A polymer brush-modified nucleic acid rapid extraction material and method

Abstract

The present invention relates to the technical field of functional materials, and in particular to a polymer brush-modified rapid nucleic acid extraction material and method. The extraction material provided by the present invention comprises a carrier surface modified with compounds having an amino functional group, a hydroxyl functional group and a sulfonic acid functional group connected to a polymer chain. Under low pH conditions, the terminal amino group carries a positive charge, and nucleic acids can be quickly adsorbed through the action of the charge. The polymer chain with the sulfonic acid group shrinks under low pH conditions, which does not affect the adsorption of nucleic acids by the amino positive charge. Under high pH conditions, the polymer chain with the sulfonic acid group stretches, repelling nucleic acids and accelerating the detachment of nucleic acids from the material surface. At the same time, the terminal hydroxyl group acts as an electrically neutral hydrophilic group to balance the positive and negative charges on the carrier surface. The three functional compounds act synergistically to shorten the nucleic acid extraction time, improve the yield and purity of nucleic acid extraction, and achieve efficient and high-quality extraction.

Description

Technical Field

[0001] The invention relates to the technical field of functional materials, and in particular to a polymer brush-modified nucleic acid rapid extraction material and method. Background Art

[0002] Modern molecular biology technology has been widely used in the fields of paternity testing, forensic identification, human genetic research, medical personal identification, disease association analysis, etc., among which the extraction and purification of nucleic acids is the most fundamental operation technology in life sciences such as clinical medicine and molecular biology. The basic steps of nucleic acid extraction and purification include 1) cell lysis and nucleic acid release: using lysis solution to lyse cells and make nucleic acids free in the lysis system; 2) nucleic acid separation and purification: removing polysaccharides, lipids, proteins and other biomacromolecules bound to nucleic acids and other impurity molecules; 3) nucleic acid enrichment and elution: using elution solution to enrich nucleic acids. Specific methods include 1) solution method: cell disruption or tissue homogenization is treated with phenol / chloroform, the aqueous phase is mainly nucleic acids, the organic phase is mainly polysaccharides and lipids, and proteins are precipitated between the two phases. After centrifugation and stratification, the water layer is taken, and after repeated operations, the aqueous phase containing nucleic acids is combined. The nucleic acids are precipitated with ethanol / isopropanol using the nature of nucleic acids being insoluble in alcohol, and then separated and purified. After dissolution, high-purity nucleic acids can be obtained; 2) column membrane method: based on silica gel membrane adsorption. The silanol groups on the surface of the silica membrane are weakly acidic and negatively charged after hydration. If there is a certain concentration of cations in the solution, the surface negative charge between the silanol groups of the nucleic acid will be neutralized, so that the nucleic acid can be firmly adsorbed on the surface of the silica membrane. On the contrary, if it is in a low-salt aqueous solution, there will be electrostatic repulsion between the phosphate groups of the nucleic acid and the silanol groups of the silica membrane, and the silica membrane will release the nucleic acid; 3) Magnetic bead method: The magnetic bead method uses nanotechnology to modify and improve the surface of superparamagnetic nanoparticles to prepare superparamagnetic silica nanomagnetic beads. The magnetic beads can specifically recognize and efficiently bind to nucleic acid molecules at the microscopic interface. The binding of nucleic acids to magnetic beads mainly relies on hydrogen bonding, electrostatic effects and hydrophobic effects. Under the action of guanidine isothiocyanate, guanidine hydrochloride and an external magnetic field, nucleic acids can be separated from the sample, and non-specifically adsorbed impurities can be washed away to obtain purified nucleic acids.

