Nucleic acid dye and application thereof in nucleic acid detection

Abstract

The invention discloses a nucleic acid dye and application thereof in nucleic acid detection, and belongs to the technical field of nanometer materials and biology, the nucleic acid dye is licorice root derived carbon dots, the carbon dots are prepared based on licorice roots or licorice root components as precursors for the first time, and the carbon dots are successfully applied to the field of nucleic acid detection. The licorice root derived carbon dots replace traditional dyes to be used in agarose gel electrophoresis, and efficient visual detection of DNA, RNA and plasmids can be achieved.

Description

[0001] This application claims priority to the Chinese patent application No. CN202411535305.2, filed on October 31, 2024, entitled "A nucleic acid dye and its application in nucleic acid detection", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of nanomaterials and biological technology, and particularly relates to a nucleic acid dye and its application in nucleic acid detection. BACKGROUND

[0003] With the deepening of biological and biochemical research, the study of nucleic acid (DNA and RNA) structure becomes particularly important. As a compound that can bind to nucleic acid, nucleic acid dye has become a key tool for observing and analyzing nucleic acid in biological samples. They can help reveal the structure and function of nucleic acid and are an indispensable part of nucleic acid research. Nucleic acid dyes can bind to the bases in nucleic acid molecules, making nucleic acid exhibit a specific color or fluorescence. This color or fluorescence change provides an intuitive observation means for experimental results. By observing the stained nucleic acid sample, researchers can observe the morphology, size and direction of nucleic acid, and thus understand the basic structural characteristics of nucleic acid molecules and the relative position of different bases in the molecule. Nucleic acid dyes play an important role in the study of nucleic acid structure and function. By comparing the color or fluorescence changes of the dye under different conditions, researchers can infer the double helix structure of nucleic acid molecules, the base pairing rules, and the supercoiled structure of DNA, etc. In addition, dyes can also be used to analyze the folding degree, sequence repeatability and other characteristics of nucleic acid, providing important information for in-depth understanding of the structure and function of nucleic acid.

[0004] Nucleic acid dyes also play a key role in nucleic acid detection technology. They can be used to detect, quantify and analyze nucleic acid samples, providing strong support for research in the fields of biology, medicine, etc. With the deepening of research, the application of nucleic acid dyes in gene detection, pathogen diagnosis, etc. gradually emerges, showing a broad application prospect. In recent years, with the progress of science and technology, nucleic acid dye technology is also constantly innovating and developing. Some new nucleic acid dyes such as BIOTIUM nucleic acid dye, SYBR series dye, etc. have emerged, which have significant advantages in sensitivity, specificity, ease of operation, etc., bringing revolutionary changes to the field of nucleic acid detection. However, at the same time, disputes about the safety and stability of nucleic acid dyes have also emerged. Therefore, how to ensure the safety of dyes while ensuring the experimental effect has become a problem to be solved.

