Preparation method and application of nucleic acid adsorption silica gel membrane

Abstract

The invention discloses a preparation method and application of a nucleic acid adsorption silica gel membrane, and belongs to the technical field of molecular biology. The invention provides a preparation method of a nucleic acid adsorption silica gel membrane, which comprises the following steps: S1, dispersing mercaptopyridine modified silicon dioxide sol, adding the dispersed mercaptopyridine modified silicon dioxide sol into a buffer salt system, and mixing and reacting with a thickening agent, a nucleic acid binding accelerant and a surfactant to obtain a modified silica gel filling system; s2, mixing and pulping the modified silica gel filling system and long-chain mixed cellulose viscose, forming a coating by using an electrostatic spinning method, demolding, pressing and dehydrating to obtain the nucleic acid adsorption silica gel membrane, the nucleic acid adsorption silica gel membrane has high mechanical strength and excellent structural uniformity, the membrane body does not collapse or peel off in the use process, the theoretical column efficiency of the obtained silica gel column is remarkably improved, and the endurance capacity of the silica gel column to dust and electrostatic interference in the environment is enhanced.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to a method for preparing and applying a nucleic acid adsorption silica membrane. Background Technology

[0002] Nucleic acid extraction is one of the most fundamental and crucial steps in molecular biology experiments, and it serves as the starting point for downstream nucleic acid detection, functional studies, and the development of related products. The purity and integrity of nucleic acids directly affect the accuracy and reliability of subsequent analyses and research. Currently, various nucleic acid extraction methods are available to isolate and purify nucleic acids from a variety of biological samples, including cells, tissues, and blood. Among these, solid-phase extraction is widely used due to its simplicity and reproducibility. This method typically uses a silica-based membrane as the adsorption carrier and includes four key steps: cell lysis, nucleic acid adsorption, rinsing, and elution. Under high-salt ion conditions, nucleic acid fragments selectively adsorb onto the surface of the silica-based membrane within a centrifuge column. Subsequently, impurities such as proteins, primers, nucleotides, and enzymes are removed through rinsing and centrifugation. Finally, the purified nucleic acids are released from the membrane using a low-salt, high-pH elution buffer.

[0003] However, existing commercial nucleic acid extraction products generally use paper fiber or glass fiber as the substrate, which has several shortcomings: First, the nucleic acid adsorption capacity is limited, making it difficult to meet the needs of high-yield extraction; second, during long-term storage, they are susceptible to the effects of static electricity and dust in the air, leading to a decrease in nucleic acid binding capacity; third, the membrane's mechanical strength is insufficient, making it prone to collapse or damage during use; furthermore, the bonding stability between paper fiber or glass fiber and silica gel is poor, and the silica gel coating is prone to peeling off, affecting repeatability and stability. Limited by substrate strength and storage conditions, the yield and finished product stability of these products are difficult to improve, resulting in high production costs.

[0004] Therefore, there is an urgent need for a nucleic acid adsorption membrane material that can maintain high nucleic acid adsorption efficiency while also possessing high mechanical strength, long-term stability, and environmental resistance. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a method for preparing and applying a nucleic acid adsorption silica membrane. The nucleic acid adsorption silica membrane prepared by this invention possesses high mechanical strength and excellent structural uniformity. The membrane does not collapse or peel off during use, significantly improving the theoretical column efficiency of the resulting silica column and enhancing its resistance to dust and electrostatic interference in the environment. During nucleic acid extraction, this membrane not only improves the adsorption and recovery efficiency of nucleic acids but also exhibits excellent stability under long-term storage conditions, demonstrating significant advantages in both yield and preservation performance compared to existing commercial products.

[0006] This invention also provides the application of the nucleic acid adsorption silica membrane prepared by the preparation method in the fields of biomedical detection equipment and environmental monitoring equipment.

[0007] According to a first aspect of the present invention, a method for preparing a nucleic acid adsorption silica membrane is provided, comprising the following steps:

[0008] S1. The modified silica sol modified with mercaptopyridine was dispersed in a buffer salt system and then mixed and reacted with a thickener, a nucleic acid binding promoter and a surfactant to obtain a modified silica gel filling system;

[0009] S2. The modified silica gel filling system is mixed with long-chain mixed cellulose viscose to form a slurry, and then coated by electrospinning. After demolding, pressing and dehydration, a nucleic acid adsorption silica gel membrane is obtained.

[0010] The raw materials for preparing the mercaptopyridine-modified silica sol include: silica sol and epoxy-based polyethylene glycol mercaptopyridine.