[0003] The extraction efficiency of nucleic acids by the column membrane method and the magnetic bead method is generally better than that by the solution method. Therefore, the column membrane method and the magnetic bead method have become the more mainstream methods for nucleic acid extraction and purification. Both the column membrane method and the magnetic bead method are based on the functional groups on the surface of the carrier, which adsorb nucleic acids on the surface of the carrier through intermolecular forces such as hydrogen bonding, electrostatics and hydrophobic effects, and then elute the nucleic acids from the surface of the carrier under elution conditions. The molecular structure and quality of the functional groups modified on the surface of the carrier determine the nucleic acid extraction effect, such as the extraction rate, purity, concentration, etc. Therefore, the modified structure of the carrier surface has become a research hotspot in the industry. For example, patents US6310199B and US6914137B2 disclose materials that generate positive charges under low pH conditions as nucleic acid extraction materials to achieve efficient extraction of nucleic acids, and specifically disclose the following molecular structures , Patents US8415277B2 and US20090005265A1 disclose the use of materials with amino and carboxyl functional groups to form positive and negative charge bifunctional groups as nucleic acid extraction materials to achieve efficient extraction of nucleic acids, and specifically disclose the following molecular structures The core principle of the technology disclosed in the above patents is to achieve nucleic acid extraction and purification based on the principle of charge switching. There are also products based on this technical principle on the market, such as ChargeSwitch@. The core of its product is the amino group modified on the surface of magnetic beads. Under low pH conditions, it is positively charged to adsorb negatively charged nucleic acids, and under high pH conditions, it is neutrally charged to desorb nucleic acids. The specific process is as follows: However, the nucleic acid extraction purity of this product is not high, and it can only be used for forensic testing, and the extraction time takes at least 15 minutes. Therefore, it is necessary to further innovate nucleic acid extraction materials to achieve high-quality and efficient extraction of nucleic acids. Summary of the invention

[0004] In order to solve the problems of the prior art, one of the objects of the present invention is to provide a polymer brush modified nucleic acid rapid extraction material, in which a pH-responsive polymer brush is modified on the surface of a carrier, and a polymer having pH-responsive positive and negative bifunctional charges and pH-responsive contraction and expansion properties is modified on the surface of the carrier to achieve high-quality and efficient nucleic acid extraction.

[0005] A second object of the present invention is to provide a method for extracting nucleic acid, which uses the nucleic acid extraction material provided by the present invention to achieve high-quality and efficient nucleic acid extraction.

[0006] At the same time, the present invention also provides a method for preparing a polymer brush-modified nucleic acid rapid extraction material.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A polymer brush-modified nucleic acid rapid extraction material comprises a solid phase carrier, characterized in that the surface of the solid phase carrier is modified with compounds represented by the following formulas (I), (II) and (III):

[0009] RP1-A1-B1-NH2(Ⅰ), RP2-A2-B2-OH(Ⅱ), RP3-A3-L4-F(Ⅲ);

[0010] Wherein F comprises the chemical structure shown in formula (IV) Wherein RP1, RP2, and RP3 are linking groups for connecting the compounds represented by formula (I), formula (II), and formula (III) to the solid phase carrier;

[0011] A1, A2, A3 are spacers; B1, B2 are water-soluble spacers; L4 is a linker between F and A3;

[0012] R1, R2, and R3 are each independently hydrogen or an optionally substituted alkyl group;

[0013] R4 is C 3～10 Alkylene or heteroalkylene.

[0014] Optionally, the F group of the compound represented by formula (III) is connected to L4 by active polymerization to form the compound represented by formula (III); wherein the monomer raw material used in the active polymerization is

[0015] Optionally, the molar ratio of the compounds of formula (I), formula (II) and formula (III) modified on the surface of each solid phase carrier unit is 2:2:1; the molar ratio of the amount of the monomer raw material for forming the F group to the compound of formula (III) is 5:1.

[0016] Optionally, A1, A2, A3 are C 3～10 Alkylene or heteroalkylene.

[0017] Optionally, B1 and B2 are n=8~12.

[0018] Optionally, the L4 is

[0019] Optionally, R1, R2, and R3 are each independently hydrogen; R4 is

[0020] Optionally, the solid phase carrier is silanol magnetic beads; the molecular structures of RP1, RP2, and RP3 are

[0021] Optionally, the particle size of the solid carrier is between 50 nm and 1 mm.