[0005] Glycyrrhizauralensis. Fisch. is a common traditional Chinese medicine, which has been accepted and used for a long time. It has the effects of detoxification, anti-inflammatory, antitussive, antitumor, antiulcer, antibacterial, etc. Glycyrrhizauralensis. Fisch. has been recorded in Chinese herbal books since ancient times. It is not only a good medicine, but also known as the "King of Medicines". It has certain curative effect on the treatment of chronic bronchitis and asthmatic bronchitis. At the same time, glycyrrhizic acid is an organic compound, which has anti-inflammatory and anti-allergic effects as a medicine, and is widely used as a sweetener in various foods. Glycyrrhizic acid is a component extracted from Glycyrrhizauralensis. Fisch. with medicinal value, which has multiple effects and functions: Anti-inflammatory effect: Glycyrrhizic acid has certain anti-inflammatory effect, which can relieve symptoms such as fever, cough, abdominal pain caused by inflammation. Hepatoprotective effect: It has certain therapeutic effect on liver inflammation caused by various reasons, which can relieve liver inflammation and promote the recovery of liver function. Immune regulation: Glycyrrhizic acid can promote the regeneration and differentiation of immune cells and enhance the function of immune cells, which has certain effect on immune regulation and can improve the body's resistance. In addition to the above effects, glycyrrhizic acid also has the effects of tonifying the spleen and replenishing qi. Glycyrrhizic acid mainly includes glycyrrhizic acid and glucuronic acid. Glycyrrhizic acid has some unique physiological activities: Mineralocorticoid-like effect: Glycyrrhizic acid can promote sodium and water retention and increase potassium excretion, showing deoxycorticosterone-like effect. Glucocorticoid-like effect: Glycyrrhizic acid has a chemical structure similar to corticosteroids, which can produce sodium retention, antidiuretic and anti-inflammatory effects. Glucuronic acid is a naturally occurring compound with wide application value: Anti-aging: Glucuronic acid has antioxidant effects, which can neutralize free radicals and prevent their damage to cells, thereby having anti-aging effects. Improve diabetes: Studies have shown that glucuronic acid can improve blood sugar control, reduce insulin resistance and postprandial glucose peak, and reduce the risk of complications of diabetes. Reduce the risk of heart disease: Glucuronic acid can reduce cholesterol levels, especially "bad" cholesterol, reduce inflammatory response, and reduce the risk of heart disease. Beauty effect: Glucuronic acid can stimulate collagen production, making the skin more elastic and reducing the production of dark spots and freckles. Glucuronic acid is a natural compound with multiple health and beauty effects, which is widely used in medicine and beauty fields.

[0006] Carbon dots (CDs) are a class of zero-dimensional carbon nanomaterials with remarkable fluorescent properties, typically with diameters ranging from 1 to 10 nm. They were first discovered in 2004 during the preparation of single-walled carbon nanotubes and have gradually gained attention in subsequent research. As a new type of nanomaterial, CDs have shown great potential in scientific research and technological applications in recent years due to their unique physical and chemical properties, such as small size, large specific surface area, high quantum efficiency, etc. There are various synthesis methods for CDs, mainly including "top-down" and "bottom-up" methods. "Top-down" methods include arc discharge, laser ablation, electrochemical oxidation, and chemical exfoliation, which typically involve exfoliating nanocarbon dots from bulk carbon materials such as graphite and graphene. "Bottom-up" methods utilize carbon-containing small molecules such as glucose and citric acid to synthesize CDs through chemical reactions. To improve the quantum yield, photostability, and adjust the emission wavelength of CDs, the synthesis process often includes steps such as passivation and doping. CDs have excellent fluorescent properties, with tunable emission wavelengths and good photostability. This makes CDs widely applicable in fields such as fluorescent display, biological imaging, and optoelectronic devices; CDs have good biocompatibility and can be combined with biological molecules to form biosensors or used in drug delivery; CDs can maintain their structure and properties under various environmental conditions, which is crucial for their reliability in practical applications; CDs can be used in wastewater treatment, air pollution control, and other fields to improve environmental quality by adsorbing harmful substances; CDs also show potential applications in quantum computing and optoelectronic devices. In recent years, with a deeper understanding of the properties and application mechanisms of CDs, significant progress has been made in the study of CDs. In the future, with the continuous improvement of synthesis technology and the development of new CDs, the application range of CDs will be more extensive. At the same time, in-depth research on the luminescence mechanism of CDs will provide new ideas and methods for their application in photoelectric conversion, biological imaging, and other fields. However, there has been no report on the application of CDs in the field of nucleic acid dyes. Nucleic acid dyes, as a compound that can bind to nucleic acids, have become a key tool for observing and analyzing nucleic acids in biological samples. They can help reveal the structure and function of nucleic acids and are an indispensable part of nucleic acid research. Traditional nucleic acid dyes, although widely used, have problems such as high cost, high toxicity, and long staining time, which pose challenges to the environment and economy. SUMMARY

[0007] To overcome these problems, the present invention proposes a nucleic acid dye and its application in nucleic acid detection. The present invention uses licorice-derived carbon dots (CDs) as a new type of nucleic acid dye to replace traditional dyes for agarose gel electrophoresis, achieving efficient and visual detection of DNA, RNA, and plasmids.

