Carbon dots for detecting activity of various cells based on different fluorescence colors as well as preparation method and application of carbon dots

Abstract

The invention discloses a carbon dot for detecting activity of multiple cells based on different fluorescence colors and a preparation method and application thereof, the carbon dot is prepared from a carbon source, a nitrogen source and a boron source, and the molar ratio of the carbon source to the nitrogen source to the boron source is (80-130): (85-180): (1-90); the carbon source is organic acid, the nitrogen source is ethidene diamine, and the boron source is boric acid, phenylboronic acid or borax. A carbon source, a nitrogen source and a boron source are used as precursor substances to prepare the boron-nitrogen co-doped carbon dots, the boron-nitrogen co-doped carbon dots are used for marking various cells, the carbon dots can enable cells with different activities to present different fluorescent colors under the same exciting light, living cells are blue under ultraviolet excitation, dead cells are green under ultraviolet excitation, and the carbon dots can be used for marking various cells. Live cells are green and dead cells are yellow under the excitation of blue light; living cells are dark red under the excitation of green light, and dead cells are bright red. The detection speed is high, the detection cost is low, the operation is simple and convenient, and the cell activity can be quickly and accurately judged clearly according to the fluorescence color.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell viability detection, and relates to a carbon dot and its preparation method and application, specifically to a carbon dot for detecting the viability of multiple cells based on different fluorescence colors, its preparation method, and its application in cell viability detection. Background Art

[0002] Detecting the viability of cells can help understand the growth of cells, the impact of the growth environment on cell viability, and the sensitivity and tolerance of cells to certain substances. In addition, detecting the viability of certain fermenting agents and probiotic products can ensure the production efficiency of fermenting agents, the functions of probiotics, and the quality of products.

[0003] There are many traditional methods for detecting cell viability. Commonly used methods for detecting cell viability include colony formation assay, chemical staining, fluorescence staining, and colorimetric assay. Among them: the colony formation assay is easily affected by culture conditions and takes a long time; the chemical staining method has strict requirements for staining time and cannot stain dead cells and live cells simultaneously; for fluorescence staining methods, such as the double staining method of carboxyfluorescein diacetate (FDA) and propidium iodide (PI), the fluorescence stability of the dyes is poor, they are highly toxic, and expensive; although the colorimetric assay has high sensitivity, it cannot detect live cells and dead cells simultaneously. Therefore, it is very meaningful to find a convenient, inexpensive, accurate, and rapid method for detecting cell viability for application in cell viability detection.

[0004] Carbon dots are a popular fluorescent nanomaterial and have been widely used in the fields of food, biology, medicine, optics, etc. due to their excellent properties. Compared with traditional fluorescent dyes, carbon dots have the advantages of simple synthesis methods, low manufacturing costs, stable fluorescence properties, and low biological toxicity. They are good alternatives to traditional fluorescent dyes and are widely used in bioimaging and detection. Summary of the Invention

[0005] The purpose of the present invention is to provide a carbon dot for detecting the viability of multiple cells based on different fluorescence colors, its preparation method, and its application, which are low-cost, high-efficiency, and storage-resistant to meet the needs of detecting the viability of multiple cells. The present invention uses a carbon source, a nitrogen source, and a boron source as precursor substances to prepare boron and nitrogen co-doped carbon dots (BN-CDs), and uses them to label multiple cells, enabling dead and live cells to emit fluorescence of different colors to quickly and accurately distinguish the dead and alive states of cells, providing a new detection method for cell viability detection.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A carbon dot for detecting the viability of multiple cells based on different fluorescence colors, which is prepared from a carbon source, a nitrogen source, and a boron source, wherein:

[0008] The molar ratio of the carbon source, nitrogen source, and boron source is 80 - 130:85 - 180:1 - 90;

[0009] The carbon source is an organic acid, such as citric acid, malic acid, etc.;

[0010] The nitrogen source is ethylenediamine, ammonia water, etc.;

[0011] The boron source is boric acid, phenylboronic acid, borax, or other boron-containing compounds, etc.

