Nucleic acid dye and application thereof in nucleic acid visual detection

Abstract

The invention discloses a nucleic acid dye and application thereof in visual detection of nucleic acid, belongs to the technical field of biochemistry, and aims to solve the problems of high price, high toxicity, long dyeing time and the like of the traditional nucleic acid dye which is widely used, which brings challenges to environment and economy. In order to overcome the problems, the m-phenylenediamine color carbon dots are used as a novel nucleic acid dye to replace a traditional dye to be used in agarose gel electrophoresis, so that efficient visual detection of DNA and RNA is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to a nucleic acid dye and an application thereof in nucleic acid visualization detection. Background Art

[0002] Nucleic acid dyes, as key chemical reagents for dyeing and labeling nucleic acid molecules, have a direct impact on the performance of nucleic acid detection. Although traditional nucleic acid dyes are widely used, they pose challenges to the environment and economy due to their high price, high toxicity, and long dyeing time. Since nucleic acid molecules are colorless and difficult to identify and detect, it is often necessary to add certain nucleic acid dyes to dye and label them, making it easier to observe, separate, and identify nucleic acids in agarose gel electrophoresis, thereby achieving the detection and quantification of nucleic acids. Nucleic acid dyes are indispensable in the fields of molecular biology, biochemistry, and genetic engineering. Nucleic acid dyes are combined with nucleic acid molecules by physical or chemical means, usually electrostatic interactions, π-π stacking, and hydrophobic interactions, so that the dyes are tightly bound to nucleic acid molecules, which helps to accurately grasp the presence and concentration of nucleic acid molecules during the experiment. Nucleic acid dyes are usually aromatic ring compounds containing benzene rings or pyridine rings in the molecules. These structures can absorb ultraviolet or blue light and emit fluorescence.

[0003] Currently, the nucleic acid dyes widely used in biological research are mainly red fluorescent ethidium bromide (EB) and green fluorescent Gold View. EB can detect DNA bands as small as 10ng, which is very sensitive and is the most effective nucleic acid dye. The binding of EB to DNA has almost no base sequence specificity. In a saturated solution with high ionic strength, approximately one ethidium bromide molecule is inserted for every 2.5 bases. This property of EB makes it a nucleic acid dye, which is often used for nucleic acid staining in agarose gel electrophoresis, but it has a fatal disadvantage that it can be embedded in base molecules to cause mismatches, induce mutations, and have potential carcinogenicity, which can cause harm to experimenters and pollute the environment. Gold View is relatively safe but lacks sensitivity. Finding a nucleic acid dye that is highly sensitive and safe is a key problem that technicians in this field have always wanted to solve.

[0004] Carbon dots (CDs) are a type of carbon-based zero-dimensional nanomaterial, which is composed of dispersed spherical carbon particles with a size of less than 10nm. It is a nanocarbon material with fluorescent properties and is also a member of the carbon family that has emerged in recent years. Since the Scrivens team first discovered photoluminescent CDs in 2004, CDs have been widely used in various fields. As a new type of fluorescent nanomaterial, CDs have shown broad application prospects in other fields such as antibacterial, photodynamic therapy, drug delivery, disease diagnosis and treatment, bioimaging, sensing and probes. CDs not only have a wide range of application value in the field of light-emitting devices, but are more suitable for application in biomedicine, sensing and bioimaging compared to traditional semiconductor quantum dots. CDs can be applied in many fields thanks to the unique properties of CDs, such as its benign chemical composition, tunable fluorescence emission, easy-to-modify surface functional groups, excellent photoluminescent properties and anti-photobleaching advantages, coupled with other advantages such as low cost, wide source of raw materials for preparation and easy synthesis, making CDs a rising star in various application fields. It still has great exploration value in different fields. The CDs synthesis methods are mainly divided into "top-down" and "bottom-up". The "top-down" method mainly includes arc discharge method and laser etching method, which decomposes larger carbon materials into smaller carbon structures by chemical, electrochemical or physical methods. This type of method is more complicated and difficult to control the structure of the product; the "bottom-up" method mainly includes microwave method, pyrolysis combustion method, template method, ultrasonic method and hydrothermal synthesis method, which connects and carbonizes small organic molecules into CDs through heat treatment. However, there has been no report on the application of CDs in the field of nucleic acid dyes. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a nucleic acid dye and its application in nucleic acid visualization detection. The present invention uses m-phenylenediamine-based colored carbon dots as a new type of nucleic acid dye to replace traditional dyes in agarose gel electrophoresis to achieve efficient visualization detection of DNA and RNA.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention:

