Phenothiazine-based near-infrared fluorescent carbon dots and their preparation method and application

Abstract

The present invention provides phenothiazine-based near-infrared fluorescent carbon dots and their preparation methods and applications. A method for preparing phenothiazine-based near-infrared fluorescent carbon dots comprises the following steps: (1) completely dissolving phenothiazine in an acetic acid solution, reacting at 160-220° C. for 5-12 hours, and then cooling to room temperature to obtain a reaction solution; (2) filtering the reaction solution, neutralizing it to neutrality, extracting it with ethyl acetate, and after layering, taking the organic phase and performing rotary evaporation. The obtained solid powder is separated and purified to obtain the phenothiazine-based near-infrared fluorescent carbon dots. The phenothiazine-based near-infrared fluorescent carbon dots of the present invention and their preparation methods and applications have a simple synthesis process and can obtain near-infrared fluorescent carbon dots with good water dispersibility, uniform particle size, stable structure, low agglomeration, good biocompatibility, and different functions.

Description

Technical Field

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

[0002] Fluorescent carbon dots have demonstrated tremendous potential in biomedicine, imaging, therapy, antibacterial, and antimicrobial applications due to their unique photostability, high yield, low toxicity, good biocompatibility, and high cost-effectiveness. Near-infrared fluorescent carbon dots exhibit low scattering and absorption in biological tissues, resulting in less phototoxicity to cells and tissues. Therefore, they can achieve deeper tissue penetration, facilitating in situ imaging, labeling, and phototherapy of living organisms.

[0003] At present, the common method to obtain near-infrared emitting fluorescent carbon dots is to adjust the fluorescent groups in carbon dots by selecting precursors, doping with heteroatoms and controlling reaction conditions. Previous studies have shown that for compounds containing aromatic rings and heteroaromatic rings, the more aromatic rings there are, the larger the conjugated system is, which is more conducive to improving the stability of the molecule and increasing the electron transfer capacity between molecules, thereby reducing the vibration relaxation caused by intermolecular vibration, enhancing its fluorescence intensity, and causing a red shift in the fluorescence emission peak. Phenothiazine is an aromatic heterocyclic compound containing both N and S elements. The two benzene ring planes fold along the SN axis, presenting a special non-planar "butterfly" structure, which can provide a large π conjugated domain size in the preparation of carbon dots and expand the sp 2 Conjugated regions reduce the band gap and also enable co-doping of N and S heteroatoms. Therefore, using phenothiazine as a precursor is expected to achieve N and S co-doping and the construction of large π-conjugated domains in carbon dots, thereby preparing near-infrared fluorescent carbon dots. However, near-infrared fluorescent carbon dots based on phenothiazine have not yet been reported.

[0004] In view of this, the present invention proposes a new phenothiazine-based near-infrared fluorescent carbon dot and its preparation method and application. The near-infrared fluorescent carbon dots are directly prepared using phenothiazine. The method is simple and the synthesized carbon dots can be used in imaging, treatment, antibacterial, preservation and other fields. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing phenothiazine-based near-infrared fluorescent carbon dots, which directly uses phenothiazine as a precursor and utilizes a one-step solvent thermal method. The method is simple and can prepare new near-infrared fluorescent carbon dots with different properties by changing the solvent ratio.

[0006] In order to achieve the above objectives, the technical solutions adopted are:

[0007] A method for preparing phenothiazine-based near-infrared fluorescent carbon dots comprises the following steps:

[0008] (1) After completely dissolving phenothiazine in acetic acid solution, reacting at 160-220°C for 5-12 hours, and then cooling to room temperature to obtain a reaction solution;

[0009] (2) The reaction solution is filtered, neutralized to neutrality, extracted with ethyl acetate, and after separation, the organic phase is subjected to rotary evaporation. The obtained solid powder is separated and purified to obtain the phenothiazine-based near-infrared fluorescent carbon dots.

[0010] Furthermore, in the step (1), the mass volume ratio of phenothiazine to acetic acid solution is 1 g: 90-110 mL;

[0011] The volume ratio of acetic acid to water in the acetic acid solution is 1:1-2.

[0012] Furthermore, in the step (1), the mass volume ratio of phenothiazine to acetic acid solution is 1 g:100 mL.

