A dye-glue-free screening medium premix composition and its preparation method and application

Abstract

The present invention provides a dye-gel-free screening medium premix composition and a preparation method and application thereof. The dye-gel-free screening medium premix composition comprises a gel-free screening medium, a nucleic acid fluorescent dye and an electrolyte buffer: the gel-free screening medium is selected from one or more of linear polyacrylamide, methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, polyvinyl alcohol and polyethylene oxide; the nucleic acid fluorescent dye is a nucleic acid fluorescent dye excited by blue light; the thermal stability temperature of the nucleic acid fluorescent dye is 10-30°C. The present invention adopts a gel-free screening medium, which is suitable for fast and efficient molecular screening, and improves the convenience and efficiency of the experiment; the nucleic acid fluorescent dye can be stored at room temperature, has good water solubility, and is compatible with the gel-free screening medium.

Description

Technical Field

[0001] The invention relates to the technical field of nucleic acid fragment analysis, and in particular to a dye-gel-free sieving medium premix composition and a preparation method and application thereof. Background Art

[0002] Electrophoresis is a technique that separates and analyzes biomolecules (such as proteins and nucleic acids) based on their charge, size, and shape under the action of an electric field. Over time, electrophoresis has evolved from traditional slab gel electrophoresis to more advanced capillary electrophoresis and then to highly integrated chip electrophoresis.

[0003] Slab gel electrophoresis is one of the most classic electrophoresis techniques, including polyacrylamide gel electrophoresis (PAGE), agarose gel electrophoresis, etc. These techniques use gel as a supporting medium and apply an electric field to make charged molecules migrate in the pores of the gel. Slab gel electrophoresis is intuitive and easy to operate, and is widely used in routine analysis in laboratories. However, this method has some limitations, such as relatively low separation efficiency, long analysis time, and difficulty in achieving high throughput and automated processing. Capillary electrophoresis (CE) is an electrophoresis process performed in a thin capillary. Compared with slab gel electrophoresis, capillary electrophoresis can provide higher separation efficiency and faster analysis speed due to the hydrodynamic effect and smaller thermal diffusion in the capillary. However, capillary electrophoresis consumes a lot of samples, takes a long time to test, and has a low throughput. With the establishment of micro-total analysis systems and the development of chip laboratories, a microfluidic chip electrophoresis (MCE) technology has been developed based on the electrophoresis theory and technology in capillaries, which miniaturizes the electrophoresis channel and integrates it on a chip. MCE has low sample consumption, fast analysis speed, high separation efficiency, and sample preparation and detection are integrated on a microchip, making it suitable for rapid analysis and on-site detection. In recent years, it has been widely used in biology, food, environment, clinical medicine, pharmacy and other fields.

[0004] However, the gel sieving medium commonly used in the past cannot meet the application requirements of chip electrophoresis. The separation channel of chip electrophoresis is extremely fine. Conventional gel solutions will only be liquid at high temperatures, and will gradually solidify at room temperature, making it difficult to pour into the chip channel. Therefore, in order to achieve more efficient, simple and flexible biomolecule analysis, chip electrophoresis generally uses gel-free sieving media. Gel-free sieving uses non-cross-linked, low-viscosity linear polymer solutions as sieving media to replace the solid gel in traditional gel electrophoresis, thereby greatly simplifying the sample processing and separation process and avoiding the cumbersome steps of gel preparation and potential channel blockage problems. This method not only significantly speeds up the electrophoresis speed and improves the experimental throughput, but also greatly reduces the cost due to the significant reduction in the volume of samples and reagents required. At the same time, the high adjustability of the gel-free system allows researchers to optimize the separation conditions by changing the type and concentration of the sieving medium to meet the analysis requirements of different biomolecules, enhancing the flexibility and scope of application of the experiment. Currently, the commonly used non-glue screening media are mainly: linear homopolymers, such as linear polyacrylamide (LPA), poly N, N-dimethylacrylamide (PDMA), polyethylene oxide (PEO), polyvinyl pyrrolidone (PVP), cellulose and its derivatives, etc.