[0008] To achieve the above object, the present application provides the following technical solutions.

[0009] One of the technical solutions of the present application is:

[0010] A nucleic acid dye is a licorice-derived carbon dot.

[0011] The present application first prepares licorice-derived carbon dots based on licorice root or licorice components as precursors, and successfully applies the licorice-derived carbon dots to the field of nucleic acid detection. In the present application, a new type of CDs is prepared by a hydrothermal method in a system containing ethylenediamine with licorice root powder as a biomass precursor. The CDs as nucleic acid dyes exhibit the potential for efficient and visualized detection of DNA, RNA and plasmid in agarose gel electrophoresis. The experimental results show that the CDs are perfectly suitable for this field.

[0012] The present application also deeply studies the main components of licorice, such as glycyrrhizic acid, which is successfully prepared into new CDs as a biomass precursor in a system containing EDA. Surprisingly, this licorice-derived carbon dot, glycyrrhizic acid carbon dot, can also be applied to nucleic acid detection.

[0013] Since glycyrrhizic acid is mainly composed of glycyrrhetic acid and glucuronic acid, the present application also prepares two new CDs with the two components as precursors, respectively. In the comparative experiment, it is found that the glucuronic acid CDs exhibit more excellent performance in nucleic acid detection. This finding reveals that the glucuronic acid in the licorice root plays a key role in preparing CDs applied to nucleic acid dyes.

[0014] The present application not only provides a new biomass source for the preparation of CDs, but also provides an efficient and visualized new method for the field of nucleic acid detection, and exhibits the great potential and application value of licorice-derived carbon dots in the field of nanomaterials and biology.

[0015] Preferably, the preparation method of the licorice-derived carbon dots comprises the following steps: dissolving licorice root or licorice components and ethylenediamine in water, performing a hydrothermal reaction, cooling to room temperature after the reaction is completed, centrifuging, washing, micro-filtering and drying the product to obtain the licorice-derived carbon dots.

[0016] Preferably, the mass-volume ratio of the licorice root or licorice components to ethylenediamine is 1g:300μL.

[0017] Preferably, the licorice components include glycyrrhizic acid, glycyrrhetic acid or glucuronic acid.

[0018] Preferably, the temperature of the hydrothermal reaction is 180℃, and the time is 6h.

[0019] As an embodiment, when the licorice-derived carbon dots are prepared from licorice root and ethylenediamine, the preparation method of the licorice-derived carbon dots is as follows: 1 g of licorice root powder and 300 μL of ethylenediamine are dissolved in 10 mL of deionized water, and then hydrothermal reaction is carried out at 180°C for 6 h in a 50 mL polytetrafluoroethylene-lined reactor. After the reaction is completed, the reaction solution is cooled to room temperature, centrifuged at 10000 rpm for 15 min, and the supernatant is collected and filtered through a microporous membrane (0.22 μm). The obtained suspension is vacuum freeze-dried to obtain the licorice-derived carbon dots.

[0020] As an embodiment, when the licorice component is glycyrrhizic acid, the preparation method of the licorice-derived carbon dots is as follows: 1 g of glycyrrhizic acid and 300 μL of ethylenediamine are dissolved in 10 mL of deionized water, and then hydrothermal reaction is carried out at 180°C for 6 h in a 50 mL polytetrafluoroethylene-lined reactor. After the reaction is completed, the reaction solution is cooled to room temperature, centrifuged at 10000 rpm for 15 min, and the supernatant is collected and filtered through a microporous membrane (0.22 μm). The obtained suspension is vacuum freeze-dried to obtain the licorice-derived carbon dots.