[0008] The invention provides a nucleic acid dye which is a m-phenylenediamine-based colored carbon dot.

[0009] Preferably, the method for preparing the m-phenylenediamine-based colored carbon dots comprises the following steps:

[0010] Mixing m-phenylenediamine and acid, reacting under hydrothermal conditions, and centrifuging and drying after the reaction to obtain the m-phenylenediamine-based colored carbon dots (CDs);

[0011] The acid includes tyrosine, phosphoric acid, L-tartaric acid or caffeic acid.

[0012] Preferably, the temperature of the hydrothermal reaction is 200° C. and the time is 6 hours.

[0013] Preferably, when the acid is tyrosine, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: weigh 0.906 g of tyrosine and 0.216 g of m-phenylenediamine and dissolve them in 10 mL of N,N-dimethylformamide solution, react at 200°C for 6 hours, and after the reaction is completed, cool to room temperature, centrifuge, collect the supernatant, wash and filter through a microporous membrane, and vacuum freeze-dry the obtained suspension to obtain the m-phenylenediamine-based colored carbon dots, which are blue fluorescent emitting carbon dots.

[0014] Preferably, when the acid is phosphoric acid, the preparation method of m-phenylenediamine-based colored carbon dots is: weigh 0.216 g of m-phenylenediamine, add it to 3 mL of phosphoric acid, and then dissolve it in 7 mL of anhydrous ethanol, react at 200°C for 6 hours, and after the reaction is completed, cool to room temperature, centrifuge, collect the supernatant, wash and filter it through a microporous membrane, and vacuum freeze-dry the obtained suspension to obtain the m-phenylenediamine-based colored carbon dots, which are green fluorescent emitting carbon dots.

[0015] Preferably, when the acid is L-tartaric acid, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: weigh 0.75 g L-tartaric acid and 0.216 g m-phenylenediamine, add them to 3 mL of phosphoric acid, and then dissolve them in 7 mL of anhydrous ethanol. React at 200°C for 6 hours. After the reaction is completed, cool to room temperature, centrifuge, collect the supernatant, wash and filter it through a microporous membrane, and vacuum freeze-dry the obtained suspension to obtain the m-phenylenediamine-based colored carbon dots, which are yellow fluorescent carbon dots.

[0016] Preferably, when the acid is caffeic acid, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: weigh 0.901 g of caffeic acid and 0.216 g of m-phenylenediamine, add them to 3 mL of phosphoric acid, and dissolve them in 7 mL of anhydrous ethanol, react at 200°C for 6 hours, and after the reaction is completed, cool to room temperature, centrifuge, collect the supernatant, wash and filter it through a microporous membrane, and vacuum freeze-dry the obtained suspension to obtain the m-phenylenediamine-based colored carbon dots, which are white fluorescent emitting carbon dots.

[0017] More preferably, when the acid is tyrosine, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: 0.906 g of tyrosine and 0.216 g of m-phenylenediamine are weighed and dissolved in 10 mL of N,N-dimethylformamide solution, and mixed thoroughly in a 50 mL polytetrafluoroethylene-lined reactor. The reactor loaded with the reactants is reacted at 200° C. for 6 hours. After the reaction is completed, it is cooled to room temperature and centrifuged at 10,000 rpm for 15 minutes. The supernatant is collected and washed and filtered through a microporous membrane (0.22 μm). The obtained suspension is vacuum freeze-dried to obtain tyrosine-m-phenylenediamine-based CDs powder, recorded as B-CDs, which is a blue fluorescent carbon dot. The excitation wavelength (Ex) and emission wavelength (Em) of B-CDs are 360 ​​nm and 418 nm, respectively.

