A kind of carbon quantum dots based on Rhodiola rosea and its preparation method and application

Abstract

The present invention relates to the technical field of carbon quantum dots, and specifically to a carbon quantum dot based on Rhodiola rosea, and a preparation method and application thereof. The present invention uses Rhodiola rosea as a raw material, mixes it with ultrapure water, performs a high-temperature reaction and cools it, and then lyophilizes it after dilution with ultrapure water, filtration, and dialysis to obtain water-soluble Rhodiola rosea carbon quantum dots. The residue obtained by filtration is diluted with ethanol, filtered, purified, and then lyophilized to obtain alcohol-soluble Rhodiola rosea carbon quantum dots. The present invention is the first to use the Tibetan medicine Rhodiola rosea as a carbon source to successfully prepare water-soluble Rhodiola rosea carbon quantum dots and alcohol-soluble Rhodiola rosea carbon quantum dots with good fluorescence properties. The prepared Rhodiola rosea carbon quantum dots are minimally affected by physiological pH and have good fluorescence stability, which is conducive to the application in cell imaging under special environments, and has a good effect when applied to cell imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of carbon quantum dots, and in particular to carbon quantum dots based on Rhodiola rosea, and a preparation method and application thereof. Background Art

[0002] Tibetan medicine boasts a long history and a well-developed theoretical framework, with a wealth of classic medical texts constituting a unique corpus of Tibetan herbal medicine. However, modern research on Tibetan medicines originating from the cold, high-altitude, and low-oxygen environment of the Qinghai-Tibet Plateau is severely lacking. To further develop and preserve traditional Tibetan medicine, it is crucial to access the treasure trove of Tibetan medicinal plants, accelerate the modernization of Tibetan medicinal plant research, and explore the synthesis and characterization of nanomaterials and study their properties.

[0003] Rhodiola rosea is the dried root and rhizome of the Crassulaceae plant Rhodiola rosea. It contains a high concentration of chemical components such as salidroside, tyrosol, gallic acid, pyrogallic acid, and β-sitosterol. Carbon quantum dots (CQDs) are carbon-based biomaterials with fluorescent properties, primarily composed of carbon, hydrogen, and oxygen. CQDs typically have a core consisting of an sp2 / sp3 hybrid atomic domain with a π-conjugated structure. Their surface is rich in functional groups such as hydroxyl, carboxyl, and amino groups. They were first discovered in 2004.

[0004] Carbon quantum dots were first artificially synthesized using laser ablation in 2006. Currently, the main methods for synthesizing carbon quantum dots are top-down and bottom-up. Carbon quantum dots (CQDs) are sometimes also referred to as carbon dots (CDs), but both names are acceptable.

[0005] The synthesis of Chinese herbal carbon quantum dots usually uses hydrothermal method and high-temperature pyrolysis method. Chinese herbal carbon quantum dots are carbon quantum dots synthesized and purified by a series of methods using Chinese herbal extracts or Chinese herbal powders, and have high antioxidant activity. With the continuous deepening of research on carbon quantum dots, the various functions of Chinese herbal carbon quantum dots have been continuously developed. In 2018, some scholars used ginkgo leaves to synthesize a type of carbon quantum dots that can be loaded with ruthenium nanoparticles as an efficient electrocatalyst for hydrogen production in alkaline media. The application of Chinese herbal carbon quantum dots is becoming more and more extensive. Currently, there are applications in metal ion detection, cell imaging, disease treatment, electrode material production, etc. Summary of the Invention

[0006] To comprehensively address the above issues, the present invention proposes carbon quantum dots based on Rhodiola rosea, their preparation method, and applications. For the first time, using the Tibetan medicinal plant Rhodiola rosea as a carbon source, water-soluble and alcohol-soluble Rhodiola rosea carbon quantum dots with excellent fluorescence properties were successfully prepared. The prepared Rhodiola rosea carbon quantum dots are minimally affected by physiological pH and exhibit excellent fluorescence stability, facilitating their application in cell imaging under specialized conditions, with excellent results.

[0007] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides carbon quantum dots based on Rhodiola rosea. Rhodiola rosea is used as a raw material, mixed with ultrapure water, subjected to high-temperature reaction and cooled, diluted with ultrapure water, filtered, dialyzed and then freeze-dried to prepare water-soluble Rhodiola rosea carbon quantum dots. The residue obtained by filtration is diluted with ethanol, filtered, purified and then freeze-dried to obtain alcohol-soluble Rhodiola rosea carbon quantum dots.