[0011] The thiol-pyridine-modified silica sol of this invention significantly enhances the specific adsorption capacity of nucleic acids through electrostatic interactions (protonated pyridine groups binding to the negatively charged phosphate backbone of nucleic acids) and hydrogen bonding. Simultaneously, the buffer salt system, along with thickeners, nucleic acid binding promoters, and surfactants, further optimizes the adsorption environment, enhancing selective nucleic acid binding and reducing impurity interference. Secondly, a uniformly interwoven fiber network is formed through electrospinning, followed by high-pressure pressing and vacuum dehydration, resulting in a denser membrane structure and significantly improved mechanical strength and anti-collapse ability. Furthermore, the thiol-pyridine groups are firmly bonded to the silica surface via covalent bonds, preventing performance degradation caused by the degradation of modified groups in traditional silica membranes. The insulating properties of the cellulose substrate effectively reduce static electricity accumulation and dust adsorption, while the vacuum dehydration process reduces the material's environmental sensitivity. Therefore, the nucleic acid adsorption membrane of this invention significantly outperforms traditional glass fiber or paper fiber-based silica membranes in terms of adsorption efficiency, mechanical properties, and long-term stability, making it suitable for demanding nucleic acid extraction applications.

[0012] According to some embodiments of the present invention, the method for preparing the silica sol includes:

[0013] A1. A precursor silica gel solution is obtained by mixing and reacting a silicon source, a pH adjuster solution, and an aqueous alcohol solution;

[0014] A2. The precursor silica gel solution, acidic catalyst, and polymeric auxiliaries are mixed and reacted to obtain a mixed solution;

[0015] A3. After removing impurities from the mixture, a silica sol is obtained.

[0016] According to some embodiments of the present invention, in step A2, the acidic catalyst includes hydrochloric acid.

[0017] According to some embodiments of the present invention, step A2 includes: adding hydrochloric acid dropwise into the precursor silica gel solution, heating to 55-65°C and stirring in a water bath for 25-50 minutes, then cooling to 30-40°C and adding a polymeric additive while stirring at room temperature for 1-2 hours.

[0018] According to some embodiments of the present invention, the impurity removal step includes: cooling the mixture to 2-8°C and letting it stand for about 1-2 hours, and filtering it through a hollow fiber tube filter to obtain a silica suspension sol.

[0019] According to some embodiments of the present invention, the pH adjusting agent solution comprises an alcoholic solution of glacial acetic acid; the alcohol in the alcoholic solution of glacial acetic acid includes at least one of methanol, ethanol and isopropanol.

[0020] According to some embodiments of the present invention, the mass ratio of the silicon source to the alcoholic solution of alcohol and glacial acetic acid is 0.5 to 1.0:1. For example, it can be about 0.5:1, 0.6:1, 0.7:1, 0.8:1 or about 1.0:1, wherein 0.7:1 is preferred.

[0021] According to some embodiments of the present invention, the aqueous solution of the alcohol includes an aqueous solution of isopropanol.

[0022] According to some embodiments of the present invention, in the aqueous solution of isopropanol, the mass ratio of isopropanol to water is 1:1.5 to 2.5. For example, it can be 1:1.5, 1:1.8, 1:2.0, 1:2.3 or 1:2.5, preferably 1:2.

[0023] According to some embodiments of the present invention, the polymeric additives include polyethylene glycol and carboxymethyl cellulose.

[0024] According to some embodiments of the present invention, the polyethylene glycol comprises an aqueous solution of polyethylene glycol.

[0025] According to some embodiments of the present invention, the mass ratio of polyethylene glycol in the aqueous solution is 8-15%. Specifically, it can be about 8%, 10%, 12%, or about 15%, with 12% being preferred.

[0026] According to some embodiments of the present invention, the carboxymethyl cellulose comprises an isopropanol solution of carboxymethyl cellulose.

[0027] According to some embodiments of the present invention, the mass ratio of carboxymethyl cellulose in the isopropanol solution of carboxymethyl cellulose is 0.5% to 1%. Specifically, it can be about 0.5%, 0.6%, 0.7%, or about 1%, with 0.7% being preferred.

[0028] According to some embodiments of the present invention, the volume ratio of the aqueous solution of polyethylene glycol to the isopropanol solution of carboxymethyl cellulose is 2:1:1.5 to 2.5. For example, it can be about 2:1:1.5, 2:1:1.8, 2:1:2 or about 2:1:2.5, with 2:1:2 being preferred.

[0029] According to some embodiments of the present invention, the raw materials for preparing the mercaptopyridine-modified silica sol further include aminosilane; the preparation method includes:

[0030] A4. The silica sol is dispersed and then modified with aminosilane;

[0031] A5. Then, the modified aminosilane silica sol is reacted with epoxy polyethylene glycol mercaptopyridine to obtain mercaptopyridine-modified silica sol.

[0032] According to some embodiments of the present invention, the aminosilane includes N-aminoethyl-3-aminopropyltriethoxysilane.