[0018] More preferably, when the acid is phosphoric acid, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: weigh 0.216 g of m-phenylenediamine, add it to 3 mL of phosphoric acid, dissolve it in 7 mL of anhydrous ethanol, and react it in a 50 mL polytetrafluoroethylene-lined reactor at 200°C for 6 hours. After the reaction is completed, cool it to room temperature, centrifuge it at 10,000 rpm for 15 minutes, collect the supernatant, wash and filter it through a microporous membrane (0.22 μm), and vacuum freeze-dry the obtained suspension to obtain phosphoric acid-m-phenylenediamine-based CDs powder, recorded as G-CDs, which is a green fluorescent carbon dot, and the Ex / Em of G-CDs is 471 / 514 nm.

[0019] More preferably, when the acid is L-tartaric acid, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: accurately weigh 0.75 g L-tartaric acid and 0.216 g m-phenylenediamine, add them to 3 mL phosphoric acid, and then dissolve them in 7 mL anhydrous ethanol. In a 50 mL polytetrafluoroethylene-lined high-pressure reactor, react at 200 ° C for 6 hours. After the reaction is completed, cool to room temperature, centrifuge at 10000 rpm for 15 minutes, collect the supernatant, wash and filter through a microporous membrane (0.22 μm), and vacuum freeze-dry the obtained suspension to obtain L-tartaric acid-m-phenylenediamine-based CDs powder, recorded as Y-CDs, which is a yellow fluorescent carbon dot, and the Ex / Em of Y-CDs is 469 / 518 nm.

[0020] More preferably, when the acid is caffeic acid, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: weigh 0.901 g of caffeic acid and 0.216 g of m-phenylenediamine, add them to 3 mL of phosphoric acid, and dissolve them in 7 mL of anhydrous ethanol, and react them in a 50 mL polytetrafluoroethylene-lined reactor at 200 ° C for 6 hours. After the reaction is completed, cool to room temperature, centrifuge at 10000 rpm for 15 minutes, collect the supernatant, wash and filter through a microporous membrane (0.22 μm), and vacuum freeze-dry the obtained suspension to obtain caffeic acid-m-phenylenediamine-based CDs powder, recorded as W-CDs, which is a carbon dot with white fluorescence emission. W-CDs has two emission peaks at 420 nm and 518 nm, and its Ex / Em is 362 / 420 nm.

[0021] The second technical solution of the present invention:

[0022] The present invention also provides application of the nucleic acid dye in nucleic acid visualization detection.

[0023] Preferably, the concentration of the m-phenylenediamine-based colored carbon dots as nucleic acid dyes is 10-100 mg / mL.

[0024] Preferably, the concentration of the m-phenylenediamine-based colored carbon dots as nucleic acid dyes is 100 mg / mL.

[0025] Compared with the difficulty in obtaining, safety and single fluorescence color of traditional nucleic acid dyes, the beneficial effects of the present invention are:

[0026] (1) Simple operation and fast synthesis speed. The entire preparation process of m-phenylenediamine-based color CDs does not involve complicated steps. m-phenylenediamine-based CDs can be prepared by using m-phenylenediamine as the main base in the CDs synthesis system.

[0027] (2) The binding mode of the nucleic acid dye prepared by the present invention with nucleic acid is different from the intercalation binding of the traditional nucleic acid dye EB, which can be embedded in the base molecule to cause mismatch, induce mutation, and has potential carcinogenicity, which can cause harm to experimenters and pollute the environment. The binding mode of CDs with nucleic acid is groove binding, which will not cause base mismatch problems and is relatively safer than EB.

