Carbon monoxide near-infrared fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a carbon monoxide near-infrared fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of fluorescent probes. The carbon monoxide near-infrared fluorescent probe provided by the invention has relatively good light stability, short response time to CO, relatively high selectivity, low detection limit and high sensitivity, and the fluorescent probe has targeting property to mitochondria and can be used for detecting and imaging CO in the mitochondria; besides, the fluorescent probe can also be used for detecting and imaging CO in living cells and subjects, and especially can be used for directly detecting CO fluctuation of eyeball blood samples of a lipopolysaccharide-induced mouse inflammation model, so that the fluorescent probe has a relatively good application prospect.

Description

Technical Field

[0001] The 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 can act 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 biological imaging 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 naphthalimide dyes, which can rapidly detect exogenous and endogenous CO in the ER through the reduction reaction of the nitro group with an amine. 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 substantial interference from intrinsic biofluorescence. These limitations have greatly restricted the application of optical imaging in biological tissues. In contrast, near-infrared fluorescent probes are known for minimizing photodamage in biological samples, facilitating deep tissue penetration, and minimal interference from biomolecular autofluorescence, making them well suited 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 purpose of the present invention is 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 as 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 groups are used as recognition units. 2+ ) When in contact with CO, Pd 2+ First, it is reduced to Pd by CO 0 , and then mediates the Tsuji-Trost reaction, resulting in the breakage of the allyl formate, releasing 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 subjecting the mixture to 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 the 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, the 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 the 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: different from 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, and 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), especially can directly detect CO fluctuations in eye blood samples of lipopolysaccharide-induced mouse inflammation models, therefore, the fluorescent probe has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0021] Figure 3 is the 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 10 μM fluorescent probe Nile-CO after the reaction with 100 μM CO and 10 μM PdCl2 in the present invention;

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

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

[0025] Figure 5B This is the fluorescence spectrum of 10 μM fluorescent probe Nile-CO after reacting with 100 μM analyte and 10 μM PdCl2 in the present invention. Each data point is obtained 30 minutes after adding the analyte and 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 result diagram of the fluorescent probe Nile-CO detecting CO in the solution system of 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 when the excitation wavelength is 580 nm in the present invention;

[0028] Figure 6 The linear relationship between the fluorescence intensity change of the fluorescent probe Nile-CO at 664 m and the CO concentration in the present invention is shown in Figure 1. Each data point is obtained 30 min after adding the analyte and repeated three times. ex =580nm;

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

[0030] Figure 7B This is a graph showing the results of LC-MS analysis of a 100 mM fluorescent probe Nile-CO solution (containing 100 μM PdCl2) after adding 500 μM CO;

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

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

[0033] Fig.10 This is a cell imaging picture of HeLa cells in the present invention after being incubated with 0, 5, 10, and 30 μM CO for 30 minutes and then incubated with 5 μM probe solution (Nile-CO+PdCl2, 1:1) for 30 minutes;

[0034] Fig.11The cell imaging images of HeLa cells in the present invention are 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] Fig.12 The cell imaging images of HeLa cells in the present invention after incubation with 100 μM heme for 2, 4, and 8 hours and then incubation with 5 μM probe solution (Nile-CO+PdCl2, 1:1) for 30 minutes;

[0036] Fig.13 The cell imaging images of HeLa cells in the present invention are first 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] Fig.14 This is a result diagram 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] Fig.15 This is the imaging picture of the mouse 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] Fig.16 This is an imaging image of mice injected with 4 mg / kg LPS intraperitoneally for 1h and 16h, and then injected with 5 mg / kg probe (Nile-CO+PdCl2, 1:1) via the tail vein in the present invention;

[0040] Fig.17 This is a graph showing the results of administering 0, 2, and 4 mg / kg LPS to the tail vein of mice for 1 h and 16 h, 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 described embodiments 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 creative work are within the scope of protection of the present invention.

[0042] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels 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 a N2 atmosphere. The resulting solution was quenched with water and extracted three times with dichloromethane. The combined organic layer was dried over anhydrous sodium sulfate and concentrated using a rotary evaporator. The crude mixture was further purified by column chromatography (dichloromethane / methanol (100: 1-10: 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 spectrum and fluorescence spectrum 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 a temperature of 24°C. The results are as follows: Figure 4A and 4B shown.

[0054] from Figure 4A As can be seen in the figure, the main absorption band of the fluorescent probe Nile-CO (10μM) is located at 534nm. After adding 10μM PdCl2, no significant changes were observed in the absorption spectrum. However, when 100μM CO (with CORM-3 as the CO source) and 10μM PdCl2 were added at the same time, the absorption band at 534nm disappeared significantly, red-shifted, and a new significantly enhanced absorption band appeared at 628nm.

[0055] from Figure 4B It can be seen that due to the quenching effect of allyl formate, after adding 10μM PdCl2, the fluorescent probe Nile-CO has no obvious near-infrared emission peak in the near infrared (664nm); after adding 100μM CO and 10μM PdCl2 at the same time, an obvious near-infrared emission peak appears at 664nm. This is because Pd 2+ First, it is reduced to Pd by CO 0 , which subsequently mediates the Tsuji-Trost reaction, leading to the cleavage of the allyl formate group and the release of the Nile blue fluorophore, resulting in 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 It can be seen that the fluorescence intensity increases slowly within 1 minute, then increases rapidly within 5 minutes and reaches the saturated fluorescence intensity. The results show that the fluorescent probe Nile-CO has a rapid response ability to 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 5B shown.