[0005] Chip electrophoresis combined with laser induced fluorescence (LIF) detection is a highly sensitive analytical technique that uses microfluidic chips for rapid separation of biomolecules and achieves accurate detection of target molecules through LIF technology. In this process, the sample is driven by an electric field through the microchannels in the chip and is efficiently separated according to the size, shape and charge differences of the molecules. Subsequently, laser irradiation is used to excite the fluorescent labeled molecules in the sample, and the generated fluorescence signal is captured by the detector to achieve qualitative and quantitative analysis of specific analytes. Among them, fluorescent dyes play a vital role. They are used to label the biomolecules to be analyzed, such as DNA, RNA or proteins. These dyes can produce bright and distinctive fluorescence signals under laser irradiation, thereby achieving real-time and highly sensitive detection of target molecules during the high-speed separation process of microfluidic chips. The selection of fluorescent dyes needs to consider factors such as their excitation and emission wavelengths, quantum yield, chemical stability and biocompatibility to ensure optimal signal intensity and minimal background interference. Common fluorescent dyes include Cy5, GelGreen, SYBRGreen, propidium iodide (PI), etc., each of which has unique optical properties and is suitable for different analytical needs. However, these fluorescent dyes usually need to be stored at low temperatures to extend their stability and shelf life, and the solvent storage system is generally DMSO, which has poor water solubility. Most of the gel-free sieving media are aqueous conditions, and some fluorescent dyes are even incompatible with the gel-free sieving media.

[0006] In the commonly used nucleic acid fragment analyzers (such as LabChip® GXTouch) developed based on the principle of chip electrophoresis on the market, the dye and the gel solution must be stored separately in a 4°C refrigerator. When using, the user needs to take the reagent out of the refrigerator half an hour in advance to restore it to room temperature. It also takes a lot of time to prepare the gel-dye reagent by yourself and add it to the chip. The testing process is time-consuming and labor-intensive. Conventional gel-free screening media (CN 117264268A, CN 105367693A) are mostly used in capillary electrophoresis, with slow detection speed, low throughput and poor sensitivity. Therefore, there is an urgent need to screen out a fluorescent dye that is perfectly compatible with the gel-free screening system and has good room temperature stability. Summary of the invention

[0007] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a dye-gel-free screening medium premix composition and its preparation method and application, so as to solve the problem that existing fluorescent dyes usually need to be stored at low temperatures, have poor water solubility, and are not compatible with gel-free screening media.

[0008] To achieve the above-mentioned purpose and other related purposes, the present invention provides a dye-gel-free sieving medium premix composition, comprising a gel-free sieving medium, a nucleic acid fluorescent dye and an electrolyte buffer; the gel-free sieving medium is selected from one or more of linear polyacrylamide, methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, polyvinyl alcohol and polyethylene oxide; the nucleic acid fluorescent dye is a blue light-excited nucleic acid fluorescent dye; the thermal stability temperature of the nucleic acid fluorescent dye is 10~30°C.

[0009] More preferably, the excitation wavelength of the blue light excited nucleic acid fluorescent dye is 488 nm.

[0010] More preferably, the nucleic acid fluorescent dye is selected from one or more of EvaGreen nucleic acid dye, RTGreen nucleic acid dye, SYBRGreen nucleic acid dye, SYBR Gold nucleic acid dye, YeaGreen nucleic acid dye, GelGreen nucleic acid dye and GeneGreen nucleic acid dye.