[0013] Furthermore, in the step (2), filtering is performed using a 0.22 μm organic filter membrane;

[0014] Neutralize with saturated sodium bicarbonate solution.

[0015] Furthermore, in the step (2), the solid powder is separated and purified by 300-400 mesh silica gel column chromatography and then rotary evaporated; the eluent in the separation and purification process is a mixed solution of dichloromethane and methanol, or a mixed solution of petroleum ether and ethyl acetate.

[0016] Furthermore, in the step (2), the volume ratio of dichloromethane to methanol is 10-20:1;

[0017] The volume ratio of petroleum ether to ethyl acetate is 2-10:1.

[0018] Another object of the present invention is to provide phenothiazine-based near-infrared fluorescent carbon dots prepared by the above-mentioned preparation method.

[0019] Another object of the present invention is to provide applications of the above-mentioned phenothiazine-based near-infrared fluorescent carbon dots, which can be used in imaging, treatment, antibacterial, preservation, and other aspects.

[0020] In order to achieve the above objectives, the technical solutions adopted are:

[0021] The above-mentioned phenothiazine-based near-infrared fluorescent carbon dots are used in biological imaging, preparation of anti-inflammatory drugs, preparation of antibacterial drugs, and preservation of fruits.

[0022] Furthermore, the biological imaging is in vitro imaging of cells, bacteria, and organisms;

[0023] The inflammation is colitis, pneumonia, mastitis, dermatitis;

[0024] The antibacterial drugs are drugs that inhibit Gram-positive bacteria, fungi, plant-infecting bacteria, and molds;

[0025] The fruits are strawberries, citrus fruits, peaches, bananas and blueberries.

[0026] Furthermore, the cells in the biological imaging are: normal cells of an animal, cancer cells of an animal, inflammatory cells of an animal;

[0027] Fungi include: Gram-positive / negative bacteria, fungi, plant-infecting fungi, and molds;

[0028] Organisms include: living animals, plants, and isolated animal organs.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. In the technical solution of the present invention, the preparation method includes a solvent thermal synthesis reaction step and a carbon dot purification step, and the synthesis method is simple.

[0031] 2. In the technical solution of the present invention, the precursors for preparing carbon dots can be purchased directly and are inexpensive. Only the ratio of water and acetic acid in the solvent needs to be adjusted. No passivating agent is required to obtain near-infrared fluorescent carbon dots with good water dispersibility, uniform particle size, stable structure, low agglomeration, good biocompatibility and different functions.

[0032] 3. In the technical solution of the present invention, the prepared phenothiazine-based near-infrared fluorescent carbon dots have strong fluorescence emission in the near-infrared region, long excitation wavelength, and rich surface groups. Therefore, they show excellent performance in in vitro imaging of cells, bacteria, and organisms, in vivo / in vitro inflammation treatment, antibacterial properties of bacteria, fungi, and molds, and fruit preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Transmission electron microscopy (a) and particle size distribution (b) of phenothiazine-based near-infrared fluorescent carbon dots 1 prepared in Example 1, and transmission electron microscopy (c) and particle size distribution (d) of phenothiazine-based near-infrared fluorescent carbon dots 2 prepared in Example 2;

[0034] Figure 2 UV absorption, fluorescence excitation, and fluorescence emission spectra of phenothiazine-based near-infrared fluorescent carbon dots 1 (a) and 2 (b) prepared in Examples 1-2;

[0035] Figure 3 This is biological imaging of the phenothiazine-based near-infrared fluorescent carbon dots 1 prepared in Example 1 and the phenothiazine-based near-infrared fluorescent carbon dots 2 prepared in Example 2; wherein a is an in vitro cell, b is a bacterium, and c is an organism.

