubation time of cells with LPS increases, the red fluorescence intensity between 603-688 nm continues to increase. The results show that the fluorescent probe Nile-CO can effectively monitor the CO concentration in living cells.

[0091] In order to verify the targeting of the fluorescent probe Nile-CO to the organelles, specifically, the HeLa cells cultured in Application Test Example 1 were incubated with 5 μM probe solution (Nile-CO + PdCl2, 1:1) for 30 minutes, and then the above cells were incubated with fresh culture medium containing 5 μM mitochondrial green fluorescent probe (Mito Tracker Green) for 20 minutes. The above treated cells were washed 2-3 times with PBS and imaged and co-localized using a confocal microscope. The results are shown in Figure 2. Figure 14 shown.

[0092] from Figure 14 It can be seen that there is a high degree of overlap between the red fluorescence emitted by the fluorescent probe Nile-CO and the green fluorescence emitted by the mitochondrial green fluorescent probe, and the Pearson colocalization coefficient is as high as 0.931. The results show that the fluorescent probe Nile-CO can target mitochondria and can detect and image CO in mitochondria.

[0093] Application Test Example 3

[0094] This application test case studies the application of the fluorescent probe Nile-CO in the detection and imaging of exogenous CO in mice.

[0095] Specifically, the hair on the back of the mouse was shaved, and the probe (100 μM Nile-CO + PdCl2, 50 μL and 10 mM PBS, 50 μL) and CORM-3 (500 μM, 50 μL) were injected into the same cavity of the mice in the experimental group. Mice injected with only CORM-3 (250 μM, 50 μL and 10 mM PBS, 50 μL) and only injected with the probe (50 μM Nile-CO + PdCl2, 50 μL and 10 mM PBS, 50 μL) were used as controls, and then continuous imaging was performed for 0-60 minutes. Fluorescence imaging was performed using a 605 nm excitation filter and a 680 nm emission filter. All mice were anesthetized during the experiment. The results are shown in Figure 2. Figure 15 shown.

[0096] from Figure 15 As can be seen, mice in the experimental group showed a significant increase in fluorescence signal within 2 minutes, and the fluorescence intensity gradually increased over time. Mice in the control group, on the other hand, showed only weak fluorescence. These results demonstrate that the fluorescent probe Nile-CO has significant tissue penetration and can therefore be used for rapid CO imaging in vivo.

[0097] Application Test Example 4

[0098] This application test case studies the application of the fluorescent probe Nile-CO in the detection and imaging of endogenous CO in mice.

[0099] Specifically, lipopolysaccharide (LPS) was injected into mice at a dose of 4 mg / kg intraperitoneally to form an inflammatory model (including mice induced for 1 hour and 16 hours). All mice were anesthetized, and 100 μl of a 5 mg / kg probe (Nile-CO + PdCl2, 1:1) mixed solution was injected into the tail vein of the LPS-pretreated mice. Control mice were not injected with the probe or treated with LPS. The fluorescence signal of the inflammatory mice was monitored in real time using a 605 nm excitation filter and a 680 nm emission filter. The results are shown in Figure 2. Figure 16 shown.

[0100] from Figure 16 As can be seen from the figure, with the increase of LPS stimulation time, the fluorescence signal observed 16 hours after stimulation was twice that observed 1 hour later. The results show that the probe can accurately detect CO2-induced fluorescence fluctuations in living animals.

[0101] Furthermore, different doses (0, 2, 4 mg / kg) of lipopolysaccharide (LPS) were directly injected into the tail vein of mice, and eye blood samples were collected at different time intervals (1, 16 h). Fluorescence analysis was then performed after adding the fluorescent probe Nile-CO for 5 min. The results are shown in Figure 2. Figure 17 shown.

[0102] from Figure 17 As shown in the figure, ocular blood samples collected one hour after intravenous injection of 2 mg / kg LPS exhibited significant fluorescence emission, and the fluorescence intensity increased twofold within 16 hours of LPS injection. Furthermore, increasing the LPS concentration from 2 mg / kg to 4 mg / kg increased the ocular blood fluorescence intensity by one-third at 16 hours. These results demonstrate that blood-based monitoring can assess CO fluctuations in vivo, thus promoting the practical application of the fluorescent probe Nile-CO.

[0103] In summary, the present invention provides a near-infrared fluorescent probe for carbon monoxide. This carbon monoxide near-infrared fluorescent probe has good photostability, a short response time to CO, high selectivity, a low detection limit, and high sensitivity. Therefore, it has good application prospects in the detection and imaging of CO in living cells and subjects.

[0104] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For any details not described in individual embodiments, reference may be made to the description in other embodiments.

[0105] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A carbon monoxide near-infrared fluorescent probe, characterized in that It has the structure shown in the following formula (1): 。 2. A method for preparing a carbon monoxide near-infrared fluorescent probe according to claim 1, characterized in that: The steps include: Nile blue is mixed with triethylamine and allyl chloroformate and then subjected to a chemical reaction. The obtained product is separated and purified to obtain a carbon monoxide near-infrared fluorescent probe.

3. The preparation method according to claim 2, characterized in that The molar ratio of Nile blue, triethylamine and allyl chloroformate is (0.3-0.7): (1.5-2): (0.5-0.8).

4. The preparation method according to claim 2, characterized in that The chemical reaction specifically comprises: stirring the reaction for 10-14 hours at a temperature of 20-25° C. under an inert atmosphere.

5. Use of the carbon monoxide near-infrared fluorescent probe according to claim 1 in the detection and / or imaging of CO in living cells for non-disease diagnosis and treatment purposes.

6. The use according to claim 5, characterized in that CO includes exogenous CO and endogenous CO.

7. The use according to claim 6, characterized in that Endogenous CO is produced by transferring hemoglobin into living cells or is induced by transferring lipopolysaccharide into living cells.

8. Use of the carbon monoxide near-infrared fluorescent probe according to claim 1 in the detection and / or imaging of CO in a subject for non-disease diagnosis and treatment purposes.

9. The use according to claim 8, characterized in that CO includes exogenous CO and endogenous CO; and endogenous CO is induced by injecting lipopolysaccharide into the subject's body.

10. The use according to claim 8 or 9, characterized in that: The subject includes a mouse.

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