CN111248190B - Application of carbon dots as antifreeze material - Google Patents
Application of carbon dots as antifreeze material Download PDFInfo
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- CN111248190B CN111248190B CN202010197719.4A CN202010197719A CN111248190B CN 111248190 B CN111248190 B CN 111248190B CN 202010197719 A CN202010197719 A CN 202010197719A CN 111248190 B CN111248190 B CN 111248190B
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A01N1/02—Preservation of living parts
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention discloses an application of carbon dots as an anti-freezing material, wherein the carbon dots are prepared by taking glucose as a raw material under the condition of high-pressure hydrothermal. The carbon dots have the advantages of rich raw materials, easy preparation, good water solubility, low toxicity, good cell compatibility and the like, so the carbon dots have wide application prospect. The carbon dots prepared by the method have the hydrophilic groups such as hydroxyl and carboxyl on the surface, so that the carbon dots have excellent water solubility. The carbon dots can be adsorbed on the surface of the ice crystal, the carbon dots and the ice interface form a micro-curve, the regrowth of the ice crystal is inhibited, the condition that the cell is damaged by the ice crystal can be effectively avoided in the process of freezing the cell, and the recovery rate of the cell is greatly improved.
Description
Technical Field
The invention belongs to the application field of nano materials, and particularly relates to an application of carbon dots as an anti-freezing material.
Background
In the field of materials, the application of carbon dots is more and more extensive, and the carbon dots are nano materials with excellent dispersity and the size of less than 10 nm. The common preparation methods are mainly divided into two types, one type is a top-down preparation method, and the method comprises the following steps: electrolytic methods, acid etching, laser etching, and the like; another class is bottom-up methods, comprising: hydrothermal method, chemical synthesis method, microwave method, etc. Because the nano-silver nano-particles have the characteristics of abundant raw materials, easiness in preparation, good dispersibility, low toxicity, good biocompatibility, environmental friendliness and the like, the nano-silver nano-particles are increasingly applied to the biological field and the industrial field, have wide application range and attract more and more attention of scientific researchers. However, the anti-freezing property has not been reported yet.
Disclosure of Invention
The invention provides an application of carbon dots as an antifreeze material. In particular, the carbon dots can be used to inhibit ice crystal growth.
The carbon dots have a graphite-like structure, have lattice defects, and have hydrophilic groups on the surface.
According to an embodiment of the invention, the lattice size of the carbon dots is 0.36 nm.
According to an embodiment of the invention, the carbon dot size is 1-20nm, such as 1-10 nm.
According to an embodiment of the invention, the carbon dots are prepared with glucose as substrate.
According to an embodiment of the present invention, the carbon dots are prepared by a hydrothermal method using glucose as a substrate;
according to an embodiment of the invention, the hydrothermal process comprises: dissolving glucose in water to form a glucose solution, stirring for dissolving, reacting at 160-220 deg.C, collecting the obtained product, purifying, and drying.
According to an embodiment of the invention, the concentration of glucose in the glucose solution is 0.1-0.8M;
according to an embodiment of the present invention, the reaction time of the hydrothermal method is between 1 and 7 h.
The invention also provides application of the carbon dots in preparation of a cell cryopreservation reagent.
The present invention also provides a method for cryopreserving cells, comprising: mixing the carbon dot solution with the cells, and freezing and storing at low temperature.
According to an embodiment of the invention, the concentration of carbon dots in the carbon dot solution is 0.1-200mg/mL, such as 1-100mg/mL, preferably 2-50mg/mL, more preferably 5-20 mg/mL.
According to an embodiment of the present invention, the carbon dot solution is a dispersion liquid in which carbon dots are dispersed in water or a buffer.
In the present invention, the cells include, but are not limited to, blood cells, germ cells, stem cells, etc., such as sheep blood cells, horse sperm cells.
The invention further provides a cell cryopreservation reagent which comprises carbon dots with the concentration of 0.1-200 mg/mL.
According to an embodiment of the invention, a buffer, such as PBS buffer, is included in the cell cryopreservation reagent.
The invention also provides an antifreeze agent which comprises the carbon dots.
The invention also provides application of the antifreeze in inhibiting ice crystal growth or freezing and storing cells.
Advantageous effects
The inventor of the invention unexpectedly discovers that the carbon dots have good effect of inhibiting the growth of ice crystals due to the specific lattice structure and a large amount of hydrophilic groups on the surface, the carbon dots can inhibit the recrystallization of ice crystals as an antifreeze material, and have the characteristic of bionic antifreeze protein in function, the antifreeze material can be well adsorbed on the surface of the ice crystals to form micro-curvature on the surface of the ice crystals, and the growth of the ice crystals is further inhibited by virtue of the Kelvin effect. The method for freezing and storing the cells can effectively store the cells, and the recovery rate of the cells after the temperature is recovered can reach 50-100%.