[0036] Figure 4 This is a diagram showing the results of in vitro inflammation treatment by the phenothiazine-based near-infrared fluorescent carbon dots 1 prepared in Example 1;

[0037] Figure 5 This is a diagram showing the results of the in vivo inflammation treatment of the phenothiazine-based near-infrared fluorescent carbon dots 1 prepared in Example 1;

[0038] Figure 6 Figure 2 is the antibacterial test result of two phenothiazine-based near-infrared fluorescent carbon dots 2 prepared in Example 2;

[0039] Figure 7 This is a graph showing the results of the preservation experiment of the two phenothiazine-based near-infrared fluorescent carbon dots 2 prepared in Example 2. DETAILED DESCRIPTION

[0040] To further illustrate the phenothiazine-based near-infrared fluorescent carbon dots, their preparation methods, and applications, and to achieve the intended purpose of the present invention, the following describes in detail the phenothiazine-based near-infrared fluorescent carbon dots, their preparation methods, and applications, along with their specific implementations, structures, features, and efficacy, in conjunction with preferred embodiments. In the following description, different references to "one embodiment" or "embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics of one or more embodiments may be combined in any suitable manner.

[0041] The following is a detailed introduction to the phenothiazine-based near-infrared fluorescent carbon dots, their preparation method, and their applications in conjunction with specific examples.

[0042] The present invention relates to phenothiazine-based near-infrared fluorescent carbon dots, and a preparation method and application thereof. The preparation method includes a solvent thermal synthesis reaction step and a carbon dot purification step. The solvent in the solvent thermal synthesis reaction includes water and acetic acid, and the precursor for preparing the carbon dots is only phenothiazine; the purification of the carbon dots includes silica gel column chromatography and developing agent preparation. The preparation method has a simple synthesis process, and the precursor can be purchased directly and is cheap. It only needs to adjust the ratio of water and acetic acid in the solvent, and no passivating agent is required to obtain near-infrared fluorescent carbon dots with good water dispersibility, uniform particle size, stable structure, not easy to agglomerate, good biocompatibility and different functions. Because this type of carbon dots has strong fluorescence emission in the near-infrared region, long excitation wavelength, and rich surface groups, they show excellent performance in in vitro imaging of cells, bacteria, and organisms, in vivo / in vitro inflammation treatment, antibacterial treatment of bacteria, fungi, and molds, and fruit preservation. The technical solution of the present invention is:

[0043] A method for preparing phenothiazine-based near-infrared fluorescent carbon dots comprises the following steps:

[0044] (1) After completely dissolving phenothiazine in acetic acid solution, reacting at 160-220°C for 5-12 hours, and then cooling to room temperature to obtain a reaction solution;

[0045] (2) The reaction solution is filtered, neutralized to neutrality, extracted with ethyl acetate, and after separation, the organic phase is subjected to rotary evaporation. The obtained solid powder is separated and purified to obtain the phenothiazine-based near-infrared fluorescent carbon dots.

[0046] Preferably, in the step (1), the mass volume ratio of phenothiazine to acetic acid solution is 1 g: 90-110 mL;

[0047] The volume ratio of acetic acid to water in the acetic acid solution is 1:1-2.

[0048] Further preferably, in the step (1), the mass volume ratio of phenothiazine to acetic acid solution is 1 g:100 mL.

[0049] Preferably, in the step (2), filtering is performed using a 0.22 μm organic filter membrane;

[0050] Neutralize with saturated sodium bicarbonate solution.

[0051] Preferably, in the step (2), the solid powder is separated and purified by 300-400 mesh silica gel column chromatography and then rotary evaporated; the eluent in the separation and purification process is a mixed solution of dichloromethane and methanol, or a mixed solution of petroleum ether and ethyl acetate.

[0052] Further preferably, in the step (2), the volume ratio of dichloromethane to methanol is 10-20:1;

[0053] The volume ratio of petroleum ether to ethyl acetate is 2-10:1.

[0054] A phenothiazine-based near-infrared fluorescent carbon dot is prepared by the above-mentioned preparation method.

[0055] The above-mentioned phenothiazine-based near-infrared fluorescent carbon dots are used in biological imaging, preparation of anti-inflammatory drugs, preparation of antibacterial drugs, and preservation of fruits.

[0056] Preferably, the biological imaging is in vitro imaging of cells, bacteria, or organisms;

[0057] The inflammation is colitis, pneumonia, mastitis, dermatitis;

[0058] The antibacterial drugs are drugs that inhibit Gram-positive bacteria, fungi, plant-infecting bacteria, and molds;

[0059] The fruits are strawberries, citrus fruits, peaches, bananas and blueberries.

[0060] Further preferably, the cells in the biological imaging are: normal cells of an animal, cancer cells of an animal, inflammatory cells of an animal;

[0061] Fungi include: Gram-positive / negative bacteria, fungi, plant-infecting fungi, and molds;

[0062] Organisms include: living animals, plants, and isolated animal organs.

