CN113372907A - Photosynthetic bacterium red light carbon dot and preparation method and application thereof - Google Patents
Photosynthetic bacterium red light carbon dot and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a photosynthetic bacteria red light carbon dot and a preparation method thereof, belonging to the field of biomedical materials. The photosynthetic bacteria red light carbon dots prepared by the method can rapidly enter cells and accumulate in lysosomes under the concentration of 1mg/ml, show a better cell developing effect and have good biological developing potential.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a photosynthetic bacteria red light carbon dot and a preparation method and application thereof.
Background
Carbon Dots (CDs) are a new class of carbon-based materials with dimensions below 10nm, usually in a spherical or hemispherical structure similar to the "core-shell" type, in sp2The hybridized conjugated carbon is a core, and a large number of organic groups containing oxygen or other heteroatoms are arranged on the surface. CDs have received much attention for their excellent optical properties, good water solubility, low toxicity, environmental protection, wide sources of raw materials, low cost and good biocompatibility. Because of these outstanding properties, CDs show great promise in the fields of sensing, bio-imaging, nanomedicine, catalysis, and the like. Bioimaging is a leading-edge field of biotechnology applications that can be used for structural and functional studies of tissues, organs and cells in vivo or in vitro. Because the traditional semiconductor quantum dots and organic fluorescent molecules have the photobleaching property, the traditional semiconductor quantum dots and organic fluorescent molecules cannot be suitable for long-term observation and research of cells and biological tissues. The carbon dots with excellent inherent fluorescence, photobleaching resistance, water solubility, low toxicity and other characteristics are expected to replace the traditional semiconductor quantum dots and organic fluorescent probes, thereby becoming a new and more ideal biological imaging material.
At present, the aspects of the synthesis raw materials, the synthesis method and the like of the carbon dots have been developed to a certain extent. However, most CDs also encounter several limitations in bioimaging applications. For example, most of the CDs reported so far show strong emission only in the region from blue light to green light, and the emission not only interferes with the autofluorescence of tissues and cells, but also causes radiation damage to the tissues and the cells, thereby greatly limiting the application of the CDs in the biomedical field. Small changes in wavelength have been reported to cause significant changes in phototoxicity. For example, the difference between 488nm and 514nm may result in a difference in survival and death of the imaged cells. Compared with these low-wavelength emission CDs, red-light emission CDs are lower in energy, more permeable to organisms, and have a greater ability to image organisms, and thus are more advantageous in the biological and medical fields. Therefore, the preparation of long-wavelength luminescent CDs has wide application prospect.
In the prior art, o-phenylenediamine is used as a precursor, and a far-red-near-infrared luminescent carbon dot with good optical performance and biocompatibility is prepared by an ethanol solvothermal method, so that the far-red-near-infrared luminescent carbon dot can be used for ultrafast lysosome imaging and polarity monitoring. However, the synthesis process of the carbon dot involves more organic reagents and the purification is more complicated, so that the further application of the carbon dot is limited.
Disclosure of Invention
In order to solve the technical problems, the invention provides a photosynthetic bacteria red light carbon dot which can generate red fluorescence, has stable structure and low cytotoxicity and can be applied to biological imaging.
The technical scheme adopted for realizing the purpose of the invention is as follows:
a preparation method of photosynthetic bacteria red light carbon dots is characterized in that photosynthetic bacteria are used as precursors, carbonized products are generated through microwave-assisted heat treatment, and the photosynthetic bacteria red light carbon dots are obtained through extraction and purification.
Specifically, the photosynthetic bacteria include, but are not limited to, Rhodospirillaceae exothiobacteria, Chromomycetaceae, Thiobacillus viridans, non-Thiobacillus violaceus, Polycytofiliform Chlorobacterium, Spirulina, obligate aerobes containing bacteriochlorophyll.
A preparation method of photosynthetic bacteria red light carbon dots specifically comprises the following steps:
1) weighing 1-5 g of photosynthetic bacteria, adding into 10-50 mL of deionized water, transferring the mixture into a 100mL beaker, and heating for 10-30 min in a microwave oven at a high fire;
2) after heating with high fire, after the beaker is gradually cooled to room temperature, adding 10-30 mL of deionized water, and carrying out ultrasonic treatment for 10-60 min to obtain a dark green solution;
3) centrifuging the dark green solution obtained in the step 2), removing large particles, collecting supernatant, carrying out suction filtration on the obtained supernatant, removing insoluble substances of the large particles, putting the obtained supernatant into a dialysis bag, dialyzing the solution in ultrapure water, and removing small molecular substances to obtain a photosynthetic bacteria red light carbon dot solution;
4) and (3) freeze-drying the obtained photosynthetic bacterium red light carbon dot solution by using a freeze-drying device to obtain a solid product, so as to obtain photosynthetic bacterium red light carbon dots.