Drawings
FIG. 1 is a transmission electron micrograph of a carbon dot prepared in example 1;
FIG. 2 is an infrared spectrum of a carbon dot prepared in example 1;
FIG. 3 is a UV spectrum of carbon dots prepared in example 1;
FIG. 4 is an X-ray diffraction pattern of carbon dots prepared in example 1;
FIG. 5 is an optical picture of ice crystal growth morphology of a) pure water and b) water-dispersed carbon dots;
FIG. 6 is an optical picture of a) PBS buffer at pH7.4 and b) carbon dot-inhibited recrystallization dispersed in PBS buffer at pH 7.4;
FIG. 7 shows the results of the carbon spot solutions of different concentrations dispersed in PBS buffer pH7.4 and PBS buffer pH7.4 for inhibiting recrystallization of the mean largest ice crystal size;
FIG. 8 is a graph showing the effect of cell cryopreservation in example 3.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Example 1
(1) Preparation of nanocarbon dots (G-CDs)
Dissolving 0.8M glucose in water, stirring thoroughly to dissolve, adding into a reaction kettle, reacting at 220 deg.C for 7h, centrifuging the obtained product three times at 12000r/min for 5min, dialyzing, and vacuum drying.
The transmission electron micrograph of the obtained carbon dots is shown in FIG. 1, the infrared spectrogram is shown in FIG. 2, the ultraviolet spectrogram is shown in FIG. 3, and the X-ray diffraction chart is shown in FIG. 4.
(2) Testing of nano carbon point anti-freezing performance
Ice crystal growth rate test: the morphology change and the growth of the ice crystals with different supercooling degrees (delta T) are observed by a nano-liter osmotic pressure instrument, and the temperature accuracy of the nano-liter osmotic pressure is 0.01 ℃. The nano-liter osmotic pressure is composed of a low-temperature circulating pump and a temperature controller. Generally, a water-dispersed carbon dot solution with a concentration of 10mg/mL is selected and injected into a temperature-controlled well containing silicone oil. The temperature was then lowered to-20 ℃ and after freezing of the carbon point solution, the temperature was subsequently raised. When a single ice crystal occurs and its size can be kept unchanged for 20s, the temperature at this point is called the melting temperature (T)mNot higher than-0.15 deg.c. Then slowly reducing the temperature to a certain value (T)f＝-0.17℃)，Tf-TmWe refer to the degree of supercooling (Δ T ═ 0.02 ℃). The same supercooling degree was tested in triplicate. The growth rate was determined by dividing the ice crystal elongation length by the time elapsed during ice crystal growth and the average was calculated from a video of three parallel experiments. FIG. 5 provides an optical picture of ice crystal growth morphology of a) pure water and b) water-dispersed carbon dots, wherein the ice crystal growth rate in a) pure water is 20 μm s-1B) a water-dispersed carbon dot having a crystal growth rate of 10 μm s-1。
Ice Recrystallisation Inhibition (IRI) activity assay: the test was carried out by the "sputtering freezing method", in which the carbon dots were dissolved in PBS buffer (carbon dot concentration: 20mg/mL) at pH7.4, and the application environment of the carbon dot solution to the cells in the frozen storage was maintained. To determine IRI activity, 10. mu.L of PBS dispersed carbon dot solution droplets 1.5m high were dropped onto a cover glass placed on a Linkam Low temperature chill station (LTS420), the temperature was previously set to-60 ℃, then 10 ℃ min-1The temperature was heated to-6 c at the heating rate of (1) for annealing for 30 minutes. Subsequently, an image of the ice recrystallization was taken with an optical microscope (AZ100, nikon), as shown in fig. 6. FIG. 6a is a recrystallization image of PBS buffer without carbon dots, and FIG. 6b is a recrystallization image of PBS buffer with carbon dots, the ice crystal size after carbon dots are added is obviously smaller than that of PBS buffer without carbon dots, so that the addition of carbon dots can obviously improve the anti-freezing performance of the PBS buffer system, and the effect of inhibiting the growth of ice crystals is achieved.
Ice crystal size was measured using Image J nanometer measurement software. Ten largest individual ice crystals were selected in each image and the process was repeated three times to obtain the average maximum grain size (MLGS). The smaller the MLGS value, the better the IRI activity, and the results are shown in FIG. 7. As can be seen from FIG. 7, the average maximum grain size in the solution system was significantly reduced after the addition of G-CDs compared to the absence of G-CDs, and the smaller the MLGS, the average maximum grain size reached around 60 μm at 20mg/mL, as the concentration of G-CDs increased within a certain range.
Example 2 preparation of a nanocarbon dot solution dispersed in PBS buffer (pH 7.4)
Dissolving 0.8M glucose in water, stirring thoroughly to dissolve, adding into a reaction kettle, reacting at 220 deg.C for 7h, centrifuging the obtained product three times at 12000r/min for 5min, dialyzing, and vacuum drying. 20mg of the carbon dots were added to PBS buffer at a concentration of 20 mg/mL.