[0028] (3) Currently, the nucleic acid dyes widely used in biological research are mainly EB with red fluorescence emission and Gold view with green fluorescence emission. The nucleic acid dye prepared by the present invention has the characteristics of blue, green, yellow and white fluorescence emission, which enriches the colors of nucleic acid dyes and makes their selection more diverse.

[0029] (4) The sensitivity of the nucleic acid dye prepared by the present invention is comparable to that of the traditional nucleic acid dye EB and can detect as little as 10 ng of the target.

[0030] (5) The novel nucleic acid dye prepared by the present invention has multiple advantages of low cost, easy synthesis and low toxicity, and has shown great potential in the field of nucleic acid detection, providing a research idea for promoting the further development and application of nanomaterials at the molecular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0032] Figure 1 This is a flow chart of the preparation and application of m-phenylenediamine-based colored carbon dots of the present invention;

[0033] Figure 2 It is a transmission electron microscope and a high-resolution transmission electron microscope image (inset) of the m-phenylenediamine-based colored carbon dot solution prepared in Examples 1-4 of the present invention, wherein a is Example 1, b is Example 2, c is Example 3, and d is Example 4;

[0034] Figure 3 is a particle size distribution histogram of the m-phenylenediamine-based colored carbon dot solution prepared in Examples 1-4 of the present invention, wherein a is Example 1, b is Example 2, c is Example 3, and d is Example 4;

[0035] Figure 4 The ultraviolet-visible spectrum, fluorescence spectrum and physical picture of the m-phenylenediamine-based colored carbon dots prepared by the present invention, wherein a is Example 1, b is Example 2, c is Example 3, and d is Example 4;

[0036] Figure 5 The diagram is a diagram showing the effect of the m-phenylenediamine-based colored carbon dot solution prepared by the present invention as the optimal concentration of nucleic acid dye, wherein a is application example 1, b is application example 2, c is application example 3, and d is application example 4;

[0037] Figure 6 The diagram is a sensitivity effect diagram of the m-phenylenediamine-based colored carbon dot solution prepared by the present invention as a nucleic acid dye, wherein a is application example 5, b is application example 6, c is application example 7, and d is application example 8;

[0038] Figure 7 1 is a diagram showing the effect of the m-phenylenediamine-based colored carbon dot solution prepared by the present invention on DNA detection, wherein a is application example 9, b is application example 10, c is application example 11, and d is application example 12;

[0039] Figure 8 3 and 4. These are diagrams showing the effect of the m-phenylenediamine-based colored carbon dot solution prepared by the present invention on RNA detection, wherein a is Application Example 13, b is Application Example 14, c is Application Example 15, and d is Application Example 16. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Unless otherwise specified, the room temperature in the present invention is 25±2°C.

[0046] All raw materials used in the examples of the present invention are commercially available.

[0047] The preparation and application flow chart of the m-phenylenediamine-based colored carbon dots of the present invention is shown in Figure 1 .

[0048] The technical solution of the present invention is further illustrated by the following embodiments.

[0049] Example 1

[0050] Accurately weigh 0.906g of tyrosine and 0.216g of m-phenylenediamine and dissolve them in 10mL of N,N-dimethylformamide solution, mix them thoroughly in a 50mL polytetrafluoroethylene-lined reactor, and react the reactor at 200°C for 6h. After the reaction is completed, cool it to room temperature, centrifuge it at 10000rpm for 15min, collect the supernatant, wash and filter it through a microporous membrane (0.22μm), and freeze-dry the obtained suspension in vacuum to obtain tyrosine-m-phenylenediamine-based CDs powder, which is recorded as B-CDs, which is a carbon dot with blue fluorescence emission. The tyrosine-m-phenylenediamine-based CDs powder is dispersed in ethanol for subsequent use.

