A fluorescent probe, preparation method and biological application thereof

Abstract

The present invention discloses a fluorescent probe, a preparation method, and its biological application. The fluorescent probe of the present invention has a simple synthesis method, inexpensive raw materials, and is easily reproducible. It can achieve real-time imaging of lysosomes in living cells, has high specificity, is localized in lysosomes, is insensitive to pH and polarity, has strong resistance to photobleaching, high biocompatibility, and low cytotoxicity. The present invention relates to the field of material synthesis and biological imaging analysis technology. The fluorescent probe, preparation method, and its biological application have lysosomal targeting specificity, are insensitive to pH and polarity, have strong resistance to photobleaching, high biocompatibility, and are essentially non-toxic to cells. Therefore, the fluorescent probe of the present invention is particularly suitable for real-time imaging of lysosomes in living cells.

Description

Technical Field

[0001] The present invention relates to the technical field of material synthesis and biological imaging analysis, and in particular to a fluorescent probe for locating lysosomes, a preparation method thereof, and biological applications thereof. Background Art

[0002] Cellular organelles enable the spatial clustering of molecules, facilitating their interactions within specialized, complex functional microenvironments. A well-known function of lysosomes is the degradation and recycling of cellular waste products. Extracellular material reaches lysosomes primarily through endocytosis and phagocytosis. Lysosomal hydrolases degrade these substances, and the resulting breakdown products are used to generate new cellular components and energy in response to the cell's nutritional needs. Lysosomes also participate in an "unconventional" secretory pathway known as lysosomal exocytosis, which plays an important role in various physiological processes, including plasma membrane repair, immune responses, and bone resorption. The lysosomal lumen has an acidic pH close to 4.5 and contains approximately 60 different soluble hydrolases that are directly involved in the degradation of metabolites. The lysosomal membrane contains proteins such as transporters, ion channels, and SNAREs that mediate various aspects of lysosomal function, as well as the vATPase complex that mediates lysosomal acidification. Notably, lysosomes have also been implicated in a variety of common diseases, such as neurodegenerative diseases, infections, obesity, and cancer. Therefore, lysosomes are considered to be important targets for disease diagnosis and treatment, and real-time imaging analysis of their fine structural changes is of great significance for the diagnosis and treatment of lysosomal stress-related diseases.

[0003] Currently, organic small molecule imaging has become a hot research topic due to its high biocompatibility and strong resistance to photobleaching, allowing for real-time monitoring of intracellular reactions. It has been applied in many fields, including cell imaging, chemical sensing, and photodynamic therapy. Although various fluorescent probes have been reported for observing lysosomal morphology and detecting their components, the pH stability, photobleaching resistance, and biocompatibility of the currently developed lysosomal fluorescent probes still need to be improved. Therefore, the development of fluorescent probes for real-time imaging of lysosomes in living cells is of great importance. Summary of the Invention

[0004] (1) Technical problems solved

[0005] In response to the deficiencies of the prior art, the present invention provides a fluorescent probe, a preparation method, and its biological application, which solve the technical problems of existing fluorescent probes such as poor resistance to photobleaching, low biocompatibility, and unstable polarity. The present invention provides a fluorescent probe for real-time imaging of lysosomes in living cells, a preparation method of the fluorescent probe, and the application of the fluorescent probe in biology.

[0006] (2) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: A fluorescent probe, whose chemical structure is as follows:

[0008]

[0009] In one aspect, the present invention further provides a method for preparing a fluorescent probe, which specifically comprises the following steps:

[0010] S1. Add 1-phenylbutane-1,3-dione and boron trifluoride into a round-bottom flask, use toluene as solvent, and react in a water bath to obtain a white precipitate, namely, intermediate 1;

[0011] S2 and intermediate 1 were mixed with 6-hydroxy-2-naphthaldehyde and added to a 100 mL three-necked round-bottom flask. The mixture was evacuated and nitrogen-filled three times using a double-row tube, and a nitrogen balloon was inserted. Piperidine was added using a syringe under nitrogen, followed by the addition of 40 mL of toluene solvent using a syringe. The mixture was nitrogen-filled for 30 minutes, and the reaction apparatus was placed in a water bath and heated under reflux to obtain a red precipitate, namely the fluorescent probe.

