阅读说明：本技术 一种核酸检测用改性硅胶膜的制备方法 (Preparation method of modified silica gel membrane for nucleic acid detection ) 是由 余满江 于 2020-07-15 设计创作，主要内容包括：本发明属于核酸提取材料制备技术领域,具体涉及一种核酸检测用改性硅胶膜的制备方法。该制备方法的步骤包括：先以正硅酸乙酯为硅源以浓盐酸、PVA和甲基纤维素等制备二氧化硅混合溶胶,然后把溶胶用羧基改性并以此为基础在二氧化硅表面添加PDA和CTS,最后在洁净室用甩胶机制得核酸检测用改性硅胶膜。本发明制备的核酸检测用改性硅胶膜在核酸提取的产量低、核酸提取时间长的上有极大的进步。(The invention belongs to the technical field of preparation of nucleic acid extraction materials, and particularly relates to a preparation method of a modified silica gel membrane for nucleic acid detection. The preparation method comprises the following steps: firstly, taking ethyl orthosilicate as a silicon source, preparing silicon dioxide mixed sol by using concentrated hydrochloric acid, PVA, methyl cellulose and the like, then modifying the sol by using carboxyl, adding PDA and CTS on the surface of the silicon dioxide on the basis of the modified sol, and finally preparing the modified silica gel film for nucleic acid detection by using a spin coater in a clean room. The modified silica gel membrane for nucleic acid detection prepared by the invention has great progress in low yield of nucleic acid extraction and long nucleic acid extraction time.)

1. A preparation method of a modified silica gel membrane for nucleic acid detection is characterized by comprising the following steps:

the method comprises the following steps: mixing anhydrous ethanol and glacial acetic acid, adding ethyl orthosilicate, ultrasonically stirring for 10-20min, then dripping a proper amount of glycerol-deionized water solution into the mixed solution, and stirring for 5-10min at room temperature; slowly dripping concentrated hydrochloric acid into the solution, stirring in a water bath at 50-60 ℃ for 30-40min, cooling the water bath to 35-40 ℃ after dripping is finished, and adding a PVA (polyvinyl alcohol) aqueous solution and a methylcellulose ethanol solution into the mixed solution in sequence and stirring at room temperature for 40-60 min; then filtering the solution by using filter paper to obtain silicon dioxide mixed sol;

step two: ultrasonically treating the silicon dioxide mixed sol for 10-15min, uniformly dispersing the silicon dioxide mixed sol in ethanol, then adding gamma-aminopropyltriethoxysilane, stirring for 2-4h at room temperature, respectively centrifugally washing for 1 time by using ethanol, acetone and tetrahydrofuran, ultrasonically treating a sample for 5-10min after washing, uniformly distributing the sample in 50ml of tetrahydrofuran, then adding trimellitic anhydride, and stirring for 3-5h in a water bath at 50-60 ℃; then centrifugally washing the mixture for 3 times by using deionized water to obtain carboxyl modified silicon dioxide mixed sol;

step three: dissolving the carboxyl modified silicon dioxide mixed sol in a dopamine solution, stirring for 5-10min, adding a chitosan solution (CTS), stirring for 12-18h at room temperature, and centrifuging; then adding the centrifugal product into a glutaraldehyde solution, stirring in a water bath at 45-50 ℃ for 2-3h, washing with deionized water for 2 times, and drying at 60-70 ℃ under argon atmosphere;

step four: mixing the modified silicon dioxide prepared in the step three with a small amount of deionized water, then placing the mixture in a 100-grade clean room, taking a silicon wafer as a substrate, forming a film by using a spin coater, carrying out heat treatment at 350 ℃ for 40-60min under the protection of nitrogen, and cooling to room temperature to obtain a modified silica gel film for nucleic acid detection;