[0021] As an embodiment, when the licorice component is glycyrrhizic acid, the preparation method of the licorice-derived carbon dots is as follows: 1 g of glycyrrhizic acid and 300 μL of ethylenediamine are dissolved in 10 mL of deionized water, and then hydrothermal reaction is carried out at 180°C for 6 h in a 50 mL polytetrafluoroethylene-lined reactor. After the reaction is completed, the reaction solution is cooled to room temperature, centrifuged at 10000 rpm for 15 min, and the supernatant is collected and filtered through a microporous membrane (0.22 μm). The obtained suspension is vacuum freeze-dried to obtain the licorice-derived carbon dots.

[0022] As an embodiment, when the licorice component is glycyrrhizic acid, the preparation method of the licorice-derived carbon dots is as follows: 1 g of glycyrrhizic acid and 300 μL of ethylenediamine are dissolved in 10 mL of deionized water, and then hydrothermal reaction is carried out at 180°C for 6 h in a 50 mL polytetrafluoroethylene-lined reactor. After the reaction is completed, the reaction solution is cooled to room temperature, centrifuged at 10000 rpm for 15 min, and the supernatant is collected and filtered through a microporous membrane (0.22 μm). The obtained suspension is vacuum freeze-dried to obtain the licorice-derived carbon dots.

[0023] The second technical solution of the present application is as follows:

[0024] The present application also provides the use of the nucleic acid dye in nucleic acid detection. The licorice-derived carbon dots of the present application can be used for efficient and visual detection of DNA, RNA and plasmids.

[0025] Preferably, the nucleic acid dye is added to an agarose gel in the form of an ethanol solution, and electrophoresis is used for nucleic acid detection. The concentration of the nucleic acid dye in the ethanol solution is 10-100 mg / mL.

[0026] More preferably, the concentration of the nucleic acid dye in the ethanol solution of the nucleic acid dye is 100 mg / mL.

[0027] The present application provides a nucleic acid dye modified agarose gel, comprising an agarose gel and a nucleic acid dye doped in the agarose gel.

[0028] The present application also provides a preparation method of the nucleic acid dye modified agarose gel as described in the technical solutions above, comprising the following steps:

[0029] After mixing the agarose and the buffer, heating and cooling are sequentially performed to obtain the agarose gel;

[0030] After mixing the ethanol solution of the glycyrrhiza-derived carbon dots and the agarose gel, solidification is performed to obtain the nucleic acid dye modified agarose gel.

[0031] Preferably, the buffer is a tricetate-acetic acid buffer; the mass concentration of the agarose solution is 1 wt.%.

[0032] The volume ratio of the ethanol solution of the glycyrrhiza-derived carbon dots to the agarose gel is 1:10.

[0033] Compared with the unobtainability and safety of traditional nucleic acid dyes, the present application has the following beneficial effects:

[0034] (1) The method of the present application is simple to operate and the carbon dots are synthesized quickly. There is no complex step in the entire preparation process of the glycyrrhiza-derived CDs. The glycyrrhiza-derived CDs are obtained by co-reaction of glycyrrhiza roots or glycyrrhiza components and ethylenediamine in the system for synthesizing the glycyrrhiza-derived CDs.

[0035] (2) The binding mode of the nucleic acid dye prepared by the present application is different from the traditional intercalation binding of nucleic acid dyes. The traditional nucleic acid dyes are inserted into base molecules, which leads to mismatch and induces mutation, has potential carcinogenicity, causes harm to experimental personnel and pollutes the environment. The binding mode of the glycyrrhiza-derived CDs of the present application is groove binding, which does not cause base mismatch and other problems. Relatively speaking, the glycyrrhiza-derived CDs are safer than traditional nucleic acid dyes.

[0036] (3) The sensitivity of the nucleic acid dye prepared by the present application is comparable to that of traditional nucleic acid dye ethidium bromide, and can detect as little as 10 ng of target.

[0037] (4) The novel nucleic acid dye prepared by the present application has multiple advantages of low cost, easy synthesis and low toxicity, and has great potential in the field of nucleic acid detection, and provides a research idea for promoting the further development and application of nanomaterials at the molecular level. BRIEF DESCRIPTION OF DRAWINGS

[0038] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this application. The illustrations, together with the description, serve to explain the application, but are not intended to limit the application in any way.

