Phenothiazinyl near-infrared fluorescent carbon dots as well as preparation method and application thereof

Abstract

The invention relates to phenothiazinyl near-infrared fluorescent carbon dots as well as a preparation method and application thereof. The invention relates to a preparation method of phenothiazine-based near-infrared fluorescent carbon dots, which comprises the following steps: (1) completely dissolving phenothiazine in an acetic acid solution, reacting at 160-220 DEG C for 5-12 hours, and cooling to room temperature to obtain a reaction solution; and (2) filtering the reaction liquid, neutralizing to be neutral, extracting with ethyl acetate, layering, carrying out rotary evaporation on an organic phase, and separating and purifying obtained solid powder to obtain the phenothiazinyl near-infrared fluorescent carbon dots. According to the phenothiazinyl near-infrared fluorescent carbon dots as well as the preparation method and application thereof, the synthesis process is simple, and the near-infrared fluorescent carbon dots which are good in water dispersibility, uniform in particle size, stable in structure, not prone to agglomeration and good in biocompatibility and have different functions can be obtained.

Description

Technical Field

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

[0002] Fluorescent carbon dots have shown great potential in biomedicine, imaging, therapy, antibacterial, and antimicrobial due to their unique photostability, high yield, low toxicity, good biocompatibility, and high cost-effectiveness. Among them, near-infrared fluorescent carbon dots have low scattering and absorption in biological tissues and low phototoxicity to cells and tissues, so they can achieve deeper tissue penetration, which is beneficial for in situ imaging, labeling, and phototherapy of organisms.

[0003] At present, the common method to obtain near-infrared 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 are folded 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 can reduce the band gap and realize co-doping of N and S heteroatoms. Therefore, using phenothiazine as a precursor is expected to realize 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 been reported yet.

[0004] In view of this, the present invention proposes a new phenothiazine-based near-infrared fluorescent carbon dot and a preparation method and application thereof. The near-infrared fluorescent carbon dots are prepared directly from phenothiazine. The method is simple and the synthesized carbon dots can be used in the fields of imaging, treatment, antibacterial, and preservation. 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, directly using phenothiazine as a precursor and utilizing a one-step solvothermal 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 purpose, the technical solution adopted is:

[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) filtering the reaction solution, neutralizing it to neutrality, extracting it with ethyl acetate, separating and purifying the solid powder, and obtaining 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 a phenothiazine-based near-infrared fluorescent carbon dot prepared by the above-mentioned preparation method.

[0019] Another object of the present invention is to provide an application 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 purpose, the technical solution adopted is:

[0021] The above-mentioned phenothiazine-based near-infrared fluorescent carbon dots are used in biological imaging, preparation of drugs for treating inflammation, 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 are: Gram-positive / negative bacteria, fungi, plant-infecting bacteria, 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 precursor for preparing carbon dots can be purchased directly and is cheap. It only needs to adjust the ratio of water and acetic acid in the solvent. No passivating agent is required to obtain near-infrared fluorescent carbon dots with good water dispersibility, uniform particle size, stable structure, non-agglomeration, good biocompatibility and different functions.

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

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

[0034] Figure 2 The ultraviolet absorption, fluorescence excitation and fluorescence emission spectra of the phenothiazine-based near-infrared fluorescent carbon dots 1 (a) and carbon dots 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 phenothiazine-based near-infrared fluorescent carbon dots 1 prepared in Example 1 in treating inflammation in vivo;

[0038] Figure 6 The antibacterial test results of two phenothiazine-based near-infrared fluorescent carbon dots 2 prepared in Example 2 are shown;

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

[0040] In order to further explain the phenothiazine-based near-infrared fluorescent carbon dots and their preparation methods and applications of the present invention and achieve the intended purpose of the invention, the following is a detailed description of the phenothiazine-based near-infrared fluorescent carbon dots and their preparation methods and applications, their specific implementation methods, structures, features and their effects proposed in accordance with the present invention in combination with preferred embodiments. In the following description, different "one embodiment" or "embodiment" does not necessarily refer to the same embodiment. In addition, specific features, structures or characteristics in one or more embodiments may be combined in any suitable form.