[0008] The second aspect of the present invention provides a method for preparing carbon quantum dots based on Rhodiola rosea, comprising:

[0009] Step 1: Prepare Rhodiola rosea powder;

[0010] Step 2: Mix the Rhodiola rosea powder prepared in step 1 with ultrapure water, react at high temperature, cool to room temperature, dilute with ultrapure water, filter, and dialyze, and freeze-dry the filtrate to obtain water-soluble Rhodiola rosea carbon quantum dots;

[0011] Step 3: The filter residue obtained after filtering in step 2 is diluted, filtered, purified, and freeze-dried to obtain alcohol-soluble Rhodiola rosea carbon quantum dots.

[0012] Preferably, the water-soluble Rhodiola rosea carbon quantum dots in step 2 have a lattice fringe spacing of 0.21 nm or 0.36 nm and are doped with nitrogen.

[0013] Preferably, the alcohol-soluble Rhodiola rosea carbon quantum dots in step 3 have a lattice fringe spacing of 0.21 nm or 0.36 nm.

[0014] Preferably, the ratio of Rhodiola rosea powder to ultrapure water in step 2 is 1:30-100.

[0015] Preferably, the temperature of the high-temperature reaction in step 2 is 160° C. to 220° C., the heating time is 6 h to 10 h, and the stirring speed is 0 r / min to 1000 r / min.

[0016] A fourth aspect of the present invention provides an alcohol-soluble Rhodiola rosea carbon quantum dots for use in cell imaging.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. This invention uses the Tibetan medicine Rhodiola rosea as a carbon source for the first time and successfully prepares water-soluble Rhodiola rosea carbon quantum dots and alcohol-soluble Rhodiola rosea carbon quantum dots with good fluorescence properties.

[0019] 2. The Rhodiola rosea carbon quantum dots prepared by the present invention are minimally affected by physiological pH and have good fluorescence stability, which is conducive to the application in cell imaging under special environments.

[0020] 3. The Rhodiola rosea carbon quantum dots prepared by the present invention are nano-fluorescent materials, and their distribution and metabolic status in the body can be tracked by fluorescence.

[0021] 4. Both Rhodiola rosea carbon quantum dots prepared in the present invention have good biocompatibility and are effective in cell imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0023] In the attached figure:

[0024] Figure 1 It is the technical route for the preparation of Rhodiola rosea carbon quantum dots;

[0025] Figure 2 It is the biological imaging technology route of Rhodiola rosea carbon quantum dots;

[0026] Figure 3 Transmission electron microscopy images of Rhodiola rosea carbon quantum dots: (a) TEM image and particle size distribution of RWW-CDs; (b) TEM image and particle size distribution of RWE-CDs; (c) HRTEM image and lattice spacing of RWW-CDs; (d) HRTEM image and lattice spacing of RWE-CDs; (e) particle size distribution of RWW-CDs; (f) particle size distribution of RWE-CDs;

[0027] Figure 4 X-ray diffraction patterns of Rhodiola rosea carbon quantum dots: (a) XRD diffraction pattern of RWW-CDs; (b) XRD diffraction pattern of RWE-CDs;

[0028] Figure 5 UV-visible spectra of Rhodiola rosea carbon quantum dots: (a) UV-visible spectrum of RWW-CDs solution; (b) UV-visible spectrum of RWE-CDs solution; (c) state of RWW-CDs solution under natural light and 365nm UV excitation; (d) state of RWE-CDs solution under natural light and 365nm UV excitation;

[0029] Figure 6 Fourier infrared spectra of Rhodiola rosea carbon quantum dots: (a) is the Fourier infrared spectrum of RWW-CDs; (b) is the Fourier infrared spectrum of RWE-CDs;

[0030] Figure 7X-ray photoelectron spectra and peak fitting spectra of Rhodiola rosea carbon quantum dots: (a) XPS spectrum of RWW-CDs; (b) XPS spectrum of RWE-CDs; (c), (e), and (g) are the peak fitting spectra of C, N, and O elements of RWW-CDs, respectively; (d), (f), and (h) are the peaks of C, N, and O elements of RWE-CDs, respectively;