[0063] Example 1.

[0064] The specific steps are as follows:

[0065] (1) Weigh 0.2 g of phenothiazine powder and dissolve it in 20 mL of acetic acid-water mixed solvent (V 乙酸 :V 水 =1:2), and after the phenothiazine is fully dissolved, transfer it to a 50 mL polytetrafluoroethylene liner. The reactor is placed in a forced air drying oven and reacted at 180°C for 10 hours. After the reaction is completed, it is naturally cooled to room temperature.

[0066] (2) After filtering the mixed solution in the liner with a 0.22 μm organic filter membrane to remove large particles, the remaining acetic acid in the filtrate was neutralized with a saturated sodium bicarbonate solution.

[0067] (3) The neutralized solution was extracted with ethyl acetate. After separation, the upper organic phase was collected and dried by rotary evaporation. The obtained solid powder was separated and purified by 300-400 mesh silica gel column chromatography to obtain phenothiazine-based near-infrared fluorescent carbon dots 1.

[0068] During the separation and purification process: the eluent is a mixed solution of dichloromethane and methanol, V 二氯甲烷 :V 甲醇 =20:1. After elution, the eluent was removed using a rotary evaporator to obtain a black powder, which was stored at 4°C in the dark.

[0069] Example 2.

[0070] The specific steps are as follows:

[0071] (1) Weigh 0.2 g of phenothiazine powder and dissolve it in 20 mL of acetic acid-water mixed solvent (V 乙酸 :V 水 =1:1), and after the phenothiazine is fully dissolved, transfer it to a 50 mL polytetrafluoroethylene liner. The reactor is placed in a forced air drying oven and reacted at 180°C for 10 hours. After the reaction is completed, it is naturally cooled to room temperature.

[0072] (2) After filtering the mixed solution in the liner with a 0.22 μm organic filter membrane to remove large particles, the remaining acetic acid in the filtrate was neutralized with a saturated sodium bicarbonate solution.

[0073] (3) The neutralized solution was extracted with ethyl acetate. After separation, the upper organic phase was collected and dried by rotary evaporation. The obtained solid powder was separated and purified by 300-400 mesh silica gel column chromatography to obtain phenothiazine-based near-infrared fluorescent carbon dots 2.

[0074] During the separation and purification process: the eluent is a mixed solution of dichloromethane and methanol, V 石油醚 :V 乙酸乙酯 After elution, the eluent was removed using a rotary evaporator to obtain a black powder, which was stored at 4°C in the dark.

[0075] Example 3.

[0076] The phenothiazine-based near-infrared fluorescent carbon dots prepared in Example 1-2 were tested:

[0077] (1) Transmission electron microscopy test and particle size distribution

[0078] High-resolution transmission electron microscopy images and particle size distribution of two phenothiazine-based near-infrared fluorescent carbon dots are shown in Figure 2. Figure 1 As shown, the phenothiazine-based near-infrared fluorescent carbon dots 1 prepared in Example 1 are shown in Figures a and b, and the phenothiazine-based near-infrared fluorescent carbon dots 2 prepared in Example 2 are shown in Figures c and d.

[0079] Depend on Figure 1 It can be seen that the technical solution of the present invention successfully prepared phenothiazine-based near-infrared fluorescent carbon dots with good dispersibility and narrow particle size distribution. Moreover, the particle size of the prepared phenothiazine-based near-infrared fluorescent carbon dots varied depending on the acetic acid solution.

[0080] (2) Optical performance

[0081] Method: Phenothiazine-based near-infrared fluorescent carbon dots were dispersed in ethanol (concentration: 4 mg / mL) to prepare a stock solution, and then 100 μL was added to 1.9 mL of PBS buffer (pH = 7.4) (final concentration was 0.2 mg / mL) to test its ultraviolet absorption and fluorescence excitation and emission spectra.