[0012] A preparation method of the above carbon dots for detecting the activities of multiple cells based on different fluorescence colors includes the following steps:

[0013] Step (1): Add the carbon source, nitrogen source, and boron source to a high-pressure reaction kettle for thermal reaction, where:

[0014] The temperature of the thermal reaction is 160 - 260 °C, and the time is 3 - 8 h;

[0015] The solvent used for the thermal reaction is water or a 33 - 66% ethanol solution;

[0016] Step (2): Centrifuge the reaction solution obtained in step (1), and take the supernatant and add it to a dialysis bag for dialysis, where: the centrifugation speed is 1000 - 5000 r / min, and the time is 5 - 10 min; dialysis is carried out using a dialysis bag with a cut-off molecular weight of 500 - 2000 Da, the dialysis solution is ultrapure water, the dialysis time is 24 - 48 h, and the dialysis solution is changed every 4 - 8 h;

[0017] Step (3): Lyophilize the dialysis solution obtained in step (2) to obtain boron and nitrogen co-doped carbon dots.

[0018] The above carbon dots can make cells with different activities show different fluorescence colors under the same excitation light, and the cell activities can be detected quickly and accurately through the fluorescence colors, where:

[0019] The cells include animal cells, plant cells, fungi, and bacteria;

[0020] The carbon dots can be imaged and detected after being mixed with cells for 1 - 10 min;

[0021] When the carbon dots stain bacteria, the imaging concentration is 50 - 1000 μg / mL; when staining animal cells, the imaging concentration is 1000 - 3000 μg / mL; when staining fungi, the imaging concentration is 2000 - 6000 μg / mL; when staining plant cells, the imaging concentration is 5000 - 20000 μg / mL;

[0022] During the imaging detection process, the different fluorescence colors presented by different living cells under the same excitation light are as follows: under ultraviolet excitation, living cells are blue and dead cells are green; under blue light excitation, living cells are green and dead cells are yellow; under green light excitation, living cells are dark red and dead cells are bright red.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. Fast detection speed, low detection cost, and simple operation;

[0025] 2. Can stain living and dead cells simultaneously, and clearly judge cell viability according to the fluorescence color;

[0026] 3. Can detect the viability of various cells, including animal cells, plant cells, fungi, and bacteria. Description of the Drawings

[0027] Figure 1 It is a schematic diagram for the preparation of BN-CDs and an effect diagram for detecting cell viability;

[0028] Figure 2 It is the 3D spectra and TEM images of BN-CDs at different concentrations (24, 240, 2400 μg / mL);

[0029] Figure 3 It is the Fourier transform infrared spectrum and X-ray diffraction spectrum of BN-CDs, a: Fourier transform infrared spectrum of BN-CDs, b: X-ray diffraction spectrum of BN-CDs;

[0030] Figure 4 It is the fluorescence stability and toxicity effect diagrams of BN-CDs, a: fluorescence intensity change of BN-CDs solution under continuous ultraviolet light irradiation for 120 min, b: fluorescence intensity change of BN-CDs solution stored in the dark for 30 d, c: growth curve of Lactobacillus cultured in medium containing different concentrations of BN-CDs, d: colony plate map of Lactobacillus cultured in medium containing different concentrations of BN-CDs;

[0031] Figure 5 It is the imaging diagram of different viable cells stained with the optimal concentration of BN-CDs. Detailed Embodiments

[0032] The technical solutions of the present invention will be further described below in conjunction with the drawings, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the protection scope of the present invention.

[0033] Example 1. Preparation and Characterization of BN-CDs:

[0034] (1) Preparation method of BN-CDs:

[0035] As Figure 1 shown, 3.3 g of citric acid, 1.2 mL of ethylenediamine, and 0.05 g of phenylboronic acid were dissolved in 30 mL of 33-66% ethanol. The mixed solution was added to a polytetrafluoroethylene-lined autoclave and heated at 180 °C for 5 h. After cooling, impurities were removed by centrifugation at 2000 r / min for 10 min. The supernatant was taken and added to a 1000 Da dialysis bag for dialysis for 24 h, and the dialysis solution was changed every 6 h. After dialysis, the BN-CDs powder was obtained by freeze-drying.

[0036] (2) Characterization of BN-CDs:

[0037] 1) Fluorescence spectrum: BN-CDs were dissolved in deionized water to prepare pure water dispersions with concentrations of 24 μg / mL, 240 μg / mL, and 2400 μg / mL. The 3D spectrum of the BN-CDs solution was measured using a fluorescence spectrophotometer. The results showed that as the concentration of BN-CDs increased, the fluorescence center would undergo a red shift ( Figure 2 a-c).

[0038] 2) Particle size measurement: BN-CDs were dissolved in deionized water to prepare pure water dispersions with concentrations of 24 μg / mL, 240 μg / mL, and 2400 μg / mL, and then TEM scanning was performed. The results showed that as the concentration of BN-CDs increased, BN-CDs aggregated, and the particle size increased from 0.5 nm to 2.3 nm ( Figure 2 d-f).