[0033] According to some embodiments of the present invention, the mass ratio of the silica sol, N-aminoethyl-3-aminopropyltriethoxysilane, and epoxy polyethylene glycol mercaptopyridine is (20-40):(1-2):(1-2). For example, it can be about 20:1:1, 24:1.5:1.5, 30:1.8:1.8, or about 40:2:2, with 24:1.5:1.5 being preferred.

[0034] According to some embodiments of the present invention, the polyethylene glycol chain length in the epoxy-based polyethylene glycol mercaptopyridine is 2 to 4. For example, it can be 2, 3 or 4, with 2 being preferred.

[0035] According to some embodiments of the present invention, step A4 includes: dispersing the silica sol in isopropanol and ultrasonically stirring for 15-30 min, then adding N-aminoethyl-3-aminopropyltriethoxysilane, stirring for 2-4 h, then adding it to ice-cold tetrahydrofuran under stirring, then adding a specified amount of epoxy polyethylene glycol mercaptopyridine, stirring in a water bath at 55-70°C for 2-4 h, cooling, and centrifuging and washing with deionized water 3-6 times to obtain the silica sol modified with aminosilane.

[0036] According to some embodiments of the present invention, in step S1, the step of dispersing the mercaptopyridine-modified silica sol includes: dispersing the mercaptopyridine-modified silica sol in a mixed solution of isopropanol and deionized water with a volume ratio of 1:1, and stirring for 30 to 60 minutes.

[0037] According to some embodiments of the present invention, the mass ratio of the mercaptopyridine-modified silica sol to the mixed solution of isopropanol and deionized water is 1:2 to 3. For example, it can be about 1:2, 1:2.3, 1:2.5 or about 1:3, wherein 1:2.5 is preferred.

[0038] According to some embodiments of the present invention, the buffer salt comprises Tris-HCl.

[0039] According to some embodiments of the present invention, the thickener includes gum arabic.

[0040] According to some embodiments of the present invention, the nucleic acid binding promoter includes guanidine isothiocyanate.

[0041] According to some embodiments of the present invention, the surfactant includes polyvinylpyrrolidone K30.

[0042] According to some embodiments of the present invention, the mass ratio of Tris-HCl, gum arabic, guanidine isothiocyanate, and polyvinylpyrrolidone K30 is (1-2):(12-16):(6.5-11.5):(4-10). For example, it can be about 1:12:6.5:4, 1.2:14:7:6, 1.5:15:9:8, or about 2:16:11.5:10, wherein 1.2:14:7:6 is preferred.

[0043] According to some embodiments of the present invention, in step S1, the mixing reaction time is 2 to 3 hours.

[0044] According to some embodiments of the present invention, the method for preparing the long-chain mixed cellulose viscose includes:

[0045] B1 is formed by dissolving cellulose raw materials after alkalization, aging, and sulfonation treatments.

[0046] B2. The viscose is mixed with a modified cellulose polymer additive and the pH is adjusted to obtain a long-chain mixed cellulose viscose.

[0047] According to some embodiments of the present invention, the vitamin raw material includes one or more of the following materials: wood, degreased cotton, bamboo, reed, hemp, wherein long-staple degreased cotton is preferred.

[0048] According to some embodiments of the present invention, the modified cellulose polymeric additive includes hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ether.

[0049] According to some embodiments of the present invention, the amount of hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ether added is 20% to 30% of the mass of the defatted cotton. For example, it can be about 20%, 22%, 25%, 28%, or about 30%, with 25% being preferred.

[0050] According to some embodiments of the present invention, in step B2, the pH value is 8.5 to 9.5. For example, it can be about 8.5, 8.8, 9.0, 9.2 or about 9.5, with 9.0 being preferred.

[0051] According to some embodiments of the present invention, step S2 includes: adding the modified silica gel filling system and long-chain mixed cellulose viscose together into an AH-NANO homogenizer and homogenizing for 2-3 hours to form a slurry; then using an EEFE-02 electrospinning machine to spray a thick and uniform coating onto a roller; after reaching a specified thickness, demolding; and after high-pressure pressing and vacuum dehydration under fixed pressure, a high-efficiency nucleic acid adsorption silica membrane of fixed thickness is obtained.

[0052] According to some embodiments of the present invention, the electrospinning process parameters include:

[0053] (1) The spinning voltage is 10 to 30 kV, for example, about 10 kV, 15 kV, 20 kV, 25 kV or about 30 kV, wherein 20 kV is preferred;

[0054] (2) The collector rotation speed is 1000 to 2000 r / min, for example, about 1000 r / min, 1500 r / min, 1800 r / min or about 2000 r / min, wherein 1800 r / min is preferred.

[0055] According to some embodiments of the present invention, the mass ratio of the modified silica gel filling system to the long-chain mixed cellulose viscose is 1:2.0 to 2.5, for example, it can be about 1:2.0, 1:2.25, 1:2.4 or about 1:2.5, wherein 1:2.25 is preferred.