[0022] The preparation method of the polymer brush modified nucleic acid rapid extraction material comprises:

[0023] 1) Silanization modification:

[0024] The compounds shown in Ⅰ-1, Ⅱ-1 and Ⅳ-1 were added to anhydrous ethanol according to the molar ratio to prepare a 5% silane reagent solution;

[0025] The solid phase carrier magnetic beads were surface activated with 0.01M sodium hydroxide, and after cleaning, a silane reagent solution was added at a solid-liquid ratio of 1:50, and the reaction was carried out at room temperature for 12 hours. The magnetic beads were separated and dried to obtain silanized magnetic beads;

[0026] 2) Living polymerization: dissolve tri[2-(dimethylamino)ethyl]amine in water, add cuprous bromide, mix at room temperature, and prepare a living polymerization reaction solution;

[0027] According to the solid-liquid ratio of 1:50, the silanized magnetic beads prepared in step 1) are added to the aqueous solution of the compound represented by formula (V), and under an inert gas protection environment, the active polymerization reaction solution is added, and the reaction is stirred at room temperature for 4 hours, and the magnetic beads are separated and washed to obtain the polymer brush modified nucleic acid rapid extraction material;

[0028]

[0029] A method for rapidly extracting nucleic acid comprises the following steps:

[0030] S1. Using the above extraction material, at a first pH value, the extraction material adsorbs and fixes the nucleic acid in the sample;

[0031] S2. At a second pH value, releasing the nucleic acid from the surface of the extraction material.

[0032] Optionally, step S1 is carried out at room temperature; step S2 is carried out at 55°C to 70°C, preferably step S2 is carried out at 65°C; the first pH is less than 6.0, more preferably, the first pH is 5.0; the second pH is greater than 7.5, preferably the second pH is 10.0.

[0033] The extraction material provided by the present invention has three compounds modified on the surface of the carrier, which respectively have an amino functional group, a hydroxyl functional group and a sulfonic acid functional group connected to a polymer chain. Under low pH conditions, the terminal amino group carries a positive charge, and can quickly adsorb nucleic acids through the action of the charge, and the polymer chain with the sulfonic acid group shrinks under low pH conditions, which does not affect the adsorption of nucleic acids by the amino positive charge. Under high pH, ​​the polymer chain with the sulfonic acid group stretches, repelling nucleic acids and accelerating the detachment of nucleic acids from the material surface; at the same time, the terminal hydroxyl group acts as an electrically neutral hydrophilic group to balance the positive and negative charges on the surface of the carrier. The three functional compounds with specific functional group structures uniformly distributed and modified on the surface of the carrier act synergistically, shortening the nucleic acid extraction time, improving the yield and purity of nucleic acid extraction, and realizing efficient and high-quality extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A schematic diagram of the structure of the extraction material provided in Example 1 of the present invention;

[0035] Figure 2 This is a schematic diagram of the synthesis process of the extraction material provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] definition:

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0038] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] The silicon hydroxy magnetic beads used in the following examples and comparative examples were purchased from Suzhou Nanovita Technology Co., Ltd., and the particle size thereof was 0.7 um.

[0040] The following embodiments of the present invention modify the extraction material provided by the present invention on the surface of silanol magnetic beads for nucleic acid extraction. This is only for illustration. It should be understood that the extraction material can also be used as a modified material to modify the surface of any material matrix that has been used for nucleic acid extraction, such as fiber membranes, polymer fillers, polymer particles, glass beads, etc.

[0041] In the following embodiments of the present invention, the extraction material provided by the present invention is modified on the surface of silanol magnetic beads to prepare magnetic beads for nucleic acid extraction and purification. The method flow used for nucleic acid extraction is the extraction flow conventionally used in the art, including the steps of binding, washing, elution, magnetic bead separation, etc., and further includes a sample pretreatment step before binding, and may also include cell lysis, etc. for different samples.