[0011] The role of adding electrolyte buffer to the premixed composition of dye-free sieving medium of the present application is to ensure its conductivity. The preparation difficulty of gel in the chip is extremely great. The present application adopts non-gel sieving medium, which is mainly hydrophilic linear or branched macromolecules, such as linear polyacrylamide, etc., and its sieving principle is mainly based on molecular size exclusion and stereochemical effect. After these substances are dissolved in water, when the concentration is greater than a certain value, the viscosity of the system will change suddenly. At this time, the corresponding concentration is the entanglement threshold, and the macromolecule in the system will automatically form a dynamic network. When molecules of different sizes pass through this network, smaller molecules can enter and travel through these micropores, while larger molecules cannot enter completely due to size restrictions and can only move along the outer surface of the cellulose network. This process makes the path of macromolecules longer and the migration speed slows down, thereby separating from small molecules in time, and realizing screening and separation based on molecular size. This characteristic makes non-gel sieving medium particularly suitable for fast and efficient molecular sieving, especially in the separation and purification of biomolecules such as proteins and nucleic acids, it shows significant advantages, without the complex preparation process of traditional gel, and improves the convenience and efficiency of the experiment. The nucleic acid fluorescent dyes added in the present application can be stored at room temperature, have good water solubility, and are compatible with the gel-free screening medium.

[0012] In chip electrophoresis technology, the working principle of nucleic acid fluorescent dyes is based on the combination of fluorescent labeling and optical detection. When biological molecules in the sample, such as DNA, RNA or protein, are specifically labeled with nucleic acid fluorescent dyes, these molecules will be separated according to their molecular size, shape and charge distribution under the action of an externally applied electric field when passing through the microchannels on the chip during electrophoresis. When the fluorescently labeled molecules pass through the detection point, they are excited by laser or ultraviolet light, and the fluorescent dyes then emit fluorescent signals of specific wavelengths. These fluorescent signals are captured by highly sensitive detectors and converted into electronic signals, which are then analyzed by a data analysis system to achieve qualitative and quantitative analysis of the sample molecules. The use of nucleic acid fluorescent dyes greatly improves the detection accuracy and sensitivity of chip electrophoresis, allowing efficient and accurate analysis of trace biological molecules in extremely small volumes of samples.

[0013] Preferably, the electrolyte buffer is a mixed solution of 2-(N-morpholine)ethanesulfonic acid and tris(hydroxymethyl)aminomethane.

[0014] Preferably, the solvent of the electrolyte buffer is selected from one or more of water, methanol, ethanol, formamide and dimethyl sulfoxide, and at least contains water.

[0015] Electrolyte buffers are generally prepared with water, but a small amount of organic solvents can be added to improve separation or separation selectivity and enable many difficult-to-dissolve samples to be analyzed by chip electrophoresis. Commonly used organic solvent additives are mainly polar organic compounds with low volatility, such as methanol, ethanol, formamide, and dimethyl sulfoxide (DMSO).

[0016] Preferably, the pH of the electrolyte buffer is 4.0-9.0.

[0017] The selection of the electrolyte buffer solution in this application is mainly determined by pH. MES is 2-(N-morpholino)ethanesulfonic acid, Tris is trishydroxymethylaminomethane, and MES-Tris is a good biological buffer system with a wide pH adjustable range from 4.0 to 9.0, and has good biocompatibility. The buffer is generally prepared with water, but a small amount of organic solvent can be added to improve separation or separation selectivity, and to enable many difficult-to-dissolve samples to be analyzed by chip electrophoresis. Commonly used organic solvent additives are mainly polar organic substances with low volatility, such as methanol, ethanol, formamide, and DMSO.

[0018] Preferably, the concentration of 2-(N-morpholine)ethanesulfonic acid in the electrolyte buffer is 10-400 mmol / L, and the concentration of tris(hydroxymethyl)aminomethane is 10-400 mmol / L. The concentration and mobility of the electrolyte buffer will affect the separation effect and need to be selected according to the sample, but a better separation effect can be produced within the above range.

[0019] Preferably, the concentration of the non-gelatin sieving medium in the dye-non-gelatin sieving medium premix composition is 0.01-10% (m / v).

[0020] Preferably, the concentration of the nucleic acid fluorescent dye in the dye-gel-free sieving medium premix composition is 0.2-60 nmol / L.

[0021] Preferably, the dye-gel-free sieving media premix composition further comprises additives.