in the first step, the addition amount of the ethyl orthosilicate is 0.05-0.1mol, the concentration of the ethyl orthosilicate in a mixed solution of absolute ethyl alcohol and glacial acetic acid is 0.6-0.85g/ml, the volume ratio of the absolute ethyl alcohol to the glacial acetic acid is 2:1-1:1, the mass ratio of the ethyl orthosilicate to the glycerol is 1:1-5:1, the mass ratio of the ethyl orthosilicate to the deionized water is 1:2-1:4, and the mass ratio of the ethyl orthosilicate to the concentrated hydrochloric acid is 20:1-33: 1;

the concentration of the ethanol solution of the silica mixed sol prepared in the second step is 0.1g/ml, the mass ratio of the silica to the gamma-aminopropyltriethoxysilane in the system is 25:1-50:1, and the mass ratio of the silica to the trimellitic anhydride is 10:1-15: 1;

the concentration of the dopamine solution of the silicon dioxide mixed sol in the third step is 0.05 g/ml; the chitosan solution is diluted to 1 wt% by acetic acid, and the volume ratio of the addition amount of the chitosan solution to the dopamine solution is 0.8:1-1: 1; the concentration of the glutaraldehyde solution is 1 wt%, and the volume ratio of the glutaraldehyde solution to the dopamine solution is 1:8-1: 10.

2. The method for preparing the modified silica gel membrane for nucleic acid detection according to claim 1, wherein the PVA aqueous solution in the first step is 10 wt%, and the volume ratio of the PVA aqueous solution to the absolute ethyl alcohol-glacial acetic acid mixed solution is 1:2-1: 4; the concentration of the ethanol solution of the methyl cellulose is 0.5 wt%, and the volume ratio of the ethanol solution of the methyl cellulose to the mixed solution of the absolute ethyl alcohol and the glacial acetic acid is 1:2-1: 4.

3. The method for preparing the modified silica gel membrane for nucleic acid detection according to claim 1, wherein the method for preparing the dopamine solution in the third step comprises: mixing dopamine hydrochloride, Tris-HCl buffer solution and deionized water according to the mass ratio of 5-10:3-6:1250, and stirring at room temperature to prepare the dopamine hydrochloride-Tris-HCl buffer solution.

4. The method according to claim 1, wherein in the first step, a proper amount of glycerol-deionized water solution is added dropwise to the mixed solution at a rate of 1 ml/min.

5. The method for preparing a modified silica gel membrane for nucleic acid detection as claimed in any one of claims 1 to 4, wherein the rotation speed of the spin coater in the fourth step is 2500-; the heating rate of the silicon membrane during heat treatment is kept at 2 ℃/min.

6. The modified silica gel membrane prepared by the preparation method of the modified silica gel membrane for nucleic acid detection according to any one of claims 1 to 5, wherein the ratio of the highest peak of nucleic acid absorption to the highest peak of protein absorption during nucleic acid detection is greater than 1.8, the nucleic acid concentration is greater than 28 μ l, and the extraction completion time is less than 45 min.

7. Use of the modified silica gel membrane of claim 6 for nucleic acid detection.

Technical Field

The invention belongs to the technical field of preparation of nucleic acid extraction materials, and particularly relates to a preparation method of a modified silica gel membrane for nucleic acid detection.

Background

Nucleic acid extraction is one of the most critical methods in molecular biology, since nucleic acid extraction is the starting point for downstream nucleic acid detection, research or product development, and the quality and integrity of the isolated nucleic acid directly affects the research or diagnostic result. Many specialized nucleic acid extraction methods are available to extract, or total nucleic acids from a variety of biological samples. Most commercial nucleic acid extraction kits of the prior art employ solid phase extraction methods, which mainly use a solid phase support as a carrier, and comprise four key steps of cell lysis, nucleic acid adsorption, rinsing and elution. However, the method is mainly divided into two methods, namely, nucleic acid extraction by a centrifugal column method and nucleic acid extraction by a magnetic bead method, according to the difference of the used solid phase support.