[0042] Example

[0043] This embodiment provides a polymer brush modified nucleic acid rapid extraction material, such as Figure 1 As shown, it includes a silicon hydroxy magnetic bead carrier, and the silicon hydroxy magnetic bead carrier shows that the compound represented by formula (I-1), formula (II-1) and formula (III-1) is uniformly modified:

[0044]

[0045]

[0046] The compound represented by formula (III-1) is prepared by living polymerization of the compound represented by formula (IV-1) and formula (V)

[0047]

[0048] The molar ratio of the compounds represented by formula (Ⅰ-1), formula (Ⅱ-1), formula (Ⅳ-1) and formula (V) is 2:2:1:5.

[0049] Figure 1 wherein X represents covalent connection with the hydroxyl group on the surface of the magnetic beads or covalent connection with the compound represented by formula (Ⅰ-1) or (Ⅱ-1) or (Ⅲ-1);

[0050] The preparation method of the polymer brush modified nucleic acid rapid extraction material provided in this embodiment is as follows: Figure 2 The process flow diagram is shown in the figure, and the specific operation steps are as follows:

[0051] 1) Silanization modification:

[0052] The compounds shown in Ⅰ-1, Ⅱ-1 and Ⅳ-1 were added to anhydrous ethanol according to the molar ratio to prepare a 5% silane reagent solution;

[0053] The solid phase carrier magnetic beads were surface activated with 0.01M sodium hydroxide, and after cleaning, a silane reagent solution was added at a solid-liquid ratio of 1:50, and the reaction was carried out at room temperature for 12 hours. The magnetic beads were separated and dried to obtain silanized magnetic beads;

[0054] 2) Living polymerization: dissolve tri[2-(dimethylamino)ethyl]amine in water, add cuprous bromide, mix at room temperature, and prepare a living polymerization reaction solution;

[0055] According to the solid-liquid ratio of 1:50, the silanized magnetic beads prepared in step 1) are added to the aqueous solution of the compound represented by formula (V), and under the protection of inert gas, the active polymerization reaction liquid is added, and the reaction is stirred at room temperature for 4 hours. The magnetic beads are separated and washed to obtain the polymer brush modified nucleic acid rapid extraction material.

[0056] Comparative Example 1

[0057] This comparative example provides a nucleic acid extraction material, which mainly differs from the nucleic acid extraction material provided in the embodiment in that the structure of the surface-modified compound is different. The modified compound in this comparative example is a compound represented by formula (Ⅰ-1) and formula (Ⅱ-1); its preparation process includes the silanization modification process of the embodiment, and the molar ratio of formula (Ⅰ-1) and formula (Ⅱ-1) is 1:1.

[0058] Comparative Example 2

[0059] This comparative example provides a nucleic acid extraction material, which is mainly different from the nucleic acid extraction material provided in the embodiment in that the structure of the surface-modified compound is different. The modified compound in this comparative example is a compound represented by formula (I-1), formula (II-1) and formula (III-2):

[0060] Wherein n=10; the molar ratio of formula (Ⅰ-1), formula (Ⅱ-1) and formula (Ⅲ-2) is 2:2:5.

[0061] Test example:

[0062] 1. Test samples: 1) Simulation sample: 50 mM acetic acid / sodium acetate buffer solution and 1 M guanidine hydrochloride lysis solution containing 1000 ng λDNA, pH = 5;

[0063] 2) Clinical samples: HBV positive clinical samples: HBV positive clinical plasma samples were added with 50 mM acetic acid / sodium acetate buffer solution and 1 M guanidine hydrochloride lysis buffer, and the pH was adjusted to 5.0;

[0064] 2. Experimental groups: Group 1: using the extraction materials provided in the examples;

[0065] The second group: using the extraction material provided in Comparative Example 1;

[0066] The third group: using the extraction material provided in Comparative Example 2;

[0067] Group 4: Using commercially available As extraction material.