[0022] Preferably, the additive is selected from one or more of glucose, glycerol, mannitol and chitosan; and the concentration of the additive in the dye-gel-free sieving medium premix composition is 0.01-10% (m / v).

[0023] The present invention also provides a method for preparing a dye-gel-free screening medium premix composition, wherein a gel-free screening medium and a nucleic acid fluorescent dye are sequentially added into an electrolyte buffer solution and mixed evenly to obtain the dye-gel-free screening medium premix composition.

[0024] The present invention also provides an application of a dye-gel-free screening medium premix composition in chip electrophoresis combined with laser induced fluorescence detection. The dye-gel-free screening medium premix composition of the present invention has the characteristics of high resolution, fast detection, and room temperature stability when applied to chip electrophoresis combined with laser induced fluorescence detection, and has broad application prospects.

[0025] As described above, the present invention has the following beneficial effects: it adopts gel-free sieving medium, which is suitable for rapid and efficient molecular sieving based on molecular size exclusion and stereochemical effects, and shows significant advantages in the separation and purification of biological molecules such as proteins, nucleic acids, etc., without the need for the complex preparation process of traditional gels, thereby improving the convenience and efficiency of the experiment; the nucleic acid fluorescent dyes used can be stored at room temperature, have good water solubility, are compatible with gel-free sieving medium, and have the characteristics of high resolution, rapid detection, and room temperature stability when used in chip electrophoresis combined with laser induced fluorescence detection, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the electrophoresis diagram of the non-gel sieving of the GeneRuler LowRange DNA Ladder standard fragment by using the dye-non-gel sieving medium premix composition prepared in Example 1.

[0027] Figure 2 The chip electrophoresis separation diagram of the mixed sample of 105bp DNA and 107bp DNA (difference of 2bp) and the mixed sample of 103bp DNA and 107bp DNA (difference of 4bp) using the dye-gel-free sieving medium premix composition prepared in Example 1.

[0028] Figure 3 This is a chip electrophoresis separation diagram of the GeneRuler Low Range DNA Ladder standard fragment using the dye-gel-free sieving medium premix composition stored at room temperature for 3 months in Example 1.

[0029] Figure 4 This is the gel-free sieving electrophoresis electrophoresis diagram of the dye-gel-free sieving medium premix composition prepared in Example 2.

[0030] Figure 5 This is the gel-free sieving electrophoresis electrophoresis diagram of the dye-gel-free sieving medium premix composition prepared in Example 3.

[0031] Figure 6 This is the gel-free sieving electrophoresis electrophoresis diagram of the dye-gel-free sieving medium premix composition prepared in Comparative Example 1.

[0032] Figure 7This is the gel-free sieving electrophoresis electrophoresis diagram of the dye-gel-free sieving medium premix composition of Comparative Example 1 after being stored at room temperature for 5 days.

[0033] Figure 8 This is the gel-free sieving electrophoresis electrophoresis diagram of the dye-gel-free sieving medium premix composition prepared in Comparative Example 2. DETAILED DESCRIPTION

[0034] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0035] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0036] In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present invention does not exclude the existence of other devices / apparatuses before or after the combination device / apparatus or the insertion of other devices / apparatuses between these explicitly mentioned two devices / apparatuses, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or the scope of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the present invention without substantially changing the technical content.

[0037] Example 1

[0038] The embodiment of the present application provides a dye-gel-free screening medium premix composition, including a gel-free screening medium, a blue light-excited GelGreen nucleic acid fluorescent dye with an excitation wavelength of 488 nm, and an electrolyte buffer (MES-Tris), wherein the electrolyte buffer is a mixed aqueous solution of 2-(N-morpholinoline)ethanesulfonic acid (MES) and tris(hydroxymethylaminomethane) (Tris) with a pH of 8.0, wherein the concentration of 2-(N-morpholinoline)ethanesulfonic acid is 40 mmol / L, and the concentration of tris(hydroxymethylaminomethane) is 80 mmol / L; the gel-free screening medium uses 2% linear polyacrylamide (2% PAM).