[0039] Figure 1 Preparation and application flow chart of glycyrrhiza-derived carbon dots CDs-4 provided for embodiment 4 of the present application;

[0040] Figure 2 Transmission electron microscope and high-resolution transmission electron microscope images (insert) of glycyrrhiza-derived carbon dots CDs-4 solution prepared in embodiment 4 of the present application;

[0041] Figure 3 Particle size distribution histogram of glycyrrhiza-derived carbon dots CDs-4 solution prepared in embodiment 4 of the present application;

[0042] Figure 4 UV-visible spectrum, fluorescence spectrum and actual object diagram of glycyrrhiza-derived carbon dots CDs-4 prepared in embodiment 4 of the present application, wherein "Day" represents under daylight, and "UV" represents under ultraviolet light;

[0043] Figure 5 Effect diagram of glycyrrhiza-derived carbon dots CDs-4 as nucleic acid dye at different concentrations in application example 1;

[0044] Figure 6 Sensitivity effect diagram of glycyrrhiza-derived carbon dots CDs-4 as nucleic acid dye for DNA detection in application example 2;

[0045] Figure 7 Effect diagram of glycyrrhiza-derived carbon dots CDs-4 for DNA, RNA and plasmid detection;

[0046] Figure 8 Effect diagram of glycyrrhiza-derived carbon dots CDs-1, CDs-2 and CDs-3 prepared in embodiments 1-3 as nucleic acid dye for DNA Marker III detection, wherein a is embodiment 1, b is embodiment 2, and c is embodiment 3. DETAILED DESCRIPTION

[0047] Various illustrative embodiments of the present application are now described in detail. The detailed description should be considered in connection with the accompanying drawings, which form a part of this disclosure, and which show, by way of illustration, specific examples of the application. The detailed description includes specific examples, and embodiments. It is to be understood that other examples can be utilized without departing from the scope of the application. For example, the described embodiments can be implemented in various ways, some of which are not described, and the application can be in other embodiments than those described and / or claimed.

[0048] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed by the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0050] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0051] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to mean including, but not limited to.

[0052] Unless otherwise specified, the room temperature in the present application is 25±2℃.

[0053] Each raw material used in the embodiments of the present application is obtained by commercial purchase.

[0054] The technical solutions of the present application are further illustrated by the following examples.

[0055] Example 1

[0056] 1 g of licorice root powder and 300 μL of ethylenediamine were dissolved in 10 mL of deionized water, and the mixture was thoroughly mixed in a 50 mL polytetrafluoroethylene-lined reaction kettle. The reaction kettle containing the reaction mixture was reacted at 180℃ for 6 h. After the reaction was completed, the reaction kettle was cooled to room temperature, centrifuged at 10000 rpm for 15 min, and the supernatant was collected and washed by filtration through a microporous membrane (0.22 μm). The obtained suspension was vacuum freeze-dried to obtain licorice-derived carbon dots, which were recorded as CDs-1. The CDs-1 were in a powder state and were dispersed in ethanol for subsequent use.

[0057] Example 2

[0058] 1 g glycyrrhizic acid and 300 μL ethylenediamine were dissolved in 10 mL deionized water, and the mixture was mixed uniformly in a 50 mL polytetrafluoroethylene-lined reaction kettle. The reaction kettle was filled with the reactants and reacted at 180 °C for 6 h. After the reaction was completed, the reaction kettle was cooled to room temperature, centrifuged at 10,000 rpm for 15 min, and the supernatant was collected. The supernatant was washed and filtered through a microporous membrane (0.22 μm). The obtained suspension was vacuum freeze-dried to obtain glycyrrhizic acid-derived carbon dots, which were recorded as CDs-2. The glycyrrhizic acid-derived carbon dots were in a powder state and were dispersed in ethanol for subsequent use.