[0051] The transmission electron microscopy and high-resolution transmission electron microscopy of the B-CDs prepared in this example are as follows: Figure 2 As shown in a (the 0.21 nm in the small figure in the upper right corner represents the distance between the two lattices, the same below), it can be seen that B-CDs were successfully prepared. The particle size distribution histogram of the B-CDs prepared in this example is shown in Figure 3 As shown in a, it can be seen that the particle size of B-CDs prepared in Example 1 is 5.56±0.05 nm.

[0052] Example 2

[0053] Accurately weigh 0.216g of m-phenylenediamine and add it to 3mL of phosphoric acid, then dissolve it in 7mL of anhydrous ethanol, and react it at 200°C for 6h in a 50mL polytetrafluoroethylene-lined reactor. After the reaction is completed, cool it to room temperature, centrifuge it at 10000rpm for 15min, collect the supernatant, wash and filter it through a microporous membrane (0.22μm), and vacuum freeze-dry the obtained suspension to obtain phosphoric acid-m-phenylenediamine-based CDs powder, which is recorded as G-CDs, which is a carbon dot emitting green fluorescence. The phosphoric acid-m-phenylenediamine-based CDs powder is dispersed in ethanol for subsequent use.

[0054] The transmission electron microscopy and high-resolution transmission electron microscopy of the G-CDs prepared in this example are as follows: Figure 2 As shown in b, the particle size distribution histogram is as follows Figure 3 As shown in b, it can be seen that G-CDs were successfully prepared, and the particle size of G-CDs was 5.79±0.05nm.

[0055] Example 3

[0056] Accurately weigh 0.75g L-tartaric acid and 0.216g m-phenylenediamine, add them to 3mL phosphoric acid, dissolve them in 7mL anhydrous ethanol, react them in a 50mL polytetrafluoroethylene-lined autoclave at 200°C for 6h, and after the reaction is completed, cool to room temperature, centrifuge at 10000rpm for 15min, collect the supernatant, wash and filter through a microporous membrane (0.22μm), and vacuum freeze-dry the obtained suspension to obtain L-tartaric acid-m-phenylenediamine-based CDs powder, denoted as Y-CDs, which is a yellow fluorescent carbon dot. The L-tartaric acid-m-phenylenediamine-based CDs powder is dispersed in ethanol for subsequent use.

[0057] The transmission electron microscopy and high-resolution transmission electron microscopy of the Y-CDs prepared in this example are as follows: Figure 2 As shown in c, the particle size distribution histogram is as follows Figure 3 As shown in c, it can be seen that Y-CDs were successfully prepared, and the particle size of Y-CDs was 8.68±0.05nm.

[0058] Example 4

[0059] Accurately weigh 0.901g of caffeic acid and 0.216g of m-phenylenediamine, add them to 3mL of phosphoric acid, and dissolve them in 7mL of anhydrous ethanol. In a 50mL polytetrafluoroethylene-lined reactor, react at 200°C for 6h. After the reaction is completed, cool to room temperature, centrifuge at 10000rpm for 15min, collect the supernatant, wash and filter through a microporous membrane (0.22μm), and vacuum freeze-dry the obtained suspension to obtain caffeic acid-m-phenylenediamine-based CDs powder, denoted as W-CDs, which is a carbon dot with white fluorescence emission. The caffeic acid-m-phenylenediamine-based CDs powder is dispersed in ethanol for subsequent use.

[0060] The transmission electron microscopy and high-resolution transmission electron microscopy of the W-CDs prepared in this example are as follows: Figure 2 As shown in (d), the particle size distribution histogram is as follows Figure 3 As shown in middle d, it can be seen that W-CDs were successfully prepared, and the particle size of W-CDs was 7.07±0.05nm.