[0056] According to some embodiments of the present invention, the high-pressure plate and frame pressing process parameters include:

[0057] (1) The pressing pressure is 1000-3500 kg, for example, about 1000 kg, 1500 kg, 2000 kg, 3000 kg or about 3500 kg, wherein 2000 kg is preferred;

[0058] (2) The thickness of the silicone film after pressing is 0.5 to 1.0 mm, for example, about 0.5 mm, 0.6 mm, 0.8 mm or about 1.0 mm, wherein 0.5 mm is preferred.

[0059] According to some embodiments of the present invention, the vacuum dehydration process parameters include:

[0060] (1) The dehydration time is 48 to 72 hours, for example, about 48 hours, 60 hours, or 72 hours, with 72 hours being preferred;

[0061] (2) The dehydration temperature is 55 to 75°C, for example, about 55°C, 60°C, 65°C, 70°C or about 75°C;

[0062] (3) The vacuum degree is 100 to 900 Pa, for example, about 100 Pa, 300 Pa, 500 Pa, 700 Pa or about 900 Pa, wherein 100 Pa is preferred.

[0063] According to some embodiments of the present invention, in step S2, the mass ratio of the modified silica gel filling system to the long-chain mixed cellulose viscose is 1:2.0 to 2.5.

[0064] According to some embodiments of the present invention, the nucleic acid adsorption silica membrane is punched in a clean room using a circular punching tool to obtain a circular disc of a specified diameter, and then pressed into an empty medical-grade PP nucleic acid extraction column using a special pressing ring according to the required number of layers, thus obtaining a usable finished nucleic acid extraction column that can be used for subsequent use and performance testing.

[0065] According to a second aspect of the present invention, the application of the nucleic acid adsorption silica membrane prepared by the aforementioned preparation method in the fields of biomedical detection equipment and environmental monitoring equipment is provided.

[0066] Unless otherwise specified, the term "about" in this invention actually means that the error is allowed to be within ±2%, for example, about 100 is actually 100 ± 2% × 100.

[0067] Unless otherwise specified, "between" in this invention includes the number itself, for example, "between 2 and 3" includes the endpoint values ​​2 and 3.

[0068] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0069] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0070] Figure 1 This is a comparison diagram of PCR amplification curves from embodiments of the present invention.

[0071] Figure 2 These are embodiments of the present invention and nucleic acid electrophoresis patterns.

[0072] Figure 3 This is a diagram of an electrospinning device according to an embodiment of the present invention.

[0073] Figure 4 This is a diagram of the AH-NANO homogenizer in an embodiment of the present invention.

[0074] Figure 5 This is a diagram of the 3.5t pneumatic paper press machine in an embodiment of the present invention. Detailed Implementation

[0075] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0076] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0077] Example 1

[0078] This embodiment provides a method for preparing a nucleic acid adsorption silica membrane, specifically as follows:

[0079] S1. The mercaptopyridyl modified silica suspension sol was dispersed in 100g of a 1:1 volume ratio mixture of isopropanol and deionized water and stirred for 30min. Then, 1.2g Tris-HCl, 14g gum arabic, 7g isothiocyanate guanidine and 6g polyvinylpyrrolidone K30 were added and stirred for 2-3h to obtain a mercaptopyridyl modified silica gel filling system.

[0080] S2. The modified silica gel filling system modified with mercaptopyridine was mixed with 400g of long-chain mixed cellulose viscose to form a slurry. The slurry was homogenized in an AH-NANO homogenizer for 2-3 hours. Then, the slurry was made into a film using an EEFE-02 electrospinner. The film was transferred to a mold and pressed under 2000kg high pressure using a 3.5t pneumatic paper press. After demolding, the film was dehydrated under vacuum at 75℃ and 100pa for 72 hours to obtain a high-efficiency nucleic acid adsorption silica gel film with a thickness of about 0.5mm.

[0081] S3. In a cleanroom, use a 7mm special circular punching tool (automatic or manual) to punch the silicone mold obtained in step 7) to obtain a 7mm diameter circular piece, and press the 8 layers into an empty medical-grade PP nucleic acid extraction column using a special pressure ring to obtain a usable finished nucleic acid extraction column.

[0082] S4. Take approximately 5 × 10 6 293T cells were washed with PBS, centrifuged at 300×g for 5 min, and the cells were collected and the supernatant was discarded.

[0083] S5. Add 20 μL of proteinase K and 200 μL of lysis buffer and vortex to mix.

[0084] Incubate at 6.56℃ for 10 minutes, inverting the container several times during the process to mix thoroughly before extraction.

[0085] S7. Add 150 μL of anhydrous ethanol, shake to mix. Flocculent precipitate may appear. Briefly centrifuge to collect the liquid on the cap and inner wall.