[0061] from Figure 5B It can be seen that only after adding CO, the fluorescence intensity can be significantly enhanced. After adding 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 did not increase significantly in various pH solutions. However, after the addition of CO, the probe was significantly enhanced in the pH range of 6-9, and the fluorescence intensity was the highest at pH 7-8. This is very beneficial for the detection and bioimaging applications of CO.

[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 It can be seen that 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 1h of excitation, it maintains 90% of its initial fluorescence intensity. The results show 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, after adding 10 μM PdCl2 and different concentrations (0-35 μM) of CO to a 10 μM fluorescent probe Nile-CO solution, the fluorescence intensity (emission wavelength is 664 nm) was measured, and a linear equation was established with the fluorescence intensity as the ordinate and the CO concentration as the abscissa. The results are as follows: Figure 6 shown.

[0069] from Figure 6 It can be seen that 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, 100 mM fluorescent probe Nile-CO solution (containing 100 μM PdCl2) was subjected to LC-MS analysis test. The results are as follows: Fig. 7A As shown, 500 μM CO was then added to a 100 mM fluorescent probe Nile-CO (containing 100 μM PdCl2) solution, and the LC-MS analysis test was continued. The results are shown in Figure 7B shown.

[0073] from Fig. 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, and its molecular weight was 318.25, which is consistent with the molecular weight result of Nile blue. Figure 8 shown.

[0074] from Figure 8 As can be seen in the detection process of CO by fluorescent probe Nile-CO, Pd 2+ First, it is reduced to Pd by CO 0 , which subsequently mediates the Tsuji-Trost reaction, leading to the cleavage of the allyl formate group and the release of the Nile blue fluorophore, resulting in 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 method. HeLa cells (1×10 4Cells / well) were seeded in a 96-well flat-bottom plate 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 fluorescent probe Nile-CO concentrations (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 DMSO was added to each well to dissolve formazan crystals; after shaking for 10 min, the absorbance value of the well was measured and read at 570 nm using enzyme-linked immunosorbent assay, 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 FIG. Fig. 9 shown.

[0078] from Fig. 9 As can be seen, 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 showed 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 The density of cells / mL was inoculated in a 15mm glass-bottomed cell culture dish for confocal microscopy cell imaging. In the cell imaging experiment, the probe (red channel) was excited at 590nm and emitted in the range of 603-688nm; Hoechst33342 (blue channel) was excited at 405nm and emitted in the range of 425-474nm; Mito-Tracker Green (green channel) was excited at 499nm and emitted in the range of 517-591nm.

[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 min. Subsequently, each group of cells was incubated with 5 μM probe solution (Nile-CO + PdCl2, 1:1) for 30 min 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. Fig.10 shown.

[0081] from Fig.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 experiment. 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 Fig.11 shown.

[0083] from Fig.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 shown in Figure 2. Fig.12 shown.

[0088] from Fig.12As can be seen, after the cells were pretreated with 100 μM heme and then incubated with 5 μM probe solution, the fluorescence intensity in the cells gradually increased with the extension of heme incubation time. The results show 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, 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 h, respectively, and then the above-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 Hoechst33342, washed 2-3 times with PBS, and imaged using a confocal microscope. The results are shown in Figure 2. Fig.13 shown.

[0090] from Fig.13 It can be seen that as the incubation time of cells with LPS increases, the red fluorescence intensity between 603-688nm 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 min, and then the above cells were incubated with fresh culture medium containing 5 μM mitochondrial green fluorescent probe (Mito Tracker Green) for 20 min. The above treated cells were washed 2-3 times with PBS, and imaged and co-localized using a confocal microscope. The results are as follows: Fig.14 shown.

[0092] from Fig.14 It can be seen that there is a high 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, and 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 imaged continuously for 0-60 min. 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. Fig.15 shown.

[0096] from Fig.15 As can be seen in the figure, the mice in the experimental group showed obvious fluorescence signal enhancement at 2 minutes, and the fluorescence intensity gradually increased over time. The mice in the control group showed only weak fluorescence. The results show that the fluorescent probe Nile-CO has significant tissue penetration ability and can therefore be used for rapid imaging of CO 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 h and 16 h), all the mice were anesthetized, and 100 μl of a mixed solution of 5 mg / kg probe (Nile-CO+PdCl2, 1:1) was injected into the tail vein of the mice pretreated with LPS. The control group 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. Fig.16 shown.

[0100] from Fig.16 It can be seen that with the increase of LPS stimulation time, the fluorescence signal observed 16 hours after stimulation is 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). Then, fluorescence analysis was performed after adding the fluorescent probe Nile-CO for 5 min. The results are shown in Fig.17 shown.

[0102] from Fig.17 As can be seen in the figure, after intravenous injection of 2 mg / kg LPS, the ocular blood samples collected 1 hour later showed obvious fluorescence emission, and the fluorescence intensity increased by 2 times within 16 hours of LPS injection. In addition, increasing the LPS concentration from 2 mg / kg to 4 mg / kg increased the fluorescence intensity of the ocular blood by one third at 16 hours. The above results show that blood-based monitoring can evaluate the fluctuation of CO in the body, thereby 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, which 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 matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0105] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached 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 as claimed in claim 1, characterized in that: The steps include: Nile blue is mixed with triethylamine and allyl chloroformate to carry out a chemical reaction, and 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 step specifically includes: 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 as claimed in claim 1 in the detection and / or imaging of CO in living cells.

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 heme into living cells, or is induced by transferring lipopolysaccharide into living cells.

8. Use of the carbon monoxide near-infrared fluorescent probe as claimed in claim 1 in the detection and / or imaging of CO in a subject.

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.

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

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