[0061] Example 1-Example 4 uses different carbon sources and different solvents to participate in the synthesis of carbon dots, and the other conditions are the same. Under the conditions of different carbon sources and different solvents, the ultraviolet-visible spectra, fluorescence spectra and physical images of the m-phenylenediamine-based colored carbon dots prepared in Example 1-Example 4 are as follows: Figure 4As shown, it can be seen that the excitation wavelength (Ex) and emission wavelength (Em) of B-CDs are 360nm and 418nm respectively; the Ex / Em of G-CDs is 471 / 514nm; the Ex / Em of Y-CDs is 469 / 518nm; and W-CDs has two emission peaks at 420nm and 518nm, and its Ex / Em is 362 / 420nm; and the m-phenylenediamine-based color CDs prepared in Examples 1 to 4 all show strong absorption ability in the ultraviolet region of 200-300nm.

[0062] Application Example 1

[0063] The B-CDs prepared in Example 1 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 used as nucleic acid dyes in agarose gel electrophoresis to detect nucleic acid products. The effect of different concentrations of B-CDs solutions as nucleic acid dyes is shown in the figure below. Figure 5 As shown in a, it can be seen that the optimal concentration of B-CDs prepared in Example 1 as a nucleic acid dye is 100 mg / mL.

[0064] Application Example 2

[0065] The G-CDs prepared in Example 2 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 used as nucleic acid dyes in agarose gel electrophoresis to detect nucleic acid products. The effect of different concentrations of G-CDs solutions as nucleic acid dyes is shown in the figure below. Figure 5 As shown in b, it can be seen that the optimal concentration of G-CDs prepared in Example 2 as a nucleic acid dye is 100 mg / mL.

[0066] Application Example 3

[0067] The Y-CDs prepared in Example 3 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 used as nucleic acid dyes in agarose gel electrophoresis to detect nucleic acid products. The effect of different concentrations of Y-CDs solutions as nucleic acid dyes is shown in the figure below. Figure 5 As shown in c, it can be seen that the optimal concentration of Y-CDs prepared in Example 3 as a nucleic acid dye is 100 mg / mL.

[0068] Application Example 4

[0069] The W-CDs 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 used as nucleic acid dyes in agarose gel electrophoresis to detect nucleic acid products. The effect of different concentrations of W-CDs solutions as nucleic acid dyes is shown in the figure below. Figure 5 As shown in Figure d, it can be seen that the optimal concentration of W-CDs prepared in Example 4 as a nucleic acid dye is 100 mg / mL.

[0070] Application Example 5

[0071] DNA Marker III (purchased from Shanghai Aladdin Reagent Co., Ltd.) was added to the sample wells at a sample volume of 1 μL, 2 μL, 3 μL and 4 μL respectively, and the voltage was maintained at 120V for 20 minutes. After bromophenol blue was electrophoresed to 1-2 cm of the gel, the staining effect of each band could be clearly observed in the fully automatic gel imaging analyzer (Bio-Rad Gel-Doc EZ, USA). This was used to determine the minimum detection limit of the B-CDs prepared in Example 1 as a nucleic acid dye. The sensitivity effect diagram of the B-CDs prepared in Example 1 as a nucleic acid dye is shown in the figure below: Figure 6 As shown in a (Marker 3 is composed of a mixture of 7 double-stranded linear DNA fragments, the indicator band is 1200bp, and the rest are non-indicator bands, the target content is 50ng / 5μL, and the loading amount is controlled at 1μL, which can be explained that the amount of target contained in the remaining non-indicator bands is 10ng / μL), it can be seen that the B-CDs prepared in Example 1 can detect 10ng / μL of DNA product.

[0072] Application Example 6

[0073] Same as Application Example 5, except that the carbon dots are G-CDs prepared in Example 2. The sensitivity effect diagram of G-CDs prepared in Example 2 as nucleic acid dyes is shown in FIG. Figure 6 As shown in b, it can be seen that the G-CDs prepared in Example 2 can detect 10 ng / μL of DNA product.

[0074] Application Example 7

[0075] Same as Application Example 5, except that the carbon dots are Y-CDs prepared in Example 3. The sensitivity effect diagram of Y-CDs prepared in Example 3 as nucleic acid dye is shown in FIG. Figure 6 As shown in c, it can be seen that the Y-CDs prepared in Example 3 can detect 10 ng / μL of DNA product.