[0041] The following will further introduce the phenothiazine-based near-infrared fluorescent carbon dots and their preparation method and application in detail in conjunction with specific embodiments:

[0042] The present invention discloses a phenothiazine-based near-infrared fluorescent carbon dot and a preparation method and application thereof. The preparation method comprises a solvent thermal synthesis reaction step and a carbon dot purification step. The solvent in the solvent thermal synthesis reaction comprises water and acetic acid, and the precursor for preparing the carbon dot is only phenothiazine; the purification of the carbon dot comprises 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 dot has strong fluorescence emission in the near-infrared region, long excitation wavelength, and rich surface groups, it exhibits excellent performance in in vitro cell, bacteria, and organism imaging, in vivo / in vitro inflammation treatment, antibacterial of bacteria, fungi, and molds, and fruit preservation. The technical scheme 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) filtering the reaction solution, neutralizing it to neutrality, extracting it with ethyl acetate, separating and purifying the solid powder, and obtaining 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 adopting the above preparation method.

[0055] The above-mentioned phenothiazine-based near-infrared fluorescent carbon dots are used in biological imaging, preparation of drugs for treating inflammation, 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 are: Gram-positive / negative bacteria, fungi, plant-infecting bacteria, 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), after the phenothiazine is fully dissolved, transfer it to a 50 mL polytetrafluoroethylene liner. Put the reactor into a forced air drying oven and react at 180°C for 10 hours. After the reaction is completed, cool it naturally 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 is neutralized with a saturated sodium bicarbonate solution to neutralize it.

[0067] (3) The neutralized solution was extracted with ethyl acetate. After layering, the upper organic phase was taken 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 by 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), 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 is neutralized with a saturated sodium bicarbonate solution to neutralize it.

[0073] (3) The neutralized solution was extracted with ethyl acetate. After layering, the upper organic phase was taken 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 by 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 diagrams 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. And based on the difference in acetic acid solution, the particle sizes of the prepared phenothiazine-based near-infrared fluorescent carbon dots are different.

[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 are shown in Figure a, and the phenothiazine-based near-infrared fluorescent carbon dots 2 prepared in Example 2 are 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 solutions. The phenothiazine-based near-infrared fluorescent carbon dots prepared by the present invention can generate near-infrared fluorescence and have the characteristics of 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), after the phenothiazine is fully dissolved, transfer it to a 50 mL polytetrafluoroethylene liner. Put the reactor into a forced air drying oven, react at 160°C for 12 hours, and cool naturally to room temperature after the reaction is completed.

[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 is neutralized with a saturated sodium bicarbonate solution to neutralize it.

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

[0088] (4) The solid powder was divided into three parts and purified by 300-400 mesh silica gel column chromatography. During the separation and purification process: the eluent used for the three parts of solid powder was a mixed solution of dichloromethane and methanol, V 二氯甲烷 :V 甲醇 The ratios 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), 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 is neutralized with a saturated sodium bicarbonate solution to neutralize it.

[0094] (3) The neutralized solution was extracted with ethyl acetate, and after layering, the upper organic phase was taken and rotary evaporated to dry to obtain a solid powder.

[0095] (4) The solid powder was divided into three parts and purified by 300-400 mesh silica gel column chromatography. During the separation and purification process: the eluent used for the three 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 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 inoculated into confocal dishes at 1×105 cells / well. The dishes 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 24h. 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 continued to incubate for 24h. After incubation in the confocal dish for 24h, 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 15min, and then observed 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, weighing 18-22 g, were used and kept in separate cages in the experimental animal center. The temperature was 20-24°C, the light-dark cycle was 12 h, and the relative humidity was 40%-70%. The mice were first adapted to feeding for 7 days. After the mice were adapted to feeding for 7 days, phenothiazine-based near-infrared fluorescent carbon dots 1 were injected intraperitoneally once a day. After 7 days, the colon of the mice was removed and placed on a non-light-absorbing blackboard, and directly placed under the laser of the small animal in vivo imaging system for fluorescence imaging.

[0102] Results: 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: RAW 264.7 mouse macrophages were used to construct an in vitro inflammatory model, and cellular inflammation was induced by lipopolysaccharide (LPS). Cell culture was the same as in Example 6. Grouping: blank group, model group (LPS: 2μg / mL); experimental group 1 (LPS: 2μg / mL, phenothiazine-based near-infrared fluorescent carbon dots 1: 50μg / mL); experimental group 2 (LPS: 2μg / mL, phenothiazine-based near-infrared fluorescent carbon dots 1: 100μg / mL); experimental group 3 (LPS: 2μg / mL, phenothiazine-based near-infrared fluorescent carbon dots 1: 200μg / mL). The model group and the experimental group were treated with LPS for 24h, and the blank group was cultured with the corresponding culture medium. Then the experimental group was added with the corresponding concentration of phenothiazine-based near-infrared fluorescent carbon dots 1 and continued to incubate for 24h. Then, the ELISA kits of TNF-α and IL-6 were used to detect the levels of inflammatory factors in the cells, and the OD values ​​were measured at a wavelength of 450nm, and the content of inflammatory factors was calculated according to the standard curve.