Specifically, the centrifugation speed in the step (3) is 10000-11000 rpm, and the centrifugation time is 10-30 min.
Specifically, in the step (3), an organic aqueous microporous filter membrane with a pore size of 0.22 μm is used for suction filtration.
Specifically, when the dialysis is carried out in ultrapure water, the cut-off molecular weight of the dialysis bag is 1000Da, and the dialysis time is 48-72 h.
Specifically, in the step (4), the freeze drying time is 24-36 h.
In addition, the invention also provides the photosynthetic bacteria red light carbon dots obtained by the preparation method.
Finally, the invention also provides the effect of the photosynthetic bacteria red light carbon dots prepared by the preparation method for cell development.
According to the technical scheme, the beneficial effects of the invention are as follows:
1) the invention adopts photosynthetic bacteria as the raw material for manufacturing the carbon dots for the first time and adopts a microwave thermal method to manufacture the carbon dots. The prepared photosynthetic bacteria red light carbon dots have high dispersibility and stability in an aqueous medium.
2) The photosynthetic bacteria red light carbon dot shows red fluorescence under the irradiation of an ultraviolet lamp, and the photosynthetic bacteria red light carbon dot has three absorption peaks which are 230nm, 280nm and 405nm respectively; the fluorescence emission peaks are different at different excitation wavelengths. Under 340nm exciting light, the photosynthetic bacteria red light carbon dot has two fluorescence emission peaks respectively positioned at 450nm and 595 nm; as the excitation wavelength increases, the fluorescence peak at 450nm is red-shifted, while the fluorescence emission peak at 595nm is unchanged. In addition, under the excitation light of 400nm, a new fluorescence emission peak appears at 642nm at the red light carbon point of the photosynthetic bacteria.
3) In the concentration range of 0.1-10000 mug/ml, the photosynthetic bacteria red light carbon dot has no obvious cytotoxicity to Huvec cells. After the photosynthetic bacteria red light carbon dots with different concentrations are incubated for 24 hours, the cell survival rate fluctuates between 100% and 108%.
4) The photosynthetic bacteria red light carbon dots prepared by the method can rapidly enter cells and accumulate in lysosomes under the concentration of 1mg/ml, show a better cell developing effect and have good biological developing potential.
Drawings
FIG. 1 shows the growth of sulfur-green bacteria in example 1 inoculated with 15% inoculum size in 250mL of photosynthetic bacteria culture medium prepared in this laboratory, cultured in a light incubator at 30 ℃ and 75% relative humidity and 7920Lux light intensity for 72 h.
FIG. 2 is a transmission electron micrograph of the photosynthetic bacteria red carbon dots prepared in example 2, and the insets are a corresponding high resolution electron micrograph and a particle size statistical chart.
FIG. 3 shows the UV-VIS absorption spectrum (A) and fluorescence emission spectrum (B,ex340nm,400nm) and the inset is a photograph under 365nm uv light.
FIG. 4 shows the cytotoxicity results of MTT method after incubation of red carbon dots of photosynthetic bacteria with different concentrations in example 2 with Huvec cells for 24 h.
FIG. 5 is a confocal microscope photograph of photosynthetic bacteria obtained in example 2 at a constant concentration (1mg/ml) at excitation wavelengths of 480nm and 540nm, after co-culturing the red-light carbon dots and Lyso-Tracker Green (lysosome green fluorescent probe) with Huvec cells for 10 min.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Example 1
The photosynthetic bacteria culture medium comprises the following components: 3.0g of sodium acetate, 1g of yeast extract, 1.25g of ammonium sulfate, 0.2g of magnesium sulfate, 0.07g of calcium chloride, 0.01g of ferrous sulfate and 0.15g of potassium dihydrogen phosphate, and the mixture is dissolved in water to reach the volume of 1000 mL. And filling the prepared liquid culture medium into a whole 500ml blue-cap reagent bottle, inoculating 10-25% of the photosynthetic bacteria stock solution into the blue-cap reagent bottle, and screwing the bottle cap. The flasks were incubated in a light incubator at 30 deg.C, 75% relative humidity and 7920Lux light intensity for 3 d. Taking 30ml of bacterial liquid under aseptic condition, subpackaging into 50ml of centrifugal tubes, centrifuging at 11000rpm for 10min, discarding supernatant, and collecting red thallus. The cultured photosynthetic bacteria liquid is shown in FIG. 1.