[0012] S3, filtering the red precipitate obtained in step S2, and washing it with ethanol several times to obtain a red solid;

[0013] S4. Dissolve the red solid obtained in step S3 with dichloromethane, purify by column chromatography to obtain a red solution, concentrate the red solution by rotary evaporation, and vacuum dry to obtain a red solid powder, i.e., the fluorescent probe.

[0014] Preferably, in step S1, the molar ratio of 1-phenylbutane-1,3-dione to boron trifluoride is 1:1, the reaction temperature is 80° C., and the reaction time is 10 hours.

[0015] Preferably, in step S2, the molar ratio of intermediate 1 to 6-hydroxy-2-naphthaldehyde is 1.5:1, the reaction temperature is 75° C., and the reaction time is 17 hours.

[0016] Preferably, the eluate during column chromatography purification in step S4 is a mixture of petroleum ether and ethyl acetate, and the volume ratio is: V 石油醚 :V 乙酸乙酯 =2:1.

[0017] Preferably, the red precipitate obtained in step S3 is cooled to room temperature and washed with ethanol 2-3 times during filtration to obtain a red solid.

[0018] Preferably, in step S2, the amount of piperidine added using a syringe under a nitrogen environment is 0.16 g, 1.86 mmol.

[0019] On the other hand, the present invention also provides an application of a fluorescent probe prepared by the method for preparing a fluorescent probe in real-time imaging of lysosomes in living cells.

[0020] (3) Beneficial effects

[0021] The present invention provides a fluorescent probe, preparation method, and biological application thereof. Compared with the prior art, it has the following advantages:

[0022] (1) The fluorescent probe, preparation method and biological application thereof: The fluorescent probe of the present invention has a simple synthesis method, cheap raw materials and is easy to repeat; it can realize real-time imaging of lysosomes in living cells.

[0023] (2) The fluorescent probe, preparation method and biological application thereof have high specificity, are localized in cell lysosomes, are insensitive to pH and polarity, have strong resistance to photobleaching, high biocompatibility and low cytotoxicity.

[0024] (3) The fluorescent probe, preparation method, and biological application thereof have lysosomal targeting specificity, are insensitive to pH and polarity, have strong resistance to photobleaching, are highly biocompatible, and are essentially non-toxic to cells. Therefore, the fluorescent probe of the present invention is particularly suitable for real-time imaging of lysosomes in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the synthesis of the fluorescent probe of the present invention;

[0026] Figure 2 is the mass spectrum of the fluorescent probe of Example 1 of the present invention;

[0027] Figure 3 Schematic diagram of the UV-visible spectrum and excitation-emission spectrum of the fluorescent probe in Example 4 of the present invention in PBS buffer (0.01 M, pH = 7.4);

[0028] Figure 4 Schematic diagram of UV-visible spectra and excitation-emission spectra of the fluorescent probe in Example 5 of the present invention in solvents of different polarities;

[0029] Figure 5 Schematic diagram of UV-visible spectra and excitation-emission spectra of the fluorescent probe at different pH values in Example 5 of the present invention;

[0030] Figure 6 is the ratio of the fluorescence intensity of the fluorescent probe at different times to the fluorescence intensity at the initial time under 460 nm laser excitation in Example 5 of the present invention;

[0031] Figure 7This is a schematic diagram of the selective analysis of various analytical substrates contained in cells by the fluorescent probe in Example 6 of the present invention: 1.1mM Cu2SO4, 2.1mM H2S, 3.1mM FeCl3, 4.1mM FeCl2, 5.1mM ZnCl2, 6.1mM MgCl2, 7.1mM NaCl, 8.1mM CaCl2, 9.1mM KCl, 10. Glutathione, 11. Threonine, 12. Cysteine, 13. Glycine, 14. Serine, 15. Glucose, 16. RNA.