[0039] 3) Fourier transform infrared spectroscopy: The surface structure of BN-CDs was analyzed using a Fourier transform infrared spectrometer. The results showed that the surface of BN-CDs had rich functional groups, which was beneficial for its binding to cells ( Figure 3 a).

[0040] 4) Electron energy spectrum: An X-ray diffraction device was used to analyze the elemental composition of BN-CDs. The results showed that the elements contained in BN-CDs were C, O, N, and B, and the element ratios were 69.59%, 17.07%, 12.95%, and 0.38% respectively ( Figure 3 b).

[0041] Example 2. Fluorescence stability and toxicity of BN-CDs:

[0042] (1) Detection of fluorescence stability of BN-CDs:

[0043] The BN-CDs solution was irradiated under an ultraviolet lamp for 120 min, and the fluorescence intensity at the fluorescence center was measured every 20 min by a fluorescence spectrophotometer. The results showed that the fluorescence intensity of the BN-CDs solution could still reach 90.9% of the fluorescence intensity of the initial sample after 120 min of continuous irradiation( Figure 4 a). The fluorescence intensity at the fluorescence center of BN-CDs was measured after storing in the dark environment for 30 d. The results showed that the fluorescence intensity at the fluorescence center after storing in the dark environment for 30 d was 65.9% of the initial sample( Figure 4 b). It indicated that BN-CDs had strong fluorescence stability and the advantage of being storable for a long time.

[0044] (2) Toxicity detection of BN-CDs:

[0045] The toxicity of BN-CDs was tested using Lactobacillus. Lactobacillus was cultured in MRS medium containing different concentrations of BN-CDs (0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL) at 37 °C for 24 h. The OD value of the culture solution was measured at 600 nm every 5 h using an ultraviolet-visible spectrophotometer, and the growth curve of Lactobacillus was plotted. The results showed that BN-CDs had almost no toxicity to Lactobacillus( Figure 4 c). 100 μL of the above-mentioned stationary-phase bacterial solution was evenly spread on the MRS medium plate and cultured at 37 °C for 24 h, and the growth situation was observed. The results showed that even when the concentration of BN-CDs reached 800 μg / mL, it would not affect the growth of Lactobacillus( Figure 4 d). Therefore, when using BN-CDs to detect the activity of Lactobacillus, the detection results would not be affected by the death of Lactobacillus caused by the toxicity of BN-CDs.

[0046] Example 3. Fluorescence imaging of BN-CDs for detecting cell activity:

[0047] (1) Preparation of cell samples:

[0048] After centrifuging to remove impurities, the Lactobacillus, Escherichia coli, Staphylococcus aureus, macrophages, and yeast cells to be detected were resuspended in phosphate buffer solution, mixed with BN-CDs, and then 10 μL was smeared on a microscope slide. After the sample was fixed, it was observed under a fluorescence microscope. The onion epidermis was placed on a microscope slide, and the BN-CDs solution was added dropwise. After the sample was fixed, it was observed under a fluorescence microscope.

[0049] (2) Optimal imaging concentration of BN-CDs:

[0050] When applying BN-CDs to stain cells, it is necessary to ensure that the concentration of BN-CDs is sufficient to satisfy the emission of two different colors of light from live and dead cells, and also to ensure that the background is not affected by too high a concentration of BN-CDs. In this example, when staining Lactobacillus, the optimal imaging concentration of BN-CDs is 200-400 μg / mL; when staining Escherichia coli, the optimal imaging concentration of BN-CDs is 50-100 μg / mL; when staining Staphylococcus aureus, the optimal imaging concentration of BN-CDs is 100-200 μg / mL; when staining macrophages, the optimal imaging concentration of BN-CDs is 1000-1500 μg / mL; when staining yeast, the optimal imaging concentration of BN-CDs is 2000-3000 μg / mL; when staining onions, the optimal imaging concentration of BN-CDs is 5000-10000 μg / mL.