The main principle of the magnetic bead method for extracting nucleic acid is to separate nucleic acid by using particles with magnetic charges; the magnetic particles are generally made of iron oxide particles with active groups coated on the surfaces, the active groups coated on the surfaces of the magnetic beads can specifically adsorb nucleic acid, and the nucleic acid can be removed later by permanent magnets in a magnetic field. The ferric oxide has large surface area and strong capability of combining nucleic acid, and can be used as a better separation carrier. After the side wall of the container is endowed with magnetism, the magnetic beads combined with the nucleic acid in the sample mixture are gathered on the wall of the container, other impurities can be removed by directly pouring the container, and repeated centrifugation is avoided. Most of the centrifugal column method for extracting nucleic acid is to use a silica gel filter membrane as a solid phase carrier and is based on the unique property that silicon dioxide is selectively combined with DNA. The principle is the high affinity between the negatively charged DNA backbone and the positively charged silica gel filter. The sodium ions act as a cation bridge, which attracts negatively charged oxygen within the phosphate backbone of the nucleic acid. Under high salt conditions (pH < 7), sodium ions can break the hydrogen bonds between hydrogen in water and negatively charged oxygen in silicon. After removing all impurities by extensive rinsing, the purified DNA is eluted with a buffer or distilled water at a low ionic strength (pH. gtoreq.7).

The centrifugal column method has extremely high potential in large-scale nucleic acid detection, but it also has the disadvantages of low yield of nucleic acid extraction and long time consumption of the whole extraction.

Disclosure of Invention

Aiming at solving the problems of low yield and long time consumption of the whole extraction of nucleic acid by the centrifugal column method in the background technology, the invention provides a modified silica gel membrane for a solid phase carrier for extracting nucleic acid by the centrifugal column method.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of a modified silica gel membrane for nucleic acid detection comprises the following steps:

the method comprises the following steps: dissolving Tetraethoxysilane (TEOS) in a mixed solution of absolute ethyl alcohol and glacial acetic acid, ultrasonically stirring for 10-20min, then slowly dripping a proper amount of glycerol-deionized water solution into the mixed solution, and stirring for 5-10min at room temperature. And then slowly dripping concentrated hydrochloric acid into the solution, stirring in a water bath at 50-60 ℃ for 30-40min, cooling the water bath to 35-40 ℃ after finishing dripping, and sequentially adding a proper amount of PVA aqueous solution and an ethanol solution of methylcellulose into the mixed solution, and stirring at room temperature for 40-60 min. The solution was then filtered through filter paper to obtain a silica mixed sol.

Step two: dispersing the silicon dioxide mixed sol in a certain amount of ethanol, ultrasonically mixing for 10-15min, adding a proper amount of gamma-aminopropyltriethoxysilane, stirring for 2-4h at room temperature, respectively centrifugally washing for 1 time by using ethanol, acetone and tetrahydrofuran, ultrasonically dispersing a sample in 50ml of tetrahydrofuran for 5-10min after washing, then adding a proper amount of trimellitic anhydride, and stirring for 3-5h in a 50-60 ℃ water bath; and then centrifugally washing with deionized water for 3 times to obtain the carboxyl modified silicon dioxide mixed sol.

Step three: dissolving the carboxyl modified silicon dioxide mixed sol into a certain amount of dopamine solution, stirring for 5-10min, adding a proper amount of chitosan solution (CTS), stirring for 12-18h at room temperature, and centrifuging. Then adding the centrifugal product into a glutaraldehyde solution, stirring in a water bath at 45-50 ℃ for 2-3h, washing with deionized water for 2 times, and drying at 60-70 ℃ under argon atmosphere.

Step four: and mixing the modified silicon dioxide prepared in the step three with a small amount of deionized water, then placing the mixture in a 100-grade clean room, taking a silicon wafer as a substrate, forming a film by using a spin coater, carrying out heat treatment on the gel silica film at 350 ℃ for 40-60min under the protection of nitrogen, and cooling to room temperature to obtain the modified silica gel film for nucleic acid detection.