[0032] Figure 8 This is a schematic diagram of the fluorescent probe cytotoxicity analysis in Example 7 of the present invention;

[0033] Figure 9 This is a schematic diagram of real-time imaging of the fluorescent probe entering cells in Example 7 of the present invention;

[0034] Figure 10 This is a schematic diagram of real-time imaging of fluorescent probes of different concentrations entering cells in Example 7 of the present invention;

[0035] Figure 11 Schematic diagram of co-localization imaging analysis of fluorescent probes in HeLa cells in Example 7 of the present invention;

[0036] Figure 12 This is the hydrogen spectrum of the fluorescent probe of Example 1 of the present invention. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0038] The raw materials in the following examples are described:

[0039] 1-Phenylbutane-1,3-dione, boron trifluoride, 6-hydroxy-2-naphthaldehyde, toluene, ethanol, piperidine, PBS buffer (0.01 M, pH = 7.4), dimethyl sulfoxide, methanol, dimethylformamide, tetrahydrofuran, dichloromethane, petroleum ether, ethyl acetate, Cu2SO4, H2S, FeCl3, FeCl2, ZnCl2, MgCl2, NaCl, CaCl2, and KCl were all purchased from Aladdin Reagent Shanghai Co., Ltd.

[0040] Glutathione, threonine, cysteine, glycine, serine, glucose, and RNA were purchased from Sigma-Aldrich Shanghai Trading Co., Ltd.

[0041] See also Figure 1-12 , a fluorescent probe, preparation method and biological application thereof specifically include the following embodiments:

[0042] Example 1: Preparation of fluorescent probe

[0043] The fluorescent probe of the present invention is a fluorescent probe for real-time imaging of lysosomes in living cells. The fluorescent probe is obtained by reacting 1-phenylbutane-1,3-dione, boron trifluoride, and 6-hydroxy-2-naphthaldehyde as raw materials. The specific preparation process is shown in Example 2.

[0044] The fluorescent probe of the present invention has lysosomal targeting specificity, strong resistance to photobleaching, essentially no toxicity to cells, high biocompatibility, and insensitivity to pH and polarity. Therefore, the fluorescent probe of the present invention is particularly suitable for real-time imaging of lysosomes in living cells.

[0045] Example 2: Preparation of fluorescent probe

[0046] (1) Please refer to Figure 1 , which is a schematic diagram of the synthesis of the fluorescent probe of the present invention. The preparation method of the fluorescent probe comprises the following steps:

[0047] 1-Phenylbutane-1,3-dione and boron trifluoride were added to a round-bottom flask and heated in a water bath using toluene as the solvent to produce a white precipitate, Intermediate 1. Intermediate 1 was then mixed with 6-hydroxy-2-naphthaldehyde, and piperidine (0.16 g, 1.86 mmol) was added via syringe under a nitrogen atmosphere, followed by the addition of 40 mL of toluene solvent. The reaction was then purged with nitrogen for 30 minutes, and the reaction apparatus was placed in a water bath and heated under reflux to produce a red precipitate, the fluorescent probe.

[0048] The molar ratio of 1-phenylbutane-1,3-dione to boron trifluoride is 1: 1. The reaction is carried out in a water bath, the reaction temperature is preferably 80° C., and the reaction time is preferably 10 hours.

[0049] The molar ratio of intermediate 1 to 6-hydroxy-2-naphthaldehyde is 1.5:1. For example, if 0.2 g of 6-hydroxy-2-naphthaldehyde is used, 0.37 g of intermediate 1 is required. The reaction is carried out in a water bath at a preferred temperature of 75°C and a preferred reaction time of 17 hours.

[0050] (2) The red precipitate obtained in step (1) was filtered and washed with ethanol several times to obtain a red solid.

[0051] (3) The red solid obtained in step (2) is dissolved in dichloromethane and purified by column chromatography to obtain a red solution. The obtained orange solution is concentrated by rotary evaporation and vacuum dried to obtain a red solid powder, namely the fluorescent probe.