[0068] 3. Test methods and results:

[0069] (1) Simulation sample:

[0070] The test methods for the first to third groups are:

[0071] 60mg of the extraction material was added to 2ml of 50mM acetic acid / sodium acetate buffer solution and 1M guanidine hydrochloride lysis solution containing 1000ngλDNA, pH=5, and the solution was stirred to allow the extraction material to adsorb nucleic acid for a certain period of time to form nucleic acid / extraction material complex particles. The nucleic acid / extraction material complex particles were transferred to 100ul of 50mM Tris elution solution with pH=10.0, and the solution was heated to 65℃ for a certain period of time to desorb nucleic acid from the extraction material, and UV detection was performed to calculate the nucleic acid extraction rate;

[0072] The fourth group of test methods is: using The matching lysis buffer, binding buffer and elution buffer were prepared with 2 ml of simulated sample containing 1000 ng λDNA, and the binding and elution operations were performed according to the operating instructions. The nucleic acid extraction yield was detected and calculated using UV.

[0073] The time conditions and nucleic acid extraction yields of each group of experiments are shown in Table 1 below:

[0074] Table 1

[0075] Grouping Adsorption treatment time Elution treatment time Nucleic acid extraction yield Group 1 30s 60s 95％ Group 2 30s 60s 70％ Group 3 30s 60s 60％ Group 4 30s 60s 75％ Group 1 30s 90s 95％ Group 2 30s 90s 80％ Group 3 30s 90s 60％ Group 4 30s 90s 78％ Group 1 60s 60s 95％ Group 2 60s 60s 70％ Group 3 60s 60s 65％ Group 4 60s 60s 75％ Group 1 60s 90s 95％ Group 2 60s 90s 80％ Group 3 60s 90s 65％ Group 4 60s 90s 78.5％

[0076] The results of the simulated sample test show that the extraction material provided by the present invention can achieve an efficient extraction with a nucleic acid extraction rate of 95% with an adsorption treatment time of 30s and an elution treatment time of 60s. The extraction material of Comparative Example 1, which changes the surface-modified compound structure of the present invention, has an adsorption performance of nucleic acids comparable to that of the present invention, but compared with the extraction material of the present invention, the extraction material of Comparative Example 1 is difficult to be eluted after adsorbing nucleic acids, and can only achieve 80% extraction even when the elution time is extended to 90s; the extraction material of Comparative Example 2 has a significantly inferior adsorption performance of nucleic acids to the extraction material provided by the present invention, and can only achieve 65% extraction even when the adsorption treatment time is extended to 60s;

[0077] Furthermore, the extraction material provided by the present invention has better nucleic acid adsorption performance than similar products on the market, and can be eluted more quickly, thereby achieving efficient and high-quality extraction of nucleic acids.

[0078] (2) Clinical plasma samples: Each group of experiments used the same clinical plasma samples

[0079] The test methods for the first to third groups are:

[0080] Lysis buffer and 50mM acetic acid / sodium acetate buffer solution were added to the clinical plasma samples for the test, the pH of the mixed system was adjusted to 5.0, and the test extraction materials of the corresponding groups were added. After a period of adsorption and binding treatment, the extraction materials were washed 2-3 times with 10mM MES solution, transferred to 50mM Tris elution solution with pH=10.0, and the solution was heated to 65°C. After a period of elution treatment, the supernatant was taken to prepare the qPCR reaction system, and the average Ct value of each group of samples was detected;

[0081] The fourth group of test methods are:

[0082] use The lysis buffer, binding buffer, washing and elution buffer are as follows After nucleic acid extraction from the clinical samples of the experiment, the qPCR reaction system was configured to detect the average Ct value of the samples;

[0083] The time conditions and average Ct values ​​of each group of experiments are shown in Table 2 below:

[0084] Table 2

[0085] Grouping Adsorption treatment time Elution treatment time Average Ct value Group 1 30s 60s 22.35 Group 2 30s 90s 29.26 Group 3 60s 60s 34.28 Group 4 30s 60s 23.89