[0082] Results: The UV absorption, fluorescence excitation and fluorescence emission spectra of the two phenothiazine-based near-infrared fluorescent carbon dots were as follows: Figure 2 As shown, the phenothiazine-based near-infrared fluorescent carbon dots 1 prepared in Example 1 is shown in Figure a, and the phenothiazine-based near-infrared fluorescent carbon dots 2 prepared in Example 2 is shown in Figure b. Figure 2 It can be seen that the optical properties of the prepared phenothiazine-based near-infrared fluorescent carbon dots are different based on the difference in acetic acid solution. The phenothiazine-based near-infrared fluorescent carbon dots prepared by the present invention can produce near-infrared fluorescence with strong fluorescence emission and long excitation wavelength.

[0083] Example 4.

[0084] The specific steps are as follows:

[0085] (1) Weigh 0.2 g of phenothiazine powder and dissolve it in 22 mL of acetic acid-water mixed solvent (V 乙酸 :V 水 =1:1.5), and after the phenothiazine was fully dissolved, the mixture was transferred to a 50 mL polytetrafluoroethylene liner. The reactor was placed in a forced air drying oven and reacted at 160°C for 12 hours. After the reaction was completed, the mixture was naturally cooled to room temperature.

[0086] (2) After filtering the mixed solution in the liner with a 0.22 μm organic filter membrane to remove large particles, the remaining acetic acid in the filtrate was neutralized with a saturated sodium bicarbonate solution.

[0087] (3) The neutralized solution was extracted with ethyl acetate. After separation, the upper organic phase was collected and dried by rotary evaporation to obtain a solid powder.

[0088] (4) The solid powder was divided into 3 parts and separated and purified by 300-400 mesh silica gel column chromatography. During the separation and purification process: the eluent used for the 3 parts of solid powder was a mixed solution of dichloromethane and methanol, V 二氯甲烷 :V 甲醇 The ratios of the carbon dots were 10:1, 15:1, and 12:1. After elution, the eluent was removed by a rotary evaporator to obtain a black powder, which was stored at 4°C in the dark to obtain phenothiazine-based near-infrared fluorescent carbon dots.

[0089] The three phenothiazine-based near-infrared fluorescent carbon dots obtained were subjected to the same transmission electron microscopy scanning and optical property testing as in Example 3. It was found that the prepared thiazine-based near-infrared fluorescent carbon dots had good dispersibility, narrow particle size distribution, and could produce near-infrared fluorescence.

[0090] Example 5.

[0091] The specific steps are as follows:

[0092] (1) Weigh 0.2 g of phenothiazine powder and dissolve it in 18 mL of acetic acid-water mixed solvent (V 乙酸 :V 水 =1:2), and after the phenothiazine is fully dissolved, transfer it to a 50 mL polytetrafluoroethylene liner. The reactor is placed in a forced air drying oven and reacted at 220°C for 5 hours. After the reaction is completed, it is naturally cooled to room temperature.

[0093] (2) After filtering the mixed solution in the liner with a 0.22 μm organic filter membrane to remove large particles, the remaining acetic acid in the filtrate was neutralized with a saturated sodium bicarbonate solution.

[0094] (3) The neutralized solution was extracted with ethyl acetate, and after separation, the upper organic phase was collected and dried by rotary evaporation to obtain a solid powder.

[0095] (4) The solid powder was divided into 3 parts and separated and purified by 300-400 mesh silica gel column chromatography. During the separation and purification process: the eluent used for the 3 parts of solid powder was a mixed solution of petroleum ether and ethyl acetate, V 石油醚 :V 乙酸乙酯 The ratios of the phosphine-containing carbon dots were 10:1, 6:1, and 2:1. After elution, the eluent was removed by a rotary evaporator to obtain a black powder, which was stored at 4°C in the dark to obtain phenothiazine-based near-infrared fluorescent carbon dots.

[0096] The three phenothiazine-based near-infrared fluorescent carbon dots obtained were subjected to the same transmission electron microscopy scanning and optical property testing as in Example 3. It was found that the prepared thiazine-based near-infrared fluorescent carbon dots had good dispersibility, narrow particle size distribution, and could produce near-infrared fluorescence.