[0059] Example 3

[0060] 1 g glycyrrhizic acid and 300 μL ethylenediamine were dissolved in 10 mL deionized water, and the mixture was mixed uniformly in a 50 mL polytetrafluoroethylene-lined reaction kettle. The reaction kettle was filled with the reactants and reacted at 180 °C for 6 h. After the reaction was completed, the reaction kettle was cooled to room temperature, centrifuged at 10,000 rpm for 15 min, and the supernatant was collected. The supernatant was washed and filtered through a microporous membrane (0.22 μm). The obtained suspension was vacuum freeze-dried to obtain glycyrrhizic acid-derived carbon dots, which were recorded as CDs-2. The glycyrrhizic acid-derived carbon dots were in a powder state and were dispersed in ethanol for subsequent use.

[0061] Example 4

[0062] 1 g glycyrrhizic acid and 300 μL ethylenediamine were dissolved in 10 mL deionized water, and the mixture was mixed uniformly in a 50 mL polytetrafluoroethylene-lined reaction kettle. The reaction kettle was filled with the reactants and reacted at 180 °C for 6 h. After the reaction was completed, the reaction kettle was cooled to room temperature, centrifuged at 10,000 rpm for 15 min, and the supernatant was collected. The supernatant was washed and filtered through a microporous membrane (0.22 μm). The obtained suspension was vacuum freeze-dried to obtain glycyrrhizic acid-derived carbon dots, which were recorded as CDs-2. The glycyrrhizic acid-derived carbon dots were in a powder state and were dispersed in ethanol for subsequent use.

[0063] The preparation and application flowchart of the glycyrrhizic acid-derived carbon dots CDs-4 provided in the example are shown in Figure 1 .

[0064] The transmission electron microscope and high-resolution transmission electron microscope images (insert) of the glycyrrhizic acid-derived carbon dots CDs-4 provided in the example are shown in Figure 2 , and the particle size distribution histogram of the glycyrrhizic acid-derived carbon dots CDs-4 provided in the example is shown in Figure 3 , which shows that the glycyrrhizic acid-derived carbon dots were successfully prepared, and the particle size of the glycyrrhizic acid-derived carbon dots was 1.71 ± 0.31 nm.

[0065] The ultraviolet-visible spectrum, fluorescence spectrum, and actual object diagram of the glycyrrhizic acid-derived carbon dots CDs-4 provided in the example are shown in Figure 4As shown, the excitation wavelength (Ex) and emission wavelength (Em) of the CDs-4 prepared in Example 4 are 420 nm and 496 nm, respectively; and the CDs-4 prepared in Example 4 exhibits strong absorption ability in the ultraviolet region of 250-300 nm.

[0066] Application Example 1

[0067] The CDs-4 prepared in Example 4 were dispersed in ethanol at concentrations of 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL, and 100 mg / mL, and were applied to agarose gel electrophoresis for detecting nucleic acid products as nucleic acid dyes. The effect of the CDs-4 prepared in Example 4 as nucleic acid dyes is shown in Figure 5 As shown, the optimal concentration of the CDs-4 prepared in Example 4 as nucleic acid dyes is 100 mg / mL.

[0068] Application Example 2

[0069] The DNA Marker III (purchased from Shanghai Aldrin Reagent Co., Ltd.) was added to the sample wells at sample amounts of 1 μL, 2 μL, 3 μL, and 4 μL, and was maintained at a voltage of 120 V for 20 min. After the bromophenol blue electrophoresed to 1-2 cm of the gel, the staining effect of each band could be clearly observed in the full-automatic gel imaging analyzer (Bio-Rad Gel-Doc EZ, USA). In this way, the lowest detection limit of the CDs-4 prepared in Example 4 as nucleic acid dyes was determined. The sensitivity effect of the CDs-4 prepared in Example 4 as nucleic acid dyes is shown in Figure 6 As shown (Marker 3 is mixed with 7 double-stranded linear DNA fragments, the indicated band is 1200 bp, and the rest are non-indicated bands, and the target object content is 50 ng / 5 μL, and the sample amount is controlled at 1 μL, which can indicate that the amount of target objects contained in the rest of the non-indicated bands is 10 ng / μL), the CDs-4 prepared in Example 4 can detect 10 ng / μL of DNA products.