[0039] The present application also provides a method for preparing the dye-glue-free screening medium premix composition, comprising:

[0040] 1) Weigh MES and Tris, add them to deionized water, and shake them evenly to make the final concentration of MES 40 mM and the final concentration of Tris 80 mM. Use a pH meter to test the specific pH value, and adjust the pH to 8.0 with a pH adjuster to prepare the electrolyte buffer;

[0041] 2) Add the weighed gel-free sieving medium to the electrolyte buffer, place it on a turntable, and rotate overnight to fully dissolve it, so that the concentration of the gel-free sieving medium is 2% (m / v);

[0042] 3) Adding fluorescent nucleic acid dye to a final concentration of 4 nmol / L to prepare a dye-gel-free sieving medium premix composition.

[0043] The embodiments of the present application also provide an application of the dye-gel-free sieving medium premix composition in chip electrophoresis combined with laser induced fluorescence detection.

[0044] First, the prepared dye-gel-free sieving medium premix composition is pumped into the channel of the chip so that the entire channel is evenly filled with the dye-gel-free sieving medium premix composition. Next, the sample is introduced into the channel containing the premix composition. When an electric field is applied, molecules of different sizes and charges are separated according to their migration rate differences. In addition, during the migration process, the molecules to be tested continuously combine with the fluorescent dye in the channel, and the separated components are detected by a laser-induced fluorescence detector, in which the bound fluorescent markers emit light under laser excitation, thereby achieving qualitative and quantitative analysis of the sample components.

[0045] Based on the above separation principle, the low-range DNA standard GeneRuler Low Range DNA Ladder was successfully separated using the dye-free sieving medium premix composition. The results are as follows Figure 1 As shown, from Figure 1 It can be seen that all 10 fragments have achieved baseline separation, and the peaks are sharp, and the separation effect is very good.

[0046] like Figure 2 As shown, the dye-gel-free sieving medium premix composition prepared above was used to successfully separate samples with a difference of 2 bp (105 bp and 107 bp), and clearly distinguish samples with a difference of 4 bp (103 bp and 107 bp), with good separation performance.

[0047] The dye-gel-free sieving medium premixed composition was stored at room temperature and continuously monitored at 0, 5, 10, 15, 20, 30 and 40 days. The premixed reagent system was still able to separate the low-range DNA standard GeneRuler Low Range DNA Ladder well up to the 90th day of storage at room temperature. Figure 3 As shown in Figure 2, all 10 fragments have achieved baseline separation, and the overall separation effect is similar to Figure 1 The prepared reagent systems shown are basically the same, indicating that the dye-gel-free sieving medium premix system described in this patent has good room temperature storage stability.

[0048] Example 2

[0049] The difference between this embodiment and embodiment 1 is that the sieving medium is different, specifically 1% linear polyacrylamide (1% PAM) is used, and the rest of the formula is exactly the same.

[0050] The low-range DNA standard GeneRuler Low Range DNA Ladder was successfully separated using this dye-free sieving medium premix. Figure 4 As shown, from Figure 4 It can be seen that the 10 fragments also achieved baseline separation, with sharp peaks and good separation effect, but the separation effect of small fragments was not as good as 2% PAM.

[0051] Example 3

[0052] The difference between this embodiment and embodiment 1 is that the sieving medium is different, specifically 4% hydroxypropyl cellulose (4% HPC) is used, and the rest of the formula is exactly the same.

[0053] This example successfully separated the low-range DNA standard GeneRuler LowRange DNA Ladder using this dye-free sieving medium premix. Figure 5 As shown, from Figure 5 It can be seen that the 10 fragments are also well separated, and the separation speed is faster.

[0054] Comparative Example 1

[0055] The difference between this comparative example and Example 1 is that the nucleic acid fluorescent dye is different, specifically SYBR Green nucleic acid dye is used, and the rest of the formula is exactly the same.