[0086] The results of clinical plasma tests show that the nucleic acid extraction material provided by the present invention has a more efficient clinical sample application effect.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polymer brush modified nucleic acid rapid extraction material, comprising a solid phase carrier, characterized in that: The surface of the solid phase carrier is modified with compounds represented by the following formula (I), formula (II) and formula (III): RP1-A1-B1-NH2(Ⅰ), RP2-A2-B2-OH(Ⅱ), RP3-A3-L4-F(Ⅲ); Wherein F comprises the chemical structure shown in formula (IV) in RP1, RP2, and RP3 are linking groups for connecting the compounds represented by formula (I), formula (II), and formula (III) to the solid phase carrier; A1, A2, A3 are spacers; B1, B2 are water-soluble spacers; L4 is a linker between F and A3; R1, R2, and R3 are each independently hydrogen or an optionally substituted alkyl group; R4 is C 3～10 Alkylene or heteroalkylene; The molar ratio of the compounds represented by formula (I), formula (II) and formula (III) modified on the surface of the solid phase carrier is 2:2:1; The F group of the compound represented by formula (III) is connected with L4 by active polymerization to form the compound represented by formula (III); wherein the monomer raw material used in the active polymerization is The molar ratio of the amount of the monomer raw material for forming the F group to the compound represented by formula (III) is 5:

1.

2. The polymer brush modified nucleic acid rapid extraction material according to claim 1, characterized in that: A1, A2, A3 are C 3～10 Alkylene or heteroalkylene.

3. The polymer brush modified nucleic acid rapid extraction material according to claim 2, characterized in that: B1 and B2 are n=8~12.

4. The polymer brush modified nucleic acid rapid extraction material according to claim 3, characterized in that: The L4 is 5. The polymer brush modified nucleic acid rapid extraction material according to claim 4, characterized in that: R1, R2, and R3 are each independently hydrogen; R4 is 6. The polymer brush modified nucleic acid rapid extraction material according to claim 5, characterized in that: The solid phase carrier is silicon hydroxy magnetic beads; RP1, RP2, RP3 are 7. The polymer brush modified nucleic acid rapid extraction material according to claim 6, characterized in that: The particle size of the solid phase carrier is between 50nm and 1mm.

8. A method for preparing a polymer brush modified nucleic acid rapid extraction material as claimed in claim 7, characterized in that: include: 1) Silanization modification: The compounds shown in Ⅰ-1, Ⅱ-1 and Ⅳ-1 were added to anhydrous ethanol according to the molar ratio to prepare a 5% silane reagent solution; The magnetic beads were surface activated with 0.01M sodium hydroxide, and after cleaning, a silane reagent solution was added at a solid-liquid ratio of 1:50, and the reaction was carried out at room temperature for 12 hours. The magnetic beads were separated and dried to obtain silanized magnetic beads; 2) Living polymerization: dissolve tri[2-(dimethylamino)ethyl]amine in water, add cuprous bromide, mix at room temperature, and prepare a living polymerization reaction solution; According to the solid-liquid ratio of 1:50, the silanized magnetic beads prepared in step 1) are added to the aqueous solution of the compound represented by formula (V), and under an inert gas protection environment, the active polymerization reaction solution is added, and the reaction is stirred at room temperature for 4 hours, and the magnetic beads are separated and washed to obtain the polymer brush modified nucleic acid rapid extraction material; 9. A method for rapid extraction of nucleic acid, characterized in that: The steps include: S1. Using the extraction material according to any one of claims 1 to 7, at a first pH value, the extraction material adsorbs and fixes nucleic acids in the sample; S2. At a second pH value, releasing the nucleic acid from the surface of the extraction material.

10. The method for rapid extraction of nucleic acid as claimed in claim 9, characterized in that: Step S1 is carried out at room temperature; step S2 is carried out at 55°C to 70°C; the first pH is less than 6.0; and the second pH is greater than 7.5.

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