[0105] Results: 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 proved 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 logarithmic growth phase and in good growth condition were taken and 1×10 5 The cells / well were inoculated in confocal dishes. The dishes were divided into four groups: blank group, model group, experimental group 1 (phenothiazine-based near-infrared fluorescent carbon dots 1: 50μg / mL), and experimental group 2 (phenothiazine-based near-infrared fluorescent carbon dots 1: 200μg / mL). The model group and the experimental group were treated with LPS (2μg / mL) for 24h. The blank group was cultured with the corresponding culture medium. The experimental group was added with the corresponding concentration of carbon dot dispersion and incubated for another 24h.

[0108] After incubation in the confocal dish for 24 hours, the levels of inflammatory factors in the cells were detected using ELISA kits of three inflammatory factors: 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. In the dark, 10 μL Hoechst 33342 (10 μg / mL) was added to each well and placed in an incubator for 15 min before observation with a confocal microscope.

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

[0111] Example 9: Antibacterial

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

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

[0114] Luria-Bertani (LB) liquid 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 medium is obtained by adding 15g of agar powder on this basis. Gram-positive bacteria, Staphylococcus aureus (S. aureus), are used for bacterial culture. A colony is taken from the LB agar plate and transferred to a shake flask containing 20mL of liquid culture medium, and 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 count. 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 group was divided into a blank group CK (bacterial solution + sterile water), a control group (bacterial solution + DMSO) (i.e. 0ug / ml), and an experimental group: (bacterial solution + DMSO + phenothiazine-based near-infrared fluorescent carbon dots 2). Five concentration gradients were set in the experimental group, and the concentrations of phenothiazine-based near-infrared fluorescent carbon dots 2 in the S. aureus group were: 0μg / mL, 0.4μg / mL, 0.6μg / mL, 0.8μg / mL, and 1μg / mL.

[0116] According to the above grouping, add diluted S. aureus and carbon dot solution to the liquid culture medium, and place the mixed system in a constant temperature shaker (37°C, 200rpm) overnight. According to the dilution plate method, take 20μL of the bacterial solution diluted a certain number of times and inoculate it on the agar plate, spread it evenly, let it stand for 10 minutes, and then put it in a constant temperature incubator (37°C), and turn the plate upside down to prevent contamination. After 12 hours, take a photo of the culture dish and record the bacterial growth on each agar plate. Finally, calculate the antibacterial rate 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 mould

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

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

[0123] Results: Figure 6 As shown, the phenothiazine-based near-infrared fluorescent carbon dots 2 have obvious inhibitory effects on S. aureus, C. albicans, and Greymould. Thus, 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] Embodiment 10: Fresh-keeping

[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] Strawberries purchased from the market were packed into transparent fresh-keeping boxes, and a blank group (i.e., NC in the figure), a control group (i.e., 0 μg / mL in the figure), and an experimental group were set up. The control group box was padded with paper soaked in water solution and dried, and the experimental group was placed with paper of different concentrations, while the blank group was not placed. After the packaging was completed, it was sealed with plastic wrap. The morphology of the strawberries on the 1st, 2nd, 3rd, 4th, 5th, and 6th days was recorded respectively.

[0127] Results: 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 the phenothiazine-based near-infrared fluorescent carbon dots in fruit preservation.

[0128] Subsequently, the same preservation experiment was carried out 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, thereby proving the application effect of phenothiazine-based near-infrared fluorescent carbon dots in fruit preservation.

[0129] The above is only a preferred embodiment of the embodiment of the present invention, and does not impose any form of limitation on the embodiment of the present invention. Any simple modification, equivalent changes and modifications made to the above embodiment based on the technical essence of the embodiment of the present invention are still within the scope of the technical solution of the embodiment 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, 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, separating and purifying the solid powder, and obtaining 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 preparation method is described in 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 are: Gram-positive / negative bacteria, fungi, plant-infecting bacteria, and molds; Organisms include: living animals, plants, and isolated animal organs.

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