[0086] S8. Transfer all of the above mixture to the adsorption column placed in the collection tube (including flocculent precipitate), and centrifuge at 13400×g for 1 min.

[0087] S9. Discard the filtrate, add 500 μL of protein removal solution along the tube wall into the adsorption column, and centrifuge at 13400×g for 1 min.

[0088] S10. Discard the filtrate, add 600 μL of washing solution along the tube wall into the adsorption column, and centrifuge at 13400 × g for 1 min.

[0089] S11. Repeat step S10.

[0090] S12. Discard the filtrate, place the adsorption column in the collection tube, and centrifuge at 13400×g for 2 min.

[0091] Note: After centrifuging the empty column, you can open the lid and let it stand for 2-5 minutes to allow the residual ethanol to evaporate.

[0092] S13. Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 50–200 μL of elution buffer to the center of the adsorption column membrane, let stand at room temperature for 2–5 min, centrifuge at 13400 × g for 1 min, and use the DNA product for subsequent detection.

[0093] The preparation method of the mercaptopyridyl modified silica suspension sol is as follows:

[0094] A1. Add 10g of tetraethyl orthosilicate to 20g of a mixture of isopropanol and glacial acetic acid in a mass ratio of 1:1, sonicate at room temperature for 20min, then slowly drop in 10g of an isopropanol-deionized water solution in a mass ratio of 1:3 and stir rapidly at 2℃ for 20min.

[0095] A2. Slowly add 15g of concentrated hydrochloric acid dropwise to the solution in step A1. Heat the solution to 55°C and stir in a water bath for 25 minutes. After the addition is complete, cool the solution to 30°C and add 25g of 12% PEG20000 aqueous solution and 50g of 0.7% carboxymethyl cellulose isopropanol solution. Stir at room temperature for 1 hour, then cool to 2°C and let stand for about 1 hour. Filter the solution through a hollow fiber tube filter to obtain a silica suspension sol.

[0096] A3. Disperse the silica suspension obtained in step A2 in 100g isopropanol and sonicate for 15min. Then add 9g of N-aminoethyl-3-aminopropyltriethoxysilane and stir for 2h. Then slowly add it to 100g of ice-cold tetrahydrofuran while stirring. Then add 9g of epoxy polyethylene glycol mercaptopyridine and stir in a 55℃ water bath for 2h. After cooling, wash three times with deionized water to obtain mercaptopyridine-modified silica suspension.

[0097] The preparation method of long-chain mixed cellulose viscose includes: adding 150g of long-staple degreased cotton, and preparing soluble cellulose xanthate through alkalization, aging, sulfonation and other processes, then dissolving it in dilute alkali solution to prepare 300g of viscose, then adding 50g of hydroxypropyl methylcellulose and 50g of hydroxypropyl methylcellulose ether, then adjusting the pH to 8.5, and stirring at low speed until uniform to obtain long-chain mixed cellulose viscose.

[0098] Example 2

[0099] This embodiment provides a method for preparing a nucleic acid adsorption silica membrane, specifically as follows:

[0100] S1. The mercaptopyridyl modified silica suspension sol was dispersed in 100g of a 1:1 volume ratio mixture of isopropanol and deionized water and stirred for 30-60min. Then, 1.5g Tris-HCl, 21g gum arabic, 10g isothiocyanate guanidine, and 9g polyvinylpyrrolidone K30 were added and stirred for 2-3h to obtain a mercaptopyridyl modified silica gel filling system.

[0101] S2. The mercaptopyridine-modified silica gel filling system was mixed with 400g of long-chain mixed cellulose viscose to form a slurry. The slurry was homogenized in an AH-NANO homogenizer for 2 hours. Then, the slurry was made using an EEFE-02 electrospinning machine. The slurry was transferred to a mold and pressed under 2000kg high pressure using a 3.5t pneumatic paper press. After demolding, the slurry was dehydrated under vacuum at 75℃ and 100pa for 72 hours to obtain a high-efficiency nucleic acid adsorption silica gel membrane with a thickness of about 0.5mm.

[0102] S3. In a cleanroom, use a 7mm special circular punching tool (automatic or manual) to punch the silicone mold obtained in step 7) to obtain a 7mm diameter circular piece, and press the 8 layers into an empty medical-grade PP nucleic acid extraction column using a special pressure ring to obtain a usable finished nucleic acid extraction column.

[0103] S4. Take approximately 5 × 10 6 293T cells were washed with PBS, centrifuged at 300×g for 5 min, and the cells were collected and the supernatant was discarded.

[0104] S5. Add 20 μL of proteinase K and 200 μL of lysis buffer and vortex to mix.

[0105] Incubate at 6.56℃ for 10 minutes, inverting the container several times during the process to mix thoroughly before extraction.