Example 3 application experiment of carbon dots
Sheep blood cells are selected as the cells. Selecting 5mL of blood cells, rotating at 4000r/min, centrifuging for 5min, and centrifuging and washing for a plurality of times until the suspension is clear. The obtained blood cells were stored in a refrigerator at 4 ℃ for further use. 50. mu.L of the previously prepared blood cells were collected, and the same volume of the carbon dot solution prepared in example 2 was added thereto at a carbon dot concentration of 20 mg/mL. The final carbon spot concentration containing the cells was 10 mg/mL. And then storing the cells in liquid nitrogen for 8 hours, rewarming the frozen cells at the water bath temperature of 45 ℃, exposing the damaged blood cells to hemoglobin, and then testing the hemoglobin content by ultraviolet to obtain the cell recovery rate, wherein the recovery rate of the cells stored in a pure PBS (phosphate buffer solution) in a freezing way is basically 0% and the recovery rate of the cells stored in a PBS (phosphate buffer solution) dispersed carbon dot in a freezing way is basically 60% in figure 8, thereby providing a new idea for future frozen storage materials.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (16)
1. Use of carbon dots as a freeze resistant material, wherein the carbon dots are used to inhibit ice crystal growth;
the carbon dots are prepared by taking glucose as a substrate through a hydrothermal method; the hydrothermal process comprises: dissolving glucose in water to form a glucose solution, stirring for dissolving, reacting at 160-220 ℃, collecting the obtained product, purifying and drying;
the concentration of glucose in the glucose solution is 0.1-0.8M;
the reaction time of the hydrothermal method is 1-7 h.
2. Use according to claim 1, wherein the carbon dots have a graphite-like structure, have lattice defects and have hydrophilic groups on their surface.
3. Use according to claim 1, wherein the carbon dot size is 1-20 nm.
4. Use according to claim 1, wherein the lattice size of the carbon dots is 0.36 nm.
5. A method of cryopreserving cells, comprising: mixing the carbon dot solution with cells, and freezing and storing at low temperature;
wherein the carbon dots are prepared by a hydrothermal method by taking glucose as a substrate; the hydrothermal process comprises: dissolving glucose in water to form a glucose solution, stirring for dissolving, reacting at 160-220 ℃, collecting the obtained product, purifying and drying;
the concentration of glucose in the glucose solution is 0.1-0.8M;
the reaction time of the hydrothermal method is 1-7 h.
6. The method of claim 5, wherein the concentration of carbon dots in the carbon dot solution is from 0.1 to 200 mg/mL.
7. The method of claim 5, wherein the concentration of carbon dots in the carbon dot solution is 1-100 mg/mL.
8. The method of claim 5, wherein the concentration of carbon dots in the carbon dot solution is 2-50 mg/mL.
9. The method of claim 5, wherein the concentration of carbon dots in the carbon dot solution is 5-20 mg/mL.
10. The method according to claim 5, wherein the carbon dot solution is a dispersion of carbon dots in water or a buffer;
the cells are blood cells, germ cells and stem cells.
11. The method of claim 5, wherein the cells are sheep blood cells, horse sperm cells.
12. The carbon dots are used for preparing a cell cryopreservation reagent, wherein the carbon dots are prepared by using glucose as a substrate through a hydrothermal method; the hydrothermal process comprises: dissolving glucose in water to form a glucose solution, stirring for dissolving, reacting at 160-220 ℃, collecting the obtained product, purifying and drying;
the concentration of glucose in the glucose solution is 0.1-0.8M;
the reaction time of the hydrothermal method is 1-7 h.
13. A cell cryopreservation reagent, wherein the cell cryopreservation reagent comprises carbon dots at a concentration of 0.1-200 mg/mL;
wherein the carbon dots are prepared by a hydrothermal method by taking glucose as a substrate; the hydrothermal process comprises: dissolving glucose in water to form a glucose solution, stirring for dissolving, reacting at 160-220 ℃, collecting the obtained product, purifying and drying;
the concentration of glucose in the glucose solution is 0.1-0.8M;
the reaction time of the hydrothermal method is 1-7 h.
14. The cell cryopreservation reagent of claim 13 wherein the cell cryopreservation reagent comprises a buffer.
15. An antifreeze agent, which is characterized by comprising carbon dots, wherein the carbon dots are prepared by a hydrothermal method by taking glucose as a substrate; the hydrothermal process comprises: dissolving glucose in water to form a glucose solution, stirring for dissolving, reacting at 160-220 ℃, collecting the obtained product, purifying and drying;
the concentration of glucose in the glucose solution is 0.1-0.8M;
the reaction time of the hydrothermal method is 1-7 h.
16. Use of an antifreeze agent according to claim 15 for inhibiting ice crystal growth or cryopreserving cells.
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