[0031] Figure 8 Toxicity test of Rhodiola rosea carbon quantum dots (the solvent for dissolving carbon quantum dots was DMSO, the initial concentration of the stock solution was 10 mg / mL, p≤0.0001 was indicated by ****; p≤0.001 was indicated by ***; p≤0.01 was indicated by **; p≤0.05 was indicated by *; p>0.05 was indicated by ns);

[0032] Figure 9 Relationship between the fluorescence intensity of Rhodiola rosea carbon quantum dots and pH: (a) Relationship between the fluorescence intensity of RWW-CDs and pH; (b) Relationship between the fluorescence intensity of RWE-CDs and pH;

[0033] Figure 10 Biological imaging of Rhodiola rosea carbon quantum dots (scale bars are 20 μm). DETAILED DESCRIPTION

[0034] The following combination Figures 1-10 The preferred embodiments of the present invention are described herein. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0035] Example 1:

[0036] The invention discloses carbon quantum dots based on Rhodiola rosea. The raw material of the carbon quantum dots is Rhodiola rosea, which is mixed with ultrapure water, subjected to high-temperature reaction and cooled. The carbon quantum dots are diluted with ultrapure water, filtered, dialyzed and then freeze-dried to obtain water-soluble Rhodiola rosea carbon quantum dots (RWW-CDs, Water-phase Rhodiola carbon quantum dots synthesized in water by hydrothermal synthesis). The residue obtained by filtration is diluted with ethanol, filtered, purified and then freeze-dried to obtain alcohol-soluble Rhodiola rosea carbon quantum dots (RWE-CDs, Ethanol-phase Rhodiola carbon quantum dots synthesized in water by hydrothermal synthesis).

[0037] Example 2:

[0038] A method for preparing carbon quantum dots based on Rhodiola rosea, comprising:

[0039] Step 1: grinding the dried Tibetan medicine Rhodiola rosea to prepare Rhodiola rosea powder;

[0040] Step 2: The Rhodiola rosea powder from step 1 is mixed with ultrapure water in a mass ratio of 1:30 to 100, and subjected to a high-temperature reaction (conditions: temperature of 160°C to 220°C, heating time of 6h to 10h, and stirring speed of 0r / min to 1000r / min. This can accelerate the polymerization reaction between small molecules). After cooling to room temperature, the mixture is diluted with ultrapure water, filtered, dialyzed, and freeze-dried to obtain water-soluble Rhodiola rosea carbon quantum dots. The lattice fringe spacing of the water-soluble Rhodiola rosea carbon quantum dots is 0.21nm or 0.36nm, that is, the lattice spacing is 0.36nm and 0.21nm, corresponding to the (002) and (100) crystal planes of hexagonal graphite, respectively. The water-soluble Rhodiola rosea contains nitrogen doping, which helps to achieve red shift, and after the excitation wavelength changes, the emission wavelength will shift to the infrared region.

[0041] Step 3: The filter residue obtained after filtering in Step 2 is diluted (ethanol, methanol, propanol, etc.), filtered, purified, and then freeze-dried to obtain alcohol-soluble Rhodiola rosea carbon quantum dots. The alcohol-soluble Rhodiola rosea carbon quantum dots have a lattice fringe spacing of 0.21 nm or 0.36 nm, i.e., lattice spacings of 0.36 nm and 0.21 nm, corresponding to the (002) and (100) crystal planes of hexagonal graphite, respectively. The alcohol-soluble Rhodiola rosea carbon quantum dots are doped with nitrogen at a content of 0.24%, which helps achieve red shift, and after the excitation wavelength is changed, the emission wavelength will shift to the infrared region.

[0042] Example 3:

[0043] A method for preparing carbon quantum dots based on Rhodiola rosea, comprising:

[0044] Step 1: Grind 2.5 g of dried Tibetan medicine Rhodiola rosea to prepare 2.5 g of Rhodiola rosea powder;

[0045] Step 2: Mix 2.5 g of Rhodiola rosea powder from step 1 with 120 mL of ultrapure water in a hydrothermal reactor ceramic lining according to the ratio, maintain a high temperature reaction at 200°C and 600 r / min for 480 min, then cool to room temperature, transfer the liquid in the reactor to a storage container, add ultrapure water to 300 mL for dilution, ultrasonically disperse for 90 min, filter through a 0.45 μm filter membrane, and collect the residue for extraction of alcohol-soluble carbon quantum dots.