[0076] Application Example 8

[0077] Same as Application Example 5, except that the carbon dots are W-CDs prepared in Example 4. The sensitivity effect diagram of W-CDs prepared in Example 4 as nucleic acid dyes is shown in FIG. Figure 6 As shown in (d), it can be seen that the W-CDs prepared in Example 4 can detect 10 ng / μL of DNA product.

[0078] Application Example 9

[0079] The B-CDs prepared in Example 1 were used as nucleic acid dyes to detect DNA. Weigh 0.25g of agarose, add 25mL 1XTAE (triacetate-acetic acid buffer), heat and wait for cooling to prepare 1wt% agarose gel, add 100mg / mL B-CDs solution and mix well, insert a comb, wait for solidification and then remove the comb, put it into the electrophoresis tank and make sure the buffer (1XTAE) covers the gel, add the obtained PCR product to the spotting wells at a loading volume of 5μL, keep at a voltage of 120V for 20min, and after bromophenol blue is electrophoresed to 1-2cm of the gel, the corresponding bands can be clearly seen in the gel imager. The effect diagram of B-CDs prepared in Example 1 on DNA detection is shown in the figure below. Figure 7 As shown in a, it can be seen that the B-CDs prepared in Example 1 can be used as a nucleic acid dye to detect the target DNA.

[0080] Application Example 10

[0081] The same as Application Example 9, the only difference is that the carbon dots in this application example are G-CDs prepared in Example 2. The effect of G-CDs prepared in Example 2 on DNA detection is shown in the figure Figure 7 As shown in b, it can be seen that the G-CDs prepared in Example 2 can be used as a nucleic acid dye to detect the target DNA.

[0082] Application Example 11

[0083] The same as Application Example 9, the only difference is that the carbon dots in this application example are Y-CDs prepared in Example 3. The effect of Y-CDs prepared in Example 3 on DNA detection is shown in the figure Figure 7 As shown in c, it can be seen that the Y-CDs prepared in Example 3 can be used as a nucleic acid dye to detect the target DNA.

[0084] Application Example 12

[0085] The same as Application Example 9, the only difference is that the carbon dots in this application example are W-CDs prepared in Example 4. The effect of W-CDs prepared in Example 4 on DNA detection is shown in the figure Figure 7 As shown in (d), it can be seen that the W-CDs prepared in Example 4 can be used as a nucleic acid dye to detect the target DNA.

[0086] Application Example 13

[0087] The B-CDs prepared in Example 1 were used as nucleic acid dyes to detect RNA. Weigh 0.25g agarose, add 25mL 1XTAE, heat and wait for cooling to prepare 1wt% agarose gel, add 100mg / mL B-CDs solution, mix well, insert a comb and wait for solidification, then remove the comb and put it into the electrophoresis tank. The buffer solution must cover the gel. Add the obtained PCR product to the spotting well at a loading volume of 5μL and keep the voltage at 120V for 20min. After bromophenol blue is electrophoresed to 1-2cm of the gel, the corresponding band can be clearly seen in the gel imager. The effect diagram of B-CDs prepared in Example 1 on RNA detection is shown in the figure. Figure 8 As shown in a, it can be seen that the B-CDs prepared in Example 1 can be used as a nucleic acid dye to detect the target RNA.

[0088] Application Example 14

[0089] The same as Application Example 13, the only difference is that the carbon dots in this application example are G-CDs prepared in Example 2. The effect of G-CDs prepared in Example 2 on RNA detection is shown in FIG. Figure 8 As shown in b, it can be seen that the G-CDs prepared in Example 2 can be used as a nucleic acid dye to detect the target RNA.

[0090] Application Example 15

[0091] The same as Application Example 13, the only difference is that the carbon dots in this application example are Y-CDs prepared in Example 3. The effect of Y-CDs prepared in Example 3 on RNA detection is shown in FIG. Figure 8 As shown in c, it can be seen that the Y-CDs prepared in Example 3 can be used as a nucleic acid dye to detect the target RNA.