[0051] (3) Detection of the viability of various cells by BN-CDs:

[0052] Fluorescence microscope images of different viable cells stained with the optimal concentration of BN-CDs are shown as Figure 5 follows. It can be seen from Figure 5 that BN-CDs can clearly perform multi-color imaging on various cells. Under ultraviolet excitation, blue light excitation, and green light excitation, cells in different viable states can emit different colors of emission light: live cells are blue and dead cells are green under ultraviolet excitation; live cells are green and dead cells are yellow under blue light excitation; live cells are dark red and dead cells are bright red under green light excitation. The viability state of cells can be judged by the color of the emission light. Among them, the color difference of the emission light between live and dead cells is the largest under ultraviolet excitation, and the detection effect is the best.

[0053] Example 4:

[0054] The difference between this example and Example 1 is that the carbon source is malic acid, and the molar ratio of malic acid, ethylenediamine, and phenylboronic acid is 125:90:2, that is, 3.35 g of malic acid, 1.2 mL of ethylenediamine, and 0.05 g of phenylboronic acid are added.

[0055] Example 5:

[0056] The difference between this example and Example 1 is that the carbon source is citric acid, the nitrogen source is ammonia water, and the boron source is phenylboronic acid. The molar ratio of citric acid, ammonia water, and phenylboronic acid is 85:180:2, that is, 3.3 g of citric acid, 1.39 mL of ammonia water, and 0.05 g of phenylboronic acid.

[0057] Example 6:

[0058] The difference between this embodiment and Embodiment 1 is that the carbon source is citric acid, the nitrogen source is ammonia water, the boron source is boric acid, the solvent is water, and the molar ratio of citric acid, ammonia water, and boric acid is 85:180:90, that is: 3.3 g of citric acid, 1.39 mL of ammonia water, and 1.1 g of boric acid are added.

Claims

1. A carbon dot for detecting multiple cell activities based on different fluorescent colors, characterized in that The carbon dots are prepared from a carbon source, a nitrogen source and a boron source, wherein the molar ratio of the carbon source, the nitrogen source and the boron source is 80-130:85-180:1-90; the carbon source is an organic acid, the nitrogen source is ethylenediamine or ammonia water, and the boron source is boric acid, phenylboric acid or borax.

2. The carbon dots for detecting multiple cell activities based on different fluorescent colors according to claim 1, characterized in that The organic acid is citric acid or malic acid.

3. A method for preparing carbon dots for detecting multiple cell activities based on different fluorescent colors according to any one of claims 1 to 2, characterized in that The method comprises the following steps: Step (1) adding a carbon source, a nitrogen source and a boron source into a high pressure reactor for thermal reaction; Step (2) centrifuging the reaction solution obtained in step (1) and adding the supernatant to a dialysis bag for dialysis; Step (3) freeze-drying the dialysate obtained in step (2) to obtain boron-nitrogen co-doped carbon dots.

4. The method for preparing carbon dots for detecting multiple cell activities based on different fluorescent colors according to claim 3, characterized in that The temperature of the thermal reaction is 160-260° C., and the time is 3-8 hours; the solvent used in the thermal reaction is water or 33-66% ethanol solution.

5. The method for preparing carbon dots for detecting multiple cell activities based on different fluorescent colors according to claim 3, characterized in that The centrifugal speed is 1000-5000 r / min, and the time is 5-10 min.

6. The method for preparing carbon dots for detecting multiple cell activities based on different fluorescent colors according to claim 3, characterized in that The dialysis is performed using a dialysis bag with a molecular weight cutoff of 500 to 2000 Da, the dialysis fluid is ultrapure water, the dialysis time is 24 to 48 hours, and the dialysis fluid is replaced every 4 to 8 hours.

7. Use of the carbon dots according to any one of claims 1 to 2 in rapid detection of cell activity.

8. The use of carbon dots in rapid detection of cell activity according to claim 7, characterized in that The cells include animal cells, plant cells, fungi and bacteria.

9. The use of carbon dots in rapid detection of cell activity according to claim 8, characterized in that When the carbon dots are used to stain bacteria, the imaging concentration is 50-1000 μg / mL; when they are used to stain animal cells, the imaging concentration is 1000-3000 μg / mL; when they are used to stain fungi, the imaging concentration is 2000-6000 μg / mL; when they are used to stain plant cells, the imaging concentration is 5000-20000 μg / mL.

10. The use of carbon dots in rapid detection of cell activity according to claim 7, characterized in that The carbon dots can make cells with different activities present different fluorescence colors under the same excitation light, and the cell activity can be detected quickly and accurately through the fluorescence color, wherein: under ultraviolet excitation, live cells are blue and dead cells are green; under blue light excitation, live cells are green and dead cells are yellow; under green light excitation, live cells are dark red and dead cells are bright red.

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