[0046] The filtrate was filtered through a 0.22 μm filter membrane and dialyzed using a 500 Da dialysis bag. The water was changed every 240 minutes until the dialyzate became transparent. The liquid in the dialysis bag was concentrated and freeze-dried at -60°C to obtain a water-soluble Rhodiola rosea carbon quantum dot powder. The water-soluble Rhodiola rosea carbon quantum dot powder had a lattice fringe spacing of 0.21 nm or 0.36 nm and was nitrogen-doped.

[0047] Step 3: The filter residue obtained after filtering in step 2 is diluted (ethanol, methanol, propanol, etc.), filtered, purified, and freeze-dried to obtain alcohol-soluble Rhodiola rosea carbon quantum dots. The alcohol-soluble Rhodiola rosea carbon quantum dots have a lattice fringe spacing of 0.21 nm or 0.36 nm.

[0048] Example 3:

[0049] Water-soluble Rhodiola rosea carbon quantum dots for cell imaging.

[0050] Example 4:

[0051] Alcohol-soluble Rhodiola rosea carbon quantum dots for cell imaging.

[0052] The water-soluble or alcohol-soluble Rhodiola rosea carbon quantum dots prepared in Example 3 or Example 4 were incubated with cells or bacteria for 1 to 12 hours, and free carbon quantum dots were washed away with PBS. The images were then imaged under a laser confocal microscope. Under UV and laser excitation, cells or bacteria that maintained membrane integrity exhibited a strong fluorescence response. Cell imaging of Rhodiola rosea carbon quantum dots revealed three colors: green, blue, and red.

[0053] test:

[0054] 1. Characterization of Rhodiola rosea Carbon Quantum Dots

[0055] The spectrum of the carbon quantum dot solution was scanned using a CARY-5000 visible-UV spectrophotometer from VARIAN, Australia; the fluorescence spectrum was scanned using a Fluorolog-QM fluorescence spectrometer from HORIBA, Canada; the TEM images were taken using a HT7700 transmission electron microscope from Hitachi, Japan; the X-ray photoelectron spectrum was measured using an ESCALAB Xi+ X-ray photoelectron spectrometer from Thermo Fisher Scientific, USA; the Fourier infrared spectrum was measured using an FTIR-5700 Fourier infrared spectrometer from Thermo Fisher Scientific, USA; and the θ / 2θ diffraction angle was measured using a SmartLab-3kW X-ray diffractometer from Rigaku Co., Ltd., Japan.

[0056] (1) Transmission electron microscopy characterization Figure 3 As shown in the figure, the average particle size of RWW-CDs is (13.17±0.26) nm, while the average particle size of RWE-CDs is (39.67±0.83) nm. The lattice fringe spacing of RWW-CD is 0.213 nm, while the lattice fringe spacing of RWE-CD is 0.22 nm.

[0057] (2) XRD characterization Figure 4 As shown in the figure, both RWW-CDs and RWE-CDs have the highest peak near 22°. The main peak of RWW-CDs appears at 20.04-21.68°, while the main peak of RWE-CDs appears at 19.05-21.35°, with a single peak at 21.42°.

[0058] (3) UV-visible spectra of RWW-CDs and RWE-CDs. Strong UV absorption peaks were observed at 250-275 nm and 360 nm for RWW-CDs, and at 250-280 nm and 370 nm for RWE-CDs. These two strong UV absorption regions are due to the π→π* transitions of sp2 conjugated electrons in aromatic C=C bonds and the n→π* transitions of C=O / C=N. Both RWW-CDs and RWE-CDs are yellow under natural light and emit strong blue-green fluorescence under 365 nm UV light.

[0059] (4) FTIR and XPS Figure 6 and Figure 7 As shown, RWW-CDs have functional groups such as hydroxyl, amino, aromatic hydrocarbon, carbonyl, carboxylic acid, and amide, while RWE-CDs have functional groups such as hydroxyl, aromatic hydrocarbon, carbonyl, and carboxylic acid.