[0052] During the column chromatography purification process, the eluent was a mixture of petroleum ether and ethyl acetate (V 石油醚 :V 乙酸乙酯 =2:1)

[0053] Example 3: Preparation of fluorescent probe

[0054] The preparation method of the fluorescent probe of Example 2 is described in detail with examples in this example.

[0055] 0.2g 1-phenylbutane-1,3-dione and 0.082g boron trifluoride were added to a round-bottom flask with toluene as solvent. The mixture was heated in a water bath for 10 hours to obtain a white precipitate. After cooling to room temperature, the white precipitate was filtered to obtain intermediate product 1. 0.37g intermediate 1 was then mixed with 0.2g 6-hydroxy-2-naphthaldehyde. Piperidine (0.16g, 1.86mmol) was added using a syringe under a nitrogen atmosphere. 40mL of solvent toluene was then added using a syringe. Nitrogen was purged for 30 minutes, and a double-row tube was used to evacuate and purify the mixture three times, and a nitrogen balloon was inserted. The reaction apparatus was placed in a water bath and heated under reflux for 17 hours to obtain a red precipitate, which was the fluorescent probe. The red precipitate was naturally cooled to room temperature, filtered, and the filter cake was washed with ethanol several times to obtain a red solid. The red solid obtained by filtration was dissolved in dichloromethane and purified by column chromatography. The eluate was a mixture of petroleum ether and ethyl acetate (V 石油醚 :V 乙酸乙酯 =2:1) to obtain a red solution, which was concentrated by rotary evaporation and then vacuum dried to obtain a red solid powder, namely the fluorescent probe.

[0056] The characterization of the fluorescent probe prepared in this example can be found in Figure 2 and Figure 12

[0057] Example 4: UV-visible spectra and excitation-emission spectra of fluorescent probes

[0058] See also Figure 3 ,like Figure 3 As shown in a, the fluorescent probe has a maximum absorption peak at 465nm in PBS buffer (0.01M, pH=7.4). Figure 3 b, When the excitation wavelength is 460 nm in PBS buffer, the fluorescent probe has the maximum fluorescence intensity at 650 nm.

[0059] Example 5:

[0060] In this embodiment, various investigations were conducted on the fluorescent probes of the above embodiments.

[0061] 1. Investigation of the polarity response of fluorescent probes

[0062] 20 μL of 10 -3 M small molecule fluorescent probe was prepared to obtain solutions containing the same concentration (10μM) of the fluorescent probe in solvents of different polarity. Refer to 4a. In the absorption range of 300nm-700nm, the UV-visible absorption spectrum of the small molecule fluorescent probe does not show any solvent polarity-dependent changes, indicating that the small molecule fluorescent probe has no obvious solvation effect and is a polarity-insensitive molecule. This also further proves that the fluorescent probe is a very stable small molecule. Refer to Figure 4 b. When excited at 460 nm, the small molecule fluorescent probe exhibited stronger fluorescence intensities in various solvents than in PBS buffer. However, the fluorescence emission spectrum did not shift with changes in solvent polarity, demonstrating no polarity dependence, further corroborating the UV-Vis absorption spectroscopy results. Solvent effects determined by UV-Vis absorption and fluorescence spectroscopy demonstrate that the small molecule fluorescent probe is relatively stable and has minimal solvent effects.

[0063] 2. Investigation of the response of fluorescent probes to different pH values

[0064] Different pH buffers (pH4, pH4.5, pH5, pH5.5, pH6, pH6.5, pH7, pH7.5, pH8, pH8.5, pH9, pH9.5, pH10) were prepared by mixing sodium hydroxide solution and sulfuric acid solution in different proportions. 2 mL of buffers with different pH values were added to 20 μL of 10 -3 M small molecule fluorescent probe, prepared at different pH containing the same concentration (10μM) of small molecule fluorescent probe solutions. Figure 5 a. The UV-visible absorption spectra of the small molecule fluorescent probe at different pH values do not show any pH-dependent changes, indicating that the small molecule fluorescent probe has no obvious pH effect and is a pH-insensitive molecule. This also further proves that the fluorescent probe is a small molecule with good stability. Figure 5 b. At room temperature, using a 460nm laser to excite the probe, we can see that the absorption intensity of the small molecule fluorescent probe varies at different pH values, but does not show polarity dependence, further corroborating the results of UV-visible absorption spectroscopy. The pH effect of UV-visible absorption and fluorescence spectroscopy indicates that the small molecule fluorescent probe is relatively stable and has no significant pH effect.