[0106] S7. Add 150 μL of anhydrous ethanol, shake to mix. Flocculent precipitate may appear. Briefly centrifuge to collect the liquid on the cap and inner wall.

[0107] S8. Transfer all of the above mixture to the adsorption column placed in the collection tube (including flocculent precipitate), and centrifuge at 13400×g for 1 min.

[0108] S9. Discard the filtrate, add 500 μL of protein removal solution along the tube wall into the adsorption column, and centrifuge at 13400×g for 1 min.

[0109] S10. Discard the filtrate, add 600 μL of washing solution along the tube wall into the adsorption column, and centrifuge at 13400 × g for 1 min.

[0110] S11. Repeat step S10.

[0111] S12. Discard the filtrate, place the adsorption column in the collection tube, and centrifuge at 13400×g for 2 min.

[0112] Note: After centrifuging the empty column, you can open the lid and let it stand for 2-5 minutes to allow the residual ethanol to evaporate.

[0113] S13. Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 50–200 μL of elution buffer to the center of the adsorption column membrane, let stand at room temperature for 2–5 min, centrifuge at 13400 × g for 1 min, and use the DNA product for subsequent detection.

[0114] The preparation method of the mercaptopyridyl modified silica suspension sol is as follows:

[0115] A1. Add 10g of tetraethyl orthosilicate to 20g of a mixture of isopropanol and glacial acetic acid in a mass ratio of 1:1, sonicate at room temperature for 20min, then slowly drop in 10g of an isopropanol-deionized water solution in a mass ratio of 1:3 and stir rapidly at 2℃ for 20min.

[0116] A2. Slowly add 15g of concentrated hydrochloric acid dropwise to the solution in step A1. Heat the solution to 55°C and stir in a water bath for 25 minutes. After the addition is complete, cool the solution to 30°C and add 25g of 12% PEG20000 aqueous solution and 50g of 0.7% carboxymethyl cellulose isopropanol solution. Stir at room temperature for 1 hour, then cool to 2°C and let stand for about 1 hour. Filter the solution through a hollow fiber tube filter to obtain a silica suspension sol.

[0117] A3. Disperse the silica suspension obtained in step A2 in 100g isopropanol and sonicate for 15min. Then add 12g of N-aminoethyl-3-aminopropyltriethoxysilane and stir for 2h. Then slowly add it to 100g of ice-cold tetrahydrofuran while stirring. Then add 12g of epoxy polyethylene glycol mercaptopyridine and stir in a 55℃ water bath for 2h. After cooling, wash three times with deionized water to obtain mercaptopyridine-modified silica suspension.

[0118] The preparation method of long-chain mixed cellulose viscose includes: adding 150g of long-staple degreased cotton, and preparing soluble cellulose xanthate through alkalization, aging, sulfonation and other processes, then dissolving it in dilute alkali solution to prepare 300g of viscose, then adding 50g of hydroxypropyl methylcellulose and 50g of hydroxypropyl methylcellulose ether, then adjusting the pH to 8.5, and stirring at low speed until uniform to obtain long-chain mixed cellulose viscose.

[0119] Example 3

[0120] This embodiment provides a method for preparing a nucleic acid adsorption silica membrane, specifically as follows:

[0121] S1. The mercaptopyridyl modified silica suspension sol was dispersed in 100g of a 1:1 volume ratio mixture of isopropanol and deionized water and stirred for 30min. Then, 0.9g Tris-HCl, 7g gum arabic, 5g isothiocyanate guanidine and 4g polyvinylpyrrolidone K30 were added and stirred for 2h to obtain a mercaptopyridyl modified silica gel filling system.

[0122] S2. The modified silica gel filling system modified with mercaptopyridine was mixed with 400g of long-chain mixed cellulose viscose to form a slurry. The slurry was homogenized in an AH-NANO homogenizer for 2-3 hours. Then, the slurry was made into a film using an EEFE-02 electrospinner. The film was transferred to a mold and pressed under 2000kg high pressure using a 3.5t pneumatic paper press. After demolding, the film was dehydrated under vacuum at 75℃ and 100pa for 72 hours to obtain a high-efficiency nucleic acid adsorption silica gel film with a thickness of about 0.5mm.

[0123] S3. In a cleanroom, use a 7mm special circular punching tool (automatic or manual) to punch the silicone mold to obtain a 7mm diameter disc. Then, press the 8 layers into an empty medical-grade PP nucleic acid extraction column using a special pressure ring to obtain a usable finished nucleic acid extraction column.

[0124] S4. Take approximately 5 × 10 6 293T cells were washed with PBS, centrifuged at 300×g for 5 min, and the cells were collected and the supernatant was discarded.

[0125] S5. Add 20 μL of proteinase K and 200 μL of lysis buffer and vortex to mix.