[0060] 2. Cytotoxicity Detection of Rhodiola Rosea Carbon Quantum Dots

[0061] After lung cancer A549 cells were plated for 12 hours, 1 μL, 0.5 μL, and 0.1 μL of water-soluble or alcohol-soluble Rhodiola rosea carbon quantum dot solution (10 mg / mL) were taken, respectively, mixed evenly with 99 μL, 99.5 μL, and 99.9 μL of cell culture medium DMEM (containing 10% FBS), and added to the cells. Shake gently and continue to culture for 24 hours. Prepare CCK-8 working solution according to the ratio of 1 part CCK-8 reagent: 9 parts DMEM culture medium (containing 10% FBS). The original culture medium was aspirated and 100 μL CCK-8 working solution was added. After incubation for 60 minutes, the absorbance at 450 nm was detected using a microplate reader.

[0062] like Figure 8 As shown in the figure, CCK-8 assay showed that RWW-CDs and RWE-CDs had no cytotoxicity at concentrations less than or equal to 100 μg / mL.

[0063] 3. Fluorescence intensity stability of Rhodiola rosea carbon quantum dots

[0064] Rhodiola rosea carbon quantum dots were added to solutions of different pH values ​​to a final concentration of 25 μg / mL. The fluorescence intensity was measured at the optimal excitation wavelength Ex and emission wavelength Em, and the data were normalized with the actual fluorescence intensity at pH = 7 being set as 1.

[0065] like Figure 9 As shown in the figure, fluorescence intensity stability experiments show that RWW-CDs and RWE-CDs have good fluorescence properties in the pH range of 5-11. RWE-CDs also exhibit strong fluorescence at pH 12, which is beneficial for cell imaging applications in special environments.

[0066] 4. Cell Imaging with Rhodiola Rosea Carbon Quantum Dots

[0067] After 24 hours of A549 plating, water-soluble or alcohol-soluble Rhodiola rosea carbon quantum dots solution was diluted to 50 μg / mL with culture medium and added to the cells. The cells were gently shaken and cultured for another 2 hours. The cells were washed three times with PBS solution and confocal microscopy images were taken at an excitation wavelength of 405 nm and an emission wavelength of 420-550 nm.

[0068] like Figure 10 As shown in Figure 3, the imaging effect of RWW-CDs is better than that of RWE-CDs at the same dose.

[0069] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A carbon quantum dot based on Rhodiola rosea, characterized in that: Rhodiola rosea is used as a raw material, mixed with ultrapure water, subjected to high-temperature reaction and cooled, diluted with ultrapure water, filtered, dialyzed and then freeze-dried to prepare water-soluble Rhodiola rosea carbon quantum dots, and the filtered residue is diluted with ethanol, filtered, purified and then freeze-dried to obtain alcohol-soluble Rhodiola rosea carbon quantum dots; The temperature of the high-temperature reaction is 160° C. to 220° C., the heating time is 6 h to 10 h, and the stirring speed is 0 r / min to 1000 r / min.

2. A method for preparing carbon quantum dots based on Rhodiola rosea, characterized in that: include Step 1: Prepare Rhodiola rosea powder; Step 2: Mix the Rhodiola rosea powder in step 1 with ultrapure water, cool to room temperature after high-temperature reaction, dilute with ultrapure water, filter, dialyze, and freeze-dry the filtrate to obtain water-soluble Rhodiola rosea carbon quantum dots; wherein the high-temperature reaction temperature is 160°C to 220°C, the heating time is 6h to 10h, and the stirring speed is 0r / min to 1000r / min; Step 3: The filter residue obtained after filtering in step 2 is diluted, filtered, purified, and freeze-dried to obtain alcohol-soluble Rhodiola rosea carbon quantum dots.

3. The method for preparing carbon quantum dots based on Rhodiola rosea according to claim 2, wherein: The lattice fringe spacing of the water-soluble Rhodiola rosea carbon quantum dots in step 2 is 0.21nm or 0.36nm, and contains nitrogen doping.

4. The method for preparing carbon quantum dots based on Rhodiola rosea according to claim 3, wherein: The alcohol-soluble Rhodiola rosea carbon quantum dots in step 3 have a lattice fringe spacing of 0.21 nm or 0.36 nm.

5. The method for preparing carbon quantum dots based on Rhodiola rosea according to claim 4, wherein: The mass ratio of the Rhodiola rosea powder in step 2 to ultrapure water is 1:30-100.

6. The alcohol-soluble Rhodiola rosea carbon quantum dots prepared by the method according to any one of claims 2 to 5 are used for cell imaging.

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