[0092] Application Example 16

[0093] The same as Application Example 13, the only difference is that the carbon dots in this application example are W-CDs prepared in Example 4. The effect of W-CDs prepared in Example 4 on RNA detection is shown in FIG. Figure 8 As shown in (d), it can be seen that the W-CDs prepared in Example 4 can be used as a nucleic acid dye to detect the target RNA.

[0094] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A nucleic acid dye, characterized in that The nucleic acid dye is m-phenylenediamine-based colored carbon dots.

2. The nucleic acid dye according to claim 1, characterized in that The method for preparing the m-phenylenediamine-based colored carbon dots comprises the following steps: mixing m-phenylenediamine and acid, reacting under hydrothermal conditions, and after the reaction is completed, centrifuging and drying to obtain the m-phenylenediamine-based colored carbon dots; The acid includes tyrosine, phosphoric acid, L-tartaric acid or caffeic acid.

3. The nucleic acid dye according to claim 2, characterized in that The temperature of the hydrothermal reaction was 200 °C and the time was 6 h.

4. The nucleic acid dye according to claim 2, characterized in that When the acid is tyrosine, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: weigh 0.906 g of tyrosine and 0.216 g of m-phenylenediamine and dissolve them in 10 mL of N,N-dimethylformamide solution, react at 200°C for 6 hours, and after the reaction is completed, cool to room temperature, centrifuge, collect the supernatant, wash and filter through a microporous membrane, and vacuum freeze-dry the obtained suspension to obtain the m-phenylenediamine-based colored carbon dots, which are blue fluorescent emitting carbon dots.

5. The nucleic acid dye according to claim 2, characterized in that When the acid is phosphoric acid, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: weigh 0.216 g of m-phenylenediamine, add it to 3 mL of phosphoric acid, and then dissolve it in 7 mL of anhydrous ethanol, react at 200°C for 6 hours, and after the reaction is completed, cool to room temperature, centrifuge, collect the supernatant, wash and filter it through a microporous membrane, and vacuum freeze-dry the obtained suspension to obtain the m-phenylenediamine-based colored carbon dots, which are green fluorescent emitting carbon dots.

6. The nucleic acid dye according to claim 2, characterized in that When the acid is L-tartaric acid, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: weigh 0.75 g L-tartaric acid and 0.216 g m-phenylenediamine, add them to 3 mL phosphoric acid, and then dissolve them in 7 mL anhydrous ethanol. React at 200°C for 6 hours. After the reaction is completed, cool to room temperature, centrifuge, collect the supernatant, wash and filter it through a microporous membrane, and vacuum freeze-dry the obtained suspension to obtain the m-phenylenediamine-based colored carbon dots, which are yellow fluorescent carbon dots.

7. The nucleic acid dye according to claim 2, characterized in that When the acid is caffeic acid, the preparation method of m-phenylenediamine-based colored carbon dots is as follows: weigh 0.901 g of caffeic acid and 0.216 g of m-phenylenediamine, add them to 3 mL of phosphoric acid, and dissolve them in 7 mL of anhydrous ethanol. React at 200°C for 6 hours. After the reaction is completed, cool to room temperature, centrifuge, collect the supernatant, wash and filter it through a microporous membrane, and vacuum freeze-dry the obtained suspension to obtain the m-phenylenediamine-based colored carbon dots, which are white fluorescent emitting carbon dots.

8. Use of the nucleic acid dye according to any one of claims 1 to 7 in nucleic acid visualization detection.

9. The use of the nucleic acid dye in nucleic acid visualization detection according to claim 8, characterized in that: The concentration of the m-phenylenediamine-based colored carbon dots as nucleic acid dyes is 10-100 mg / mL.

10. The use of the nucleic acid dye in nucleic acid visualization detection according to claim 9, characterized in that: The concentration of the m-phenylenediamine-based colored carbon dots as nucleic acid dyes is 100 mg / mL.

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