[0126] Incubate at 6.56℃ for 10 minutes, inverting the container several times during the process to mix thoroughly before extraction.

[0127] S7. Add 150 μL of anhydrous ethanol, shake to mix. Flocculent precipitate may appear. Briefly centrifuge to collect the liquid on the cap and inner wall.

[0128] S8. Transfer all of the above mixture to the adsorption column placed in the collection tube (including flocculent precipitate), and centrifuge at 13400×g for 1 min.

[0129] S9. Discard the filtrate, add 500 μL of protein removal solution along the tube wall into the adsorption column, and centrifuge at 13400×g for 1 min.

[0130] S10. Discard the filtrate, add 600 μL of washing solution along the tube wall into the adsorption column, and centrifuge at 13400 × g for 1 min.

[0131] S11. Repeat step S10.

[0132] S12. Discard the filtrate, place the adsorption column in the collection tube, and centrifuge at 13400×g for 2 min.

[0133] Note: After centrifuging the empty column, you can open the lid and let it stand for 2-5 minutes to allow the residual ethanol to evaporate.

[0134] S13. Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 50–200 μL of elution buffer to the center of the adsorption column membrane, let stand at room temperature for 2–5 min, centrifuge at 13400 × g for 1 min, and use the DNA product for subsequent detection.

[0135] The preparation method of the mercaptopyridyl modified silica suspension sol is as follows:

[0136] A1. Add 10g of tetraethyl orthosilicate to 20g of a mixture of isopropanol and glacial acetic acid in a mass ratio of 1:1, sonicate at room temperature for 20min, then slowly drop in 10g of an isopropanol-deionized water solution in a mass ratio of 1:3 and stir rapidly at 2℃ for 20min.

[0137] A2. Slowly add 15g of concentrated hydrochloric acid dropwise to the solution in step A1. Heat the solution to 55°C and stir in a water bath for 25 minutes. After the addition is complete, cool the solution to 30°C and add 25g of 12% PEG20000 aqueous solution and 50g of 0.7% carboxymethyl cellulose isopropanol solution. Stir at room temperature for 1 hour, then cool to 2°C and let stand for about 1 hour. Filter the solution through a hollow fiber tube filter to obtain a silica suspension sol.

[0138] A3. Disperse the silica suspension obtained in step A2 in 100g isopropanol and sonicate for 15min. Then add 6g of N-aminoethyl-3-aminopropyltriethoxysilane and stir for 2h. Then slowly add it to 100g of ice-cold tetrahydrofuran while stirring. Then add 6g of epoxy polyethylene glycol mercaptopyridine and stir in a 55℃ water bath for 2h. After cooling, wash three times with deionized water to obtain mercaptopyridine-modified silica suspension.

[0139] The preparation method of long-chain mixed cellulose viscose includes: adding 150g of long-staple degreased cotton, and preparing soluble cellulose xanthate through alkalization, aging, sulfonation and other processes, then dissolving it in dilute alkali solution to prepare 300g of viscose, then adding 50g of hydroxypropyl methylcellulose and 50g of hydroxypropyl methylcellulose ether, then adjusting the pH to 8.5, and stirring at low speed until uniform to obtain long-chain mixed cellulose viscose.

[0140] Comparative Example 1

[0141] Using the same steps as the competitor's XX reagent kit from Company T for cellular genomic DNA extraction:

[0142] 1) Follow the instructions to perform all preparatory work before extraction.

[0143] 2) Take approximately 5 × 10 6 293T cells were washed with PBS, centrifuged at 300×g for 5 min, and the cells were collected and the supernatant was discarded.

[0144] 3) Add 20 μL of proteinase K and 200 μL of lysis buffer and vortex to mix.

[0145] 4) Incubate at 56℃ for 10 minutes, inverting the container several times during the process to mix thoroughly before extraction.

[0146] 5) Add 150 μL of anhydrous ethanol, shake to mix. Flocculent precipitate may appear. Briefly centrifuge to collect the liquid on the cap and inner wall.

[0147] 6) Transfer all of the above mixture to the adsorption column placed in the collection tube (including flocculent precipitate), and centrifuge at 13400×g for 1 min.

[0148] 7) Discard the filtrate, add 500 μL of protein removal solution along the tube wall into the adsorption column, and centrifuge at 13400×g for 1 min.

[0149] 8) Discard the filtrate, add 600 μL of washing solution along the tube wall into the adsorption column, and centrifuge at 13400×g for 1 min.

[0150] 9) Repeat step 8.

[0151] 10) Discard the filtrate, place the adsorption column in the collection tube, and centrifuge at 13400×g for 2 min.

[0152] Note: After centrifuging the empty column, you can open the lid and let it stand for 2-5 minutes to allow the residual ethanol to evaporate.