Example 2
A preparation method of photosynthetic bacteria red light carbon dots is characterized in that green sulfur bacteria are used as a precursor, a carbonized product is generated through microwave-assisted heat treatment, and the photosynthetic bacteria red light carbon dots are obtained through extraction and purification.
The preparation method of the specific green sulfur bacteria red light carbon dot comprises the following steps:
1) weighing 2g of green sulfur bacteria, adding into 20mL of deionized water, transferring the mixture into a 100mL beaker, and heating for 10min with high fire in a microwave oven;
2) heating with high fire, gradually cooling the beaker to room temperature, adding 10mL of deionized water, and performing ultrasonic treatment for 30min to obtain a dark green solution;
3) centrifuging the dark green solution obtained in the step 2) at 11000rpm for 10min, removing large particles, collecting supernatant, carrying out suction filtration on the obtained supernatant, removing large-particle insoluble substances by using an organic water-based microporous filter membrane with the aperture of 0.22 mu m during suction filtration, filling the filter membrane into a dialysis bag after removing large-particle insoluble substances, dialyzing the filter membrane in ultrapure water for 72h, and removing small-molecular substances to obtain a green sulfur bacteria red light carbon dot solution, wherein the cut-off molecular weight of the dialysis bag is 1000Da during dialysis in the ultrapure water;
4) and (3) freeze-drying the obtained green sulfur bacterium red light carbon dot solution for 30h by using a freeze-drying device to obtain a solid product, so as to obtain the green sulfur bacterium red light carbon dot.
The transmission electron microscope image of the red-light carbon dot of the green sulfur bacterium prepared in the embodiment is shown in fig. 2, it can be seen that the carbon dot is in a quasi-spherical structure, the size of the carbon dot is about 3.9nm, and the inside of the carbon dot has lattice stripes with a lattice spacing of 0.242nm, which indicates that the red-light carbon dot of the green sulfur bacterium has good crystallinity.
In addition, the ultraviolet-visible absorption spectrum (A) and the fluorescence emission spectrum (B,ex340nm and 400nm), the analysis shows that the fluorescent material shows bright red fluorescence under an ultraviolet lamp of 365nm, and under the excitation of 340nm, the red carbon dot aqueous solution of the green sulfur bacteria has a fluorescence emission peak at 595 nm.
Example 3
The application of the green sulfur bacteria red carbon dots prepared in the embodiment 2 in the biomedical field has a good biological development effect.
Preliminary studies found that, at a concentration of 1mg/ml, cells can rapidly enter and accumulate in the cells, and a good cell visualization effect is shown.
Huvec cell culture conditions: adding 10mL DMEM culture solution containing 10% fetal calf serum and 1% double antibody into 60mm cell culture dish, placing at 37 deg.C and 5% CO2Culturing in a constant temperature incubator. Huvec is adherent cells, when the cells grow to 80%, 1mL of trypsin solution is used for digesting for 2min30s, 4mL of culture solution containing 10% fetal calf serum and 1% double antibody is used for stopping the action of the trypsin, and the cells at the bottom of the bottle are repeatedly blown and beaten to be fully dispersed. Centrifuging at 1000rpm for 5min, discarding supernatant, adding fresh culture medium into cell precipitate, blowing uniformly, and mixing at a speed of 1: 4, transferring the mixture to a new culture flask to continue culturing for later use.
And detecting the cytotoxicity of the prepared green sulfur bacteria red light carbon dots by adopting an MTT method. The MTT method is a common method for detecting cell survival and growth, and the detection principle is as follows: succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT (thiazole blue) into water-insoluble blue-purple crystalline formazan to be deposited in the cells, dead cells do not have the function, then formazan in the cells is dissolved out by DMSO (dimethyl sulfoxide), and the absorbance of the formazan is measured by a microplate reader under the condition of 560nm and 630nm wavelength, so that the survival rate of the cells is reflected.