[0070] Application Example 3

[0071] The CDs-4 prepared in Example 4 was used as a nucleic acid dye to detect DNA. 0.25 g of agarose was weighed and added to 25 mL of 1XTAE (Tris-acetate buffer) and heated until cooled to prepare a 1 wt% agarose gel. A 100 mg / mL solution of CDs-4 was added (volume ratio of the CDs-4 solution to the agarose gel was 1:10) and mixed, and a comb was inserted and left to solidify. After removing the comb, the gel was placed in an electrophoresis tank with buffer to cover the gel. The resulting PCR product was added to the sample well in an amount of 5 μL, and electrophoresis was performed at 120 V for 20 min. After bromophenol blue was electrophoresed to a position 1-2 cm from the gel, the corresponding bands could be clearly seen in a gel imager. The effect of the CDs-4 prepared in Example 4 on the detection of DNA is shown in FIG. 1, and it can be seen that the CDs-4 prepared in Example 4 can be used as a nucleic acid dye to detect the target DNA. Figure 7 The CDs-4 prepared in Example 4 was used as a nucleic acid dye to detect DNA. 0.25 g of agarose was weighed and added to 25 mL of 1XTAE (Tris-acetate buffer) and heated until cooled to prepare a 1 wt% agarose gel. A 100 mg / mL solution of CDs-4 was added (volume ratio of the CDs-4 solution to the agarose gel was 1:10) and mixed, and a comb was inserted and left to solidify. After removing the comb, the gel was placed in an electrophoresis tank with buffer to cover the gel. The resulting PCR product was added to the sample well in an amount of 5 μL, and electrophoresis was performed at 120 V for 20 min. After bromophenol blue was electrophoresed to a position 1-2 cm from the gel, the corresponding bands could be clearly seen in a gel imager. The effect of the CDs-4 prepared in Example 4 on the detection of DNA is shown in FIG. 1, and it can be seen that the CDs-4 prepared in Example 4 can be used as a nucleic acid dye to detect the target DNA.

[0072] Application Example 4

[0073] The CDs-4 prepared in Example 4 was used as a nucleic acid dye to detect RNA. 0.25 g of agarose was weighed and added to 25 mL of 1XTAE and heated until cooled to prepare a 1 wt% agarose gel. A 100 mg / mL solution of CDs-4 was added (volume ratio of the CDs-4 solution to the agarose gel was 1:10) and mixed, and a comb was inserted and left to solidify. After removing the comb, the gel was placed in an electrophoresis tank with buffer to cover the gel. The resulting PCR product was added to the sample well in an amount of 5 μL, and electrophoresis was performed at 120 V for 20 min. After bromophenol blue was electrophoresed to a position 1-2 cm from the gel, the corresponding bands could be clearly seen in a gel imager. The effect of the CDs-4 prepared in Example 4 on the detection of RNA is shown in FIG. 2, and it can be seen that the CDs-4 prepared in Example 4 can be used as a nucleic acid dye to detect the target RNA. Figure 7 The CDs-4 prepared in Example 4 was used as a nucleic acid dye to detect DNA. 0.25 g of agarose was weighed and added to 25 mL of 1XTAE (Tris-acetate buffer) and heated until cooled to prepare a 1 wt% agarose gel. A 100 mg / mL solution of CDs-4 was added (volume ratio of the CDs-4 solution to the agarose gel was 1:10) and mixed, and a comb was inserted and left to solidify. After removing the comb, the gel was placed in an electrophoresis tank with buffer to cover the gel. The resulting PCR product was added to the sample well in an amount of 5 μL, and electrophoresis was performed at 120 V for 20 min. After bromophenol blue was electrophoresed to a position 1-2 cm from the gel, the corresponding bands could be clearly seen in a gel imager. The effect of the CDs-4 prepared in Example 4 on the detection of DNA is shown in FIG. 1, and it can be seen that the CDs-4 prepared in Example 4 can be used as a nucleic acid dye to detect the target DNA.