[0056] The dye-free sieving medium premix composition prepared in this comparative example has the following contents during separation: Figure 6The problem of tailing of large fragments is shown in the figure. This is because the molecular structure of SYBR Green nucleic acid dye itself is relatively large. After binding to large fragments, it will produce larger aggregates, resulting in tailing phenomenon.

[0057] The dye-free sieving medium premixed composition prepared in this comparative example was stored at room temperature for 5 days and then tested. The test results are as follows: Figure 7 As shown, from Figure 7 It can be seen that the fluorescence signal of the standard is severely attenuated and the separation effect is poor. This is because the dye has poor water solubility and poor room temperature stability and cannot support premixing with the gel-free sieving medium.

[0058] Comparative Example 2

[0059] The difference between this comparative example and Example 1 is that the screening medium is different, specifically 3% polyvinyl pyrrolidone (3% PVP), and the rest of the formula is exactly the same.

[0060] like Figure 8 As shown, the dye-gel-free sieving medium premix composition prepared in this comparative example has poor sieving ability. This is because PVP has a large molecular weight, generally between several thousand and several million Daltons, and it is difficult to form a microporous structure that allows nucleic acid molecules of a specific size to pass through like other electrophoretic polymers, and it is impossible to achieve separation based on the length of the fragments. PVP is generally more suitable for protecting nucleic acids and improving electrophoresis conditions in electrophoresis.

[0061] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A dye-free sieving medium premix composition, characterized in that: It comprises a gel-free sieving medium, a nucleic acid fluorescent dye and an electrolyte buffer; the gel-free sieving medium adopts 2% linear polyacrylamide; the nucleic acid fluorescent dye is a GelGreen nucleic acid dye excited by blue light with an excitation wavelength of 488nm; the electrolyte buffer is a mixed aqueous solution of 2-(N-morpholine)ethanesulfonic acid and tris(hydroxymethylaminomethane) with a pH of 8.0, wherein the concentration of 2-(N-morpholine)ethanesulfonic acid is 40mmol / L, and the concentration of tris(hydroxymethylaminomethane) is 80mmol / L; The preparation method of the dye-non-glue screening medium premix composition comprises: 1) Weigh 2-(N-morpholino)ethanesulfonic acid and tris(hydroxymethyl)aminomethane, add them to deionized water, and shake them evenly to make the final concentration of 2-(N-morpholino)ethanesulfonic acid 40 mmol / L and the final concentration of tris(hydroxymethyl)aminomethane 80 mmol / L. Use a pH meter to test the specific pH value, and adjust the pH to 8.0 with a pH adjuster to prepare an electrolyte buffer; 2) Add the weighed linear polyacrylamide to the electrolyte buffer, place it on a turntable, and rotate overnight to fully dissolve it so that the concentration of linear polyacrylamide is 2% m / v; 3) Adding fluorescent nucleic acid dye to a final concentration of 4 nmol / L to prepare a dye-gel-free sieving medium premix composition.

2. A method for preparing the dye-non-glue screening medium premix composition as claimed in claim 1, characterized in that: include: 1) Weigh 2-(N-morpholino)ethanesulfonic acid and tris(hydroxymethyl)aminomethane, add them to deionized water, and shake them evenly to make the final concentration of 2-(N-morpholino)ethanesulfonic acid 40 mmol / L and the final concentration of tris(hydroxymethyl)aminomethane 80 mmol / L. Use a pH meter to test the specific pH value, and adjust the pH to 8.0 with a pH adjuster to prepare an electrolyte buffer; 2) Add the weighed linear polyacrylamide to the electrolyte buffer, place it on a turntable, and rotate overnight to fully dissolve it so that the concentration of linear polyacrylamide is 2% m / v; 3) Adding fluorescent nucleic acid dye to a final concentration of 4 nmol / L to prepare a dye-gel-free sieving medium premix composition.

3. Use of the dye-gel-free sieving medium premix composition as claimed in claim 1 in chip electrophoresis combined with laser induced fluorescence detection.

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