[0153] 11) Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 50–200 μL of elution buffer to the center of the adsorption column membrane, let stand at room temperature for 2–5 min, centrifuge at 13400 × g for 1 min, and use the DNA product for subsequent detection.

[0154] Detection method:

[0155] The samples were tested using a Biotek LX microplate reader and a Take3 microplate. The first two wells were used as background subtraction. 2.5 μL of each sample was taken and the concentration and 260 / 280 ratio of the sample were measured using the nucleic acid adsorption internal control standard method document.

[0156] Test results:

[0157] Table 1. Performance Testing

[0158]

[0159]

[0160] Table 2. Stability Tests

[0161]

[0162]

[0163] As shown in Table 1, analysis of the absorption peaks of nucleic acids and proteins using the OD260 / 280 ratio revealed that the nucleic acid purity (OD260 / 280 ≈ 1.90–1.91) of Examples 1–3 was significantly better than that of Comparative Example 1 (1.895), and the nucleic acid yield was also higher. This indicates that the silica membrane of the present invention has superior adsorption efficiency for nucleic acids. Figure 1 As can be seen from the comparison of PCR amplification curves, the amplification efficiency of Example 1 is significantly higher than that of Comparative Example 1, indicating that the nucleic acid eluted from this membrane material has better integrity and is suitable for high-sensitivity amplification; Figure 2As can be seen from the electrophoresis patterns, the nucleic acid bands of Examples 1-3 are clear and free of degradation impurities, while Comparative Example 1 shows a slight tailing phenomenon, which further verifies the advantages of the material of the present invention in nucleic acid recovery quality. As can be seen from Table 2, in terms of storage stability, after being placed under constant temperature, humidity and light-proof conditions for 6 months, Examples 1-3 showed significantly lower reductions in nucleic acid purity and adsorption capacity compared to Comparative Example 1. Figure 3 This is a diagram of an electrospinning device according to an embodiment of the present invention. Figure 4 This is a diagram of the AH-NANO homogenizer in an embodiment of the present invention. Figure 5 This is a diagram of the 3.5t pneumatic paper press machine in an embodiment of the present invention.

[0164] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing a nucleic acid adsorption silica membrane, characterized in that, Includes the following steps: S1. The modified silica sol modified with mercaptopyridine was dispersed in a buffer salt system and then mixed and reacted with a thickener, a nucleic acid binding promoter and a surfactant to obtain a modified silica gel filling system; S2. The modified silica gel filling system is mixed with long-chain mixed cellulose viscose to form a slurry, and then coated by electrospinning. After demolding, pressing and dehydration, a nucleic acid adsorption silica gel membrane is obtained. The raw materials for preparing the mercaptopyridine-modified silica sol include: silica sol and epoxy-based polyethylene glycol mercaptopyridine.

2. The preparation method according to claim 1, characterized in that, The method for preparing the silica sol includes: A1. A precursor silica gel solution is obtained by mixing and reacting a silicon source, a pH adjuster solution, and an aqueous alcohol solution; A2. The precursor silica gel solution, acidic catalyst, and polymeric auxiliaries are mixed and reacted to obtain a mixed solution; A3. After removing impurities from the mixture, a silica sol is obtained.

3. The preparation method according to claim 2, characterized in that, The pH adjusting agent solution includes an alcoholic solution of glacial acetic acid; the alcohol in the glacial acetic acid alcoholic solution includes at least one of methanol, ethanol and isopropanol.

4. The preparation method according to claim 2, characterized in that, The polymeric additives include polyethylene glycol and carboxymethyl cellulose.

5. The preparation method according to claim 1, characterized in that, The raw materials for preparing the mercaptopyridine-modified silica sol also include aminosilanes; the preparation method includes: A4. The silica sol is dispersed and then modified with aminosilane; A5. Then, the modified aminosilane silica sol is reacted with epoxy polyethylene glycol mercaptopyridine to obtain mercaptopyridine-modified silica sol.

6. The preparation method according to claim 1, characterized in that, The preparation method of the long-chain mixed cellulose viscose includes: B1 is formed by dissolving cellulose raw materials after alkalization, aging, and sulfonation treatments. B2. The viscose is mixed with a modified cellulose polymer additive and the pH is adjusted to obtain a long-chain mixed cellulose viscose.

7. The preparation method according to claim 4, characterized in that, In step S2, the voltage for electrospinning is 10 to 30 kilovolts.

8. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of the modified silica gel filling system to the long-chain mixed cellulose viscose is 1:2.0 to 2.

5.

9. The preparation method according to claim 1, characterized in that, In step S2, the dehydration time is 48-72 hours, the dehydration temperature is 55-75°C, and the dehydration pressure is a vacuum of 100-900 Pa.

10. The application of a nucleic acid adsorption silica membrane prepared by the preparation method according to any one of claims 1 to 9 in the fields of biomedical detection equipment and environmental monitoring equipment.

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