[0097] Example 6: Biological Imaging

[0098] method:

[0099] (1) In vitro cells: RAW264.7 cells in the logarithmic growth phase and in good growth condition were taken and inoculated into confocal microplates at 1×105 cells / well. The microplates were divided into four groups: blank group, model group, and experimental group (200μg / mL). The model group and the experimental group were treated with LPS (2μg / mL) for 24 hours. The blank group was cultured with the corresponding culture medium, and the experimental group was added with the corresponding concentration of fluorescent carbon dot 1 dispersion and incubated for another 24 hours. After incubation in the confocal microplates for 24 hours, the cells were washed twice with PBS, and complete culture medium was added. Under light-proof conditions, 10μL Hoechst 33342 (10μg / mL) was added to each well and placed in an incubator for 15 minutes before observation with a confocal microscope. The Ex / Em of Hoechst is 350nm / 461nm; the Ex / Em of PTZ-R is 590nm / 612nm.

[0100] (2) Bacteria: The cultured Staphylococcus aureus and Candida albicans were incubated with fluorescent carbon dots 2 for 4 hours. Then a drop was placed on a glass slide, covered with a coverslip, and placed upside down in a live cell imager for fluorescence imaging.

[0101] (3) Organisms: Healthy SPF-grade BALB / c female mice, 6 weeks old and weighing 18-22 g, were housed in separate cages at the Laboratory Animal Center. The temperature was 20-24°C, with a 12-h light-dark cycle and a relative humidity of 40%-70%. The mice were acclimated for 7 days. After 7 days of acclimation, phenothiazine-based near-infrared fluorescent carbon dots 1 were injected intraperitoneally once daily. After 7 days, the colons of the mice were removed and placed on a non-light-absorbing blackboard. Fluorescence imaging was performed directly under the laser of a small animal in vivo imaging system.

[0102] Results: As Figure 3As shown, phenothiazine-based near-infrared fluorescent carbon dots can achieve good imaging effects in cells, bacteria and tissues.

[0103] Example 7: Inflammation in vivo

[0104] Methods: An in vitro inflammatory model was established using RAW 264.7 mouse macrophages. Cellular inflammation was induced by lipopolysaccharide (LPS). Cell culture was performed as in Example 6. The cells were divided into the following groups: blank group, model group (LPS: 2 μg / mL); experimental group 1 (LPS: 2 μg / mL, phenothiazine-based near-infrared fluorescent carbon dots 1: 50 μg / mL); experimental group 2 (LPS: 2 μg / mL, phenothiazine-based near-infrared fluorescent carbon dots 1: 100 μg / mL); and experimental group 3 (LPS: 2 μg / mL, phenothiazine-based near-infrared fluorescent carbon dots 1: 200 μg / mL). The model and experimental groups were treated with LPS for 24 hours. The blank group was then incubated with the corresponding culture medium. The experimental groups were then treated with the corresponding concentrations of phenothiazine-based near-infrared fluorescent carbon dots 1 and incubated for an additional 24 hours. Inflammatory cytokine levels in the cells were then assessed using ELISA kits for TNF-α and IL-6. OD values ​​were measured at a wavelength of 450 nm, and the inflammatory cytokine levels were calculated based on the standard curve.

[0105] Results: As Figure 4 As shown in the figure, (--) is the blank group, (+-) is the model group, (+50) is the experimental group 1, (+100) is the experimental group 2, and (+200) is the experimental group 3. Figure 5 It can be seen that the contents of interleukin 6 and TNF-α in the experimental group were significantly lower than those in the model group, and the contents of interleukin 6 and TNF-α in the experimental group decreased with the increase of the amount of carbon dots added, which proves the effectiveness of phenothiazine-based near-infrared fluorescent carbon dots 1 in the treatment of inflammation in mice, and can effectively reduce the content of inflammatory factors and achieve inflammation treatment.

[0106] Example 8: In vitro inflammation

[0107] Methods: RAW264.7 cells in the logarithmic growth phase and in good growth condition were taken and 1×10 5 Cells were seeded per well in confocal microplates. The microplates were divided into four groups: blank, model, experimental group 1 (1:50 μg / mL of phenothiazine-based near-infrared fluorescent carbon dots), and experimental group 2 (1:200 μg / mL of phenothiazine-based near-infrared fluorescent carbon dots). Both the model and experimental groups were treated with LPS (2 μg / mL) for 24 hours. The blank group was incubated with the corresponding culture medium. The experimental groups were incubated with the corresponding concentrations of carbon dot dispersions for a further 24 hours.