MTT cytotoxicity test results showed that the results are shown in FIG. 4 after 24h co-culture of Huvec cells (vascular epithelial cells) with different concentrations of the red carbon spot of the green sulfur bacterium. As can be seen from FIG. 4, in the concentration range of 0.1-10000g/ml, the red carbon dot of the green sulfur bacterium has no obvious cytotoxicity to Huvec cells, which indicates that the prepared red carbon dot of the green sulfur bacterium has very low toxicity.
Collecting logarithmic growth phase Huvec cells according to 2X 104The cells/dish were seeded in a glass-bottom culture dish dedicated to laser scanning confocal microscopy (CLSM). Is placed in CO2CulturingAfter culturing for 24h at 37 ℃ in the incubator, absorbing the supernatant of the CLSM culture dish, washing with PBS twice, adding the prepared Lyso-Tracker Green, incubating for 30min, discarding the culture medium, washing with PBS twice, adding the prepared 1mg/ml green sulfur bacteria red light carbon dot solution, and placing back to the incubator to continue culturing for 10 min. After the incubation was completed, the supernatant was removed from the dish, washed three times with PBS and the cells were fixed with 300. mu.L of 4% paraformaldehyde at room temperature for 15 min. PBS was washed three times, stored at 4 ℃ in the dark, observed with a laser confocal microscope and photographed. The results are shown in fig. 5, which indicates that the red carbon spot of the green sulfur bacterium has good biological development potential, and the carbon spot is gathered in the lysosome of the cells.
In conclusion, the preparation method has the advantages of simple reaction steps, low cost, environmental protection and the like, and the prepared photosynthetic bacteria have uniform red light carbon dot particle size, stable structure and fluorescent property.
The photosynthetic bacteria red light carbon point has little influence on the cell activity, simultaneously shows red fluorescence and has good cell developing effect. The photosynthetic bacteria red light carbon dots prepared by taking the photosynthetic bacteria as the starting carbon source have very low cytotoxicity and good cell developing capability, and have potential application prospects in biomedicine.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (9)
1. A preparation method of photosynthetic bacteria red light carbon dots is characterized in that photosynthetic bacteria are used as precursors, carbonized products are generated through microwave-assisted heat treatment, and the photosynthetic bacteria red light carbon dots are obtained through extraction and purification.
2. The method of claim 1, wherein the photosynthetic bacteria include but are not limited to the family Exiguobacteriaceae, Phaeobacterium, Chlorella, Violet, Polyfilamentous Green, Spirobacteriaceae, obligate aerobes containing bacteriochlorophyll.
3. The preparation method of photosynthetic bacteria red light carbon dots according to claim 1, which is characterized by comprising the following steps:
1) weighing 1-5 g of photosynthetic bacteria, adding into 10-50 mL of deionized water, transferring the mixture into a 100mL beaker, and heating for 10-30 min in a microwave oven at a high fire;
2) after heating with high fire, after the beaker is gradually cooled to room temperature, adding 10-30 mL of deionized water, and carrying out ultrasonic treatment for 10-60 min to obtain a dark green solution;
3) centrifuging the dark green solution obtained in the step 2), removing large particles, collecting supernatant, carrying out suction filtration on the obtained supernatant, removing insoluble substances of the large particles, putting the obtained supernatant into a dialysis bag, dialyzing the solution in ultrapure water, and removing small molecular substances to obtain a photosynthetic bacteria red light carbon dot solution;
4) and (3) freeze-drying the obtained photosynthetic bacterium red light carbon dot solution by using a freeze-drying device to obtain a solid product, so as to obtain photosynthetic bacterium red light carbon dots.
4. The method for preparing photosynthetic bacteria red light carbon dots according to claim 1, wherein the centrifugation speed in the step (3) is 10000-11000 rpm, and the centrifugation time is 10-30 min.
5. The method for preparing photosynthetic bacteria red light carbon dots according to claim 1, wherein in the step (3), an organic aqueous microporous filter membrane with a pore size of 0.22 μm is used for suction filtration.
6. The method for preparing photosynthetic bacteria red light carbon dots according to claim 1, wherein in the step (3), when dialyzing in ultrapure water, the cut-off molecular weight of a dialysis bag is 1000Da, and the dialysis time is 48-72 h.
7. The method for preparing photosynthetic bacteria red light carbon dots according to claim 1, wherein in the step (4), the freeze drying time is 24-36 h.
8. A photosynthetic bacterium red light carbon dot obtained by the preparation method of any one of claims 1 to 7.
9. Application of the photosynthetic bacteria red light carbon dots obtained by the preparation method of any one of claims 1 to 7 in the field of biological imaging.
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