[0074] Application Example 5

[0075] The CDs-4 prepared in Example 4 was used as a nucleic acid dye to detect plasmid. 0.25 g of agarose was weighed and added to 25 mL of 1XTAE and heated until cooled to prepare a 1 wt% agarose gel. A 100 mg / mL solution of CDs-4 was added (volume ratio of the CDs-4 solution to the agarose gel was 1:10) and mixed, and a comb was inserted and left to solidify. After removing the comb, the gel was placed in an electrophoresis tank with buffer to cover the gel. The resulting PCR product was added to the sample well in an amount of 5 μL, and electrophoresis was performed at 120 V for 20 min. After bromophenol blue was electrophoresed to a position 1-2 cm from the gel, the corresponding bands could be clearly seen in a gel imager. The effect of the CDs-4 prepared in Example 4 on the detection of plasmid is shown in FIG. 3, and it can be seen that the CDs-4 prepared in Example 4 can be used as a nucleic acid dye to detect the target plasmid. Figure 7As shown, it can be seen that the CDs-4 prepared in Example 4 can be used as a nucleic acid dye to detect the target substance DNA Marker III.

[0076] The detection effect diagrams of the glycyrrhiza-derived carbon dots CDs-1, CDs-2 and CDs-3 prepared in Examples 1-3 as nucleic acid dyes on DNA Marker III are as follows Figure 8 Wherein (a) is Example 1, (b) is Example 2, and (c) is Example 3, it can be seen that the glycyrrhiza-derived carbon dots CDs-1, CDs-2 and CDs-3 as nucleic acid dyes can achieve efficient detection of DNA.

[0077] The above merely provides the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, any changes or replacements easily thought of by those skilled in the art within the technical scope disclosed by the present application should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Application of licorice-derived carbon dots as nucleic acid dyes.

2. The use according to claim 1, characterized in that The preparation method of the licorice-derived carbon dots comprises the following steps: dissolving licorice root or licorice components and ethylenediamine in water, performing a hydrothermal reaction, cooling to room temperature after the reaction is completed, centrifuging the product, washing and filtering the supernatant through a microporous membrane, and drying the suspension to obtain the licorice-derived carbon dots.

3. The use according to claim 2, characterized in that The mass volume ratio of the liquorice root or liquorice component to ethylenediamine is 1 g:300 μL.

4. The use according to claim 2, characterized in that The licorice components include glycyrrhizic acid, glycyrrhetinic acid or glucuronic acid.

5. The use according to claim 2, characterized in that The temperature of the hydrothermal reaction is 180° C. and the time is 6 hours.

6. Application of licorice-derived carbon dots in nucleic acid detection.

7. The use according to claim 6, characterized in that During the application, an ethanol solution of licorice-derived carbon dots is added to an agarose gel, and nucleic acid detection is performed using electrophoresis; The concentration of the licorice-derived carbon dots in the ethanol solution of the licorice-derived carbon dots is 10-100 mg / mL.

8. A modified agarose gel, characterized in that: The invention comprises agarose gel and nucleic acid dye, wherein the nucleic acid dye is licorice-derived carbon dots.

9. The method for preparing the modified agarose gel according to claim 8, comprising the following steps: After mixing agarose and buffer, heating and cooling are performed in sequence to obtain agarose gel; The ethanol dispersion of the licorice-derived carbon dots and the agarose gel were mixed and solidified to obtain a modified agarose gel.

10. The preparation method according to claim 9, characterized in that The buffer solution is triacetate-acetic acid buffer solution; the mass concentration of the agarose solution is 1wt.%; The volume ratio of the ethanol solution of the licorice-derived carbon dots to the agarose gel is 1:10.

Images