[0065] 3. Photostability Experiment of Fluorescent Probe

[0066] See also Figure 6, Figure 6 It is the ratio of the fluorescence intensity of the small molecule probe after continuous ultraviolet light (365nm) irradiation for 5min, 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, and 100min to the initial fluorescence intensity of the probe. There is no obvious decrease in the fluorescence intensity, which indicates that the small molecule fluorescent probe has excellent photostability.

[0067] Example 6: Probe selectivity exploration

[0068] Fluorescence response of the fluorescent probe prepared in Test Example 1 to common substances in organisms

[0069] To test the selectivity of the fluorescent probe for intracellular biomolecules such as inorganic salts, amino acids, peptides, and other proteins or enzymes, the fluorescent probe (10 μM) was added to PBS buffer (0.01 M, pH 7.4). Then, solutions of common intracellular biomolecules such as Cu2SO4, H2S, FeCl3, FeCl2, ZnCl2, MgCl2, NaCl, CaCl2, KCl, glutathione, threonine, cysteine, glycine, serine, glucose, and RNA were added to measure the changes in fluorescence intensity after reaction with the probe. Please refer to the reference page. Figure 7 ,Depend on Figure 7 It can be seen that under 460nm excitation, the fluorescence intensity of the fluorescent probe does not change significantly in PBS buffer solutions (0.01M, pH 7.4) containing different biomolecules, indicating that the probe has good stability in vivo.

[0070] Example 7: Application of probes in cell imaging

[0071] 1. Cytotoxicity Analysis

[0072] Mouse embryonic fibroblasts (3T3-L1) and human cervical cancer cells (HeLa) were plated at 1×10 4 Cells were seeded into a 96-well plate and placed in a cell culture incubator under the following culture conditions: 37°C, 5% CO2, saturated humidity, and cultured for 24 hours to allow them to completely adhere to the wall. Fresh culture medium was then replaced, and 10 μL of fluorescent probe dispersion at different concentrations was added. After 24 hours of culture, MTT solution (5 mg / mL) was added to each well and incubated for another 4 hours. Then, 100 μL of DMSO was added to each well, and the 96-well plate was placed on a horizontal shaking shaker for 10 minutes. The wavelength was set to 492 nm on the microplate reader, and the absorbance (OD value) of the solution in each well of the 96-well plate was measured. The cell survival rate was calculated according to the following formula: Cell survival rate = (OD 待测组 -OD 空白组 ) / (OD 细胞组 -OD空白组 ) × 100%. Please refer to Figure 8 ,from Figure 8 As can be seen from the figure, the fluorescent probe has almost no cytotoxicity in the range of 0-60 μM.

[0073] 2. Real-time imaging of the fluorescent probe entering the cell.

[0074] Human cervical cancer cells (HeLa) were seeded into glass-bottomed culture dishes and cultured for 2 days at 37°C, 5% CO2, and saturated humidity. Subsequently, the probe (5 μM) was added to the cells and imaged using a laser confocal microscope. Figure 9 As shown, when the excitation wavelength is 470nm and the emission wavelength is 490nm-650nm, the fluorescent probe begins to enter the cell within one minute, and the fluorescence intensity reaches the highest after 30 minutes.

[0075] 3. Real-time imaging of different concentrations of fluorescent probes entering cells.

[0076] Human cervical cancer cells (HeLa) were seeded into glass-bottomed culture dishes and cultured for 2 days at 37°C, 5% CO2, and saturated humidity. Subsequently, small molecule fluorescent probes of different concentrations (0.5μM, 1μM, 2μM, 4μM, and 5μM) were added to the HeLa cells, incubated in an incubator for 30 minutes, and then imaged using a laser confocal microscope. Figure 10 As shown, when the excitation wavelength is 470nm and the emission wavelength is 490nm-650nm, there is weak fluorescence in the cells when the concentration of the fluorescent probe is 0.5μM. As the concentration of the probe increases, the fluorescence intensity gradually increases.