[0108] After incubation in the confocal microplate for 24 hours, the levels of inflammatory factors in the cells were detected using ELISA kits for TNF-α, IL-6, and IL-1β. The OD value was measured at a wavelength of 450 nm, and the content of inflammatory factors was calculated according to the standard curve.

[0109] After incubation in the confocal dish for 24 h, the cells were washed twice with PBS and complete culture medium was added. 10 μL of Hoechst 33342 (10 μg / mL) was added to each well and placed in an incubator for 15 min under light-proof conditions before observation with a confocal microscope.

[0110] Results: As Figure 5 As shown in the results, (1) the levels of TNF-α, IL-6, and IL-1β in the experimental group were significantly lower than those in the model group, and the levels of TNF-α, IL-6, and IL-1β in the experimental group decreased with the increase of the amount of carbon dots 1 added; (2) under the microscope, the cellular inflammation in the experimental group was significantly lower. This proves that phenothiazine-based near-infrared fluorescent carbon dots can effectively reduce the content of inflammatory factors in in vitro cell culture and achieve inflammation treatment.

[0111] Example 9: Antibacterial

[0112] Experimental method, the specific steps are as follows:

[0113] (1) Staphylococcus aureus (S. aureus):

[0114] Luria-Bertani (LB) liquid culture medium is prepared by adding 10g of trypsin, 5g of yeast powder, and 10g of NaCl to 1L of ultrapure water, mixing well, and sterilizing. Solid culture medium is obtained by adding 15g of agar powder to this basis. Gram-positive bacteria - Staphylococcus aureus (S.aureus) are cultured. A colony is taken from the LB agar plate and transferred to a shaking flask containing 20mL of liquid culture medium. It is then placed in a constant temperature shaker for culture (37°C, 200rpm). After inoculation in a constant temperature shaker overnight, a preliminary bacterial suspension is obtained, and the bacterial concentration is estimated by coating the plate and counting. The obtained bacterial suspension is diluted to 10 with sterile water. 6 Phenothiazine-based near-infrared fluorescent carbon dots 2 were dispersed in dimethyl sulfoxide (DMSO).

[0115] The antibacterial test was divided into a blank group (CK) (bacteria solution + sterile water), a control group (bacteria solution + DMSO) (i.e., 0 μg / mL), and an experimental group (bacteria solution + DMSO + phenothiazine-based near-infrared fluorescent carbon dots 2). Five concentration gradients were set for the experimental groups: the concentrations of phenothiazine-based near-infrared fluorescent carbon dots 2 in the S. aureus group were 0 μg / mL, 0.4 μg / mL, 0.6 μg / mL, 0.8 μg / mL, and 1 μg / mL.

[0116] According to the aforementioned grouping, diluted S. aureus and carbon dot solutions were added to the liquid culture medium and the mixture was placed in a constant temperature shaker (37°C, 200 rpm) overnight. Using the dilution spread plate method, 20 μL of the diluted bacterial solution was inoculated onto an agar plate. The plate was evenly spread and allowed to rest for 10 minutes before being placed in a constant temperature incubator (37°C). The plates were inverted to prevent contamination. After 12 hours, the culture plates were photographed and bacterial growth on each agar plate was recorded. Finally, the inhibition rate was calculated by counting the number of colonies.

[0117] (2) Candida albicans

[0118] The experimental method is the same as (1) for Staphylococcus aureus, except that Candida albicans was used, and the experimental group settings were:

[0119] Five concentration gradients were set in the experimental groups: the concentrations of phenothiazine-based near-infrared fluorescent carbon dots 2 in the C. albicans group were: 0 μg / mL, 1 μg / mL, 1.25 μg / mL, 1.5 μg / mL, and 2 μg / mL;

[0120] (3) Grey mold

[0121] The experimental method is the same as (1) for Staphylococcus aureus, except that gray mold was used, and the experimental group settings were:

[0122] Five concentration gradients were set in the experimental group. The concentrations of phenothiazine-based near-infrared fluorescent carbon dots 2 in the grey mold group were: 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL. The DMSO content in both the control and experimental groups was 1%.

[0123] Results: As Figure 6 As shown, the phenothiazine-based near-infrared fluorescent carbon dots 2 have obvious inhibitory effects on S. aureus, C. albicans, and Greymould. Therefore, the phenothiazine-based near-infrared fluorescent carbon dots prepared in the present invention have obvious inhibitory effects on bacteria, fungi, and molds, and can be used for antibacterial purposes.