In the first step, the addition amount of the ethyl orthosilicate is 0.05-0.1mol, the concentration of the ethyl orthosilicate in a mixed solution of absolute ethyl alcohol and glacial acetic acid is 0.6-0.85g/ml, the volume ratio of the absolute ethyl alcohol to the glacial acetic acid is 2:1-1:1, the mass ratio of the ethyl orthosilicate to the glycerol is 1:1-5:1, the mass ratio of the ethyl orthosilicate to the deionized water is 1:2-1:4, and the mass ratio of the ethyl orthosilicate to the concentrated hydrochloric acid is 20:1-33: 1.

The concentration of the ethanol solution of the silica mixed sol prepared in the second step is 0.1g/ml, the mass ratio of the silica to the gamma-aminopropyltriethoxysilane in the system is 25:1-50:1, and the mass ratio of the silica to the trimellitic anhydride is 10:1-15: 1.

The preparation method of the dopamine solution in the third step comprises the following steps: mixing dopamine hydrochloride, Tris-HCl buffer solution and deionized water according to the mass ratio of 5-10:3-6:1250, and stirring at room temperature to obtain the dopamine-containing solution, wherein the concentration of the dopamine-containing solution of the silicon dioxide mixed sol is 0.05 g/ml; diluting chitosan solution to 1 wt% with acetic acid, wherein the volume ratio of the chitosan solution to the dopamine solution is 0.8:1-1: 1; the concentration of the glutaraldehyde solution is 1 wt%, and the volume ratio of the glutaraldehyde solution to the dopamine solution is 1:8-1: 10.

Preferably, in the first step, the PVA aqueous solution accounts for 10 wt%, and the volume ratio of the PVA aqueous solution to the absolute ethyl alcohol-glacial acetic acid mixed solution is 1:2-1: 4; the concentration of the ethanol solution of the methyl cellulose is 0.5 wt%, and the volume ratio of the methyl cellulose solution to the absolute ethyl alcohol-glacial acetic acid mixed solution is 1:2-1: 4.

Preferably, the speed of dropping a proper amount of glycerol-deionized water solution into the mixed solution in the first step is 1 ml/min.

Preferably, the rotating speed of the spin coater in the fourth step is 2500-; the heating rate of the silicon membrane during heat treatment is kept at 2 ℃/min.

The invention also provides the modified silica gel membrane prepared by the preparation method, the ratio of the highest peak of nucleic acid absorption to the highest peak of protein absorption is more than 1.8, the nucleic acid concentration is more than 28 mu l, and the extraction completion time is less than 45 min. On the other hand, the application of the modified silica gel membrane in nucleic acid detection is also provided.

Has the advantages that: the modified silica gel membrane for nucleic acid detection provided by the invention effectively solves the problems of low yield of nucleic acid extraction and long time consumption of the whole extraction in the prior art. According to the invention, by mixing the methyl cellulose and the silicon dioxide, the internal structure of the prepared silica gel membrane is optimized, the contact between the silica gel membrane and the nucleic acid detection liquid is increased, the binding rate of the silicon dioxide to the nucleic acid is improved, and a site is provided for the subsequent grafting of dopamine and CTS; the surface of the silica is modified by carboxyl subsequently, and PDA and CTS are grafted on the basis of the carboxyl, so that the selective adsorption of the silica gel membrane on nucleic acid can be further enhanced; but also enhances the adsorption strength of nucleic acid to a certain extent, thereby reducing the time for centrifugation. Through the modification, the nucleic acid extraction yield is effectively improved under the combined action of PDA, CTS and methylcellulose, and the time length of the whole process is reduced.

Drawings

FIG. 1 is a scanning electron microscope image of 100 μm of the modified silica gel film obtained in example 1 of the present invention.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.