[0077] IV. Intracellular Colocalization Imaging

[0078] In the colocalization experiment, human cervical cancer cells (HeLa) were incubated with fluorescent probes (5 μM excitation wavelength 470 nm, emission wavelength 490-590) for 30 minutes, and then incubated with 1 μM lysosomal stain (Lyso-Tracker Red DND-99 excitation wavelength 577 nm, emission wavelength 590 nm-630 nm) and 1 μM mitochondrial stain (Mito-Tracker Deep Red excitation wavelength 644 nm, emission wavelength 660 nm-690 nm) reagents for 10 minutes. Finally, the cells were washed twice with PBS buffer solution (0.01 M pH 7.4) and imaged using a laser confocal microscope. The results are shown in Figure 2. Figure 11 As shown in the figure, the Pearson overlap coefficient of the green channel of the fluorescent probe and the red channel of the lysosome commercial dye is 88.82%, and the Pearson overlap coefficient of the fluorescent probe with the mitochondria is 62%, which proves that it is well localized in the lysosome organelles.

[0079] In summary, the fluorescent probe of the present invention is a method for real-time imaging of lysosomes in living cells. The fluorescent probe of the present invention can selectively localize to lysosomes in cells. The fluorescent probe of the present invention overcomes the photobleaching of conventional lysosomal probes and the serious cytotoxicity, biocompatibility, polarity sensitivity, and pH sensitivity associated with other lysosomal fluorescent probe imaging methods.

[0080] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0081] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0082] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A fluorescent probe, characterized in that: Its chemical structure is as follows: 。 2. The method for preparing a fluorescent probe according to claim 1, wherein: The specific steps include: S1. Add 1-phenylbutane-1,3-dione and boron trifluoride into a round-bottom flask, use toluene as solvent, and react under heating in a water bath to obtain a white precipitate, namely, intermediate 1; S2 and intermediate 1 were mixed with 6-hydroxy-2-naphthaldehyde and added to a 100 mL three-necked round-bottom flask. The mixture was evacuated and nitrogen-filled three times using a double-row tube, and a nitrogen balloon was inserted. Piperidine was added using a syringe under nitrogen, followed by the addition of 40 mL of toluene solvent using a syringe. The mixture was nitrogen-filled for 30 minutes, and the reaction apparatus was placed in a water bath and heated under reflux to obtain a red precipitate, namely the fluorescent probe. S3, filtering the red precipitate obtained in step S2, and washing it with ethanol several times to obtain a red solid; S4. Dissolve the red solid obtained in step S3 with dichloromethane, purify by column chromatography to obtain a red solution, concentrate the red solution by rotary evaporation, and vacuum dry to obtain a red solid powder, i.e., the fluorescent probe.

3. The method for preparing a fluorescent probe according to claim 2, wherein: In step S1, the molar ratio of 1-phenylbutane-1,3-dione to boron trifluoride is 1:1, the reaction temperature is 80° C., and the reaction time is 10 hours.

4. The method for preparing a fluorescent probe according to claim 2, wherein: In step S2, the molar ratio of the intermediate 1 to 6-hydroxy-2-naphthaldehyde is 1.5:1, the reaction temperature is 75° C., and the reaction time is 17 hours.

5. The method for preparing a fluorescent probe according to claim 2, wherein: The eluate during column chromatography purification in step S4 is a mixture of petroleum ether and ethyl acetate, and the volume ratio is: V 石油醚 :V 乙酸乙酯 =2:

1.

6. The method for preparing a fluorescent probe according to claim 2, wherein: The red precipitate obtained in step S3 is cooled to room temperature and washed with ethanol 2-3 times during filtration to obtain a red solid.

7. The method for preparing a fluorescent probe according to claim 2, wherein: In the step S2, 0.16 g (1.86 mmol) of piperidine was added using a syringe under nitrogen.

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