[0124] Example 10: Freshness preservation

[0125] Method: Prepare different concentrations of phenothiazine-based near-infrared fluorescent carbon dots 2 aqueous solutions (0, 2, 10 μg / mL), soak kitchen paper in the phenothiazine-based near-infrared fluorescent carbon dots 2 aqueous solution for 2 minutes, take out and dry naturally for later use.

[0126] Commercially purchased strawberries were packaged into transparent fresh-keeping containers. A blank control group (NC, shown in the figure), a control group (0 μg / mL, shown in the figure), and an experimental group were set up. The control group's container was lined with dried paper soaked in the aqueous solution. The experimental groups were filled with paper of varying concentrations, while the blank group was left empty. After packaging, the containers were sealed with plastic wrap. The morphology of the strawberries was recorded on days 1, 2, 3, 4, 5, and 6.

[0127] Results: As Figure 7 As shown, the phenothiazine-based near-infrared fluorescent carbon dots prepared in the present invention have a preservation effect on strawberries, thereby proving the application effect of phenothiazine-based near-infrared fluorescent carbon dots in fruit preservation.

[0128] Subsequently, the same preservation experiment was conducted on citrus, peaches, bananas, and blueberries, and the results were the same as those of the strawberry preservation experiment. The phenothiazine-based near-infrared fluorescent carbon dots prepared by the present invention have a preservation effect on strawberries, thus proving the application effect of phenothiazine-based near-infrared fluorescent carbon dots in fruit preservation.

[0129] The above is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the embodiments of the present invention. Any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the embodiments of the present invention are still within the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing phenothiazine-based near-infrared fluorescent carbon dots, characterized in that: The following steps are involved: (1) After completely dissolving phenothiazine in acetic acid solution, react at 160-220°C for 5-12 hours, and then cool to room temperature to obtain a reaction solution; (2) The reaction solution is filtered, neutralized to neutrality, and extracted with ethyl acetate. After separation, the organic phase is subjected to rotary evaporation, and the obtained solid powder is separated and purified to obtain the phenothiazine-based near-infrared fluorescent carbon dots.

2. The preparation method according to claim 1, characterized in that In the step (1), the mass volume ratio of phenothiazine to acetic acid solution is 1 g: 90-110 mL; The volume ratio of acetic acid to water in the acetic acid solution is 1:1-2.

3. The preparation method according to claim 2, characterized in that In the step (1), the mass volume ratio of phenothiazine to acetic acid solution is 1 g:100 mL.

4. The preparation method according to claim 1, characterized in that In the step (2), filtering with a 0.22 μm organic filter membrane; Neutralize with saturated sodium bicarbonate solution.

5. The preparation method according to claim 1, characterized in that In the step (2), the solid powder is separated and purified by 300-400 mesh silica gel column chromatography and then rotary evaporated; the eluent in the separation and purification process is a mixed solution of dichloromethane and methanol, or a mixed solution of petroleum ether and ethyl acetate.

6. The preparation method according to claim 5, characterized in that In the step (2), the volume ratio of dichloromethane to methanol is 10-20:1; The volume ratio of petroleum ether to ethyl acetate is 2-10:

1.

7. A phenothiazine-based near-infrared fluorescent carbon dot, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the phenothiazine-based near-infrared fluorescent carbon dots according to claim 7 in biological imaging, preparation of drugs for treating inflammation, preparation of antibacterial drugs, and preservation of fruits.

9. The use according to claim 8, characterized in that The biological imaging is in vitro imaging of cells, bacteria, and organisms; The inflammation is colitis, pneumonia, mastitis, dermatitis; The antibacterial drugs are drugs that inhibit Gram-positive bacteria, fungi, plant-infecting bacteria, and molds; The fruits are strawberries, citrus fruits, peaches, bananas and blueberries.

10. The use according to claim 9, characterized in that The cells in the biological imaging are: normal cells of an animal, cancer cells of an animal, and inflammatory cells of an animal; Fungi include: Gram-positive / negative bacteria, fungi, plant-infecting fungi, and molds; Organisms include: living animals, plants, and isolated animal organs.

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