Paper-based nucleic acid extraction material based on synergistic effect of aperture and functional group as well as preparation method and application of paper-based nucleic acid extraction material

Abstract

The invention provides a paper-based nucleic acid extraction material based on the synergistic effect of pore diameters and functional groups and a preparation method and application thereof.The preparation method of the paper-based nucleic acid extraction material comprises the steps that S1, cotton pulp is dissolved in water, stirred and mixed, and a mixture 1 is obtained; carrying out suction filtration on the mixture 1 to form paper to obtain CF paper; s2, taking CF paper as a substrate, pouring the mixture 2 on the CF paper, and performing suction filtration and drying to obtain a CF / CNF paper-based material; wherein the mixture 2 is an aqueous solution of nano cellulose fibers; and S3, immersing a CF / CNF paper-based material into the mixture 3, carrying out oscillation incubation, then taking out the CF / CNF paper-based material, and carrying out washing and drying so as to obtain the CF / CNF / L-DOPA paper-based nucleic acid extraction material. Wherein the mixture 3 is prepared by dissolving the levodopa in a Tris-HCl solution. The paper-based nucleic acid extraction material can realize simple, rapid and efficient extraction of nucleic acid.

Description

Technical Field

[0001] The invention relates to the field of nucleic acid detection, and in particular to a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional group, and a preparation method and application thereof. Background Art

[0002] As a detection technology with good specificity and high sensitivity, nucleic acid detection has been widely used in the screening, prevention and diagnosis of diseases. Traditional nucleic acid detection requires professional experimenters to use large instruments in the laboratory. It has the disadvantages of complex operation, long cycle and high cost, which is difficult to meet the needs of modern people's fast pace of life. Therefore, the development of rapid nucleic acid detection technology has important practical significance and application value. Paper-based materials have played an important role in rapid nucleic acid detection in the field of in vitro diagnosis due to their rich sources, low cost, good biocompatibility, easy modification and portability. Nucleic acid extraction is the first and key link in nucleic acid detection, and plays a vital role in the sensitivity of the entire detection. Therefore, paper-based nucleic acid extraction technology is also crucial for rapid nucleic acid detection. At present, paper-based nucleic acid extraction technology has been applied to the extraction of nucleic acid from a variety of biological samples such as whole blood, cell samples, saliva and bacteria, but its extraction efficiency is low, which limits its widespread use in clinical applications. Summary of the invention

[0003] In order to solve the above problems of the prior art, the present invention provides a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional group, and a preparation method and application thereof, which can realize simple, rapid and efficient extraction of nucleic acid.

[0004] The present invention is achieved through the following technical solutions: A method for preparing a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional groups, comprising: S1, dissolving cotton pulp in water, stirring and mixing, to obtain a mixture 1; filtering the mixture 1 into paper, to obtain CF paper; S2, using CF paper as a substrate, pouring mixture 2 on the CF paper, filtering, and drying to obtain a CF / CNF paper-based material; wherein the mixture 2 is an aqueous solution of nanocellulose fibers; S3, immersing the CF / CNF paper-based material into the mixture 3, shaking and incubating, then taking out the CF / CNF paper-based material, rinsing and drying, to obtain the CF / CNF / L-DOPA paper-based nucleic acid extraction material; wherein the mixture 3 is prepared by dissolving L-DOPA in a Tris-HCl solution.

[0005] Preferably, in S1, the beating degree of the cotton pulp is 30~50° SR.

[0006] Preferably, in S2, the mass of nanocellulose fibers in the CF / CNF paper-based material accounts for 1% to 10% of the mass of the CF paper.

[0007] Preferably, in S3, the mass ratio of the levodopa to the mass of the CF paper is (0.03-0.09):0.64.

[0008] Preferably, in S3, the shaking incubation temperature is room temperature.

[0009] Preferably, in S3, the shaking incubation time is 10 to 12 hours.

[0010] Preferably, in S2, the drying temperature is 35-45°C; in S3, the drying temperature is 35-45°C.

[0011] The present invention also provides a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional group obtained by the preparation method as described above.

[0012] The present invention also provides the use of the paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional group in extracting nucleic acid in biological samples.

[0013] Preferably, the application is specifically as follows: dripping the lysed biological sample mixture onto the paper-based nucleic acid extraction material, and the paper-based nucleic acid extraction material adsorbs nucleic acids; then, using the paper-based nucleic acid extraction material adsorbed with nucleic acids as a template for PCR amplification, and then using gel electrophoresis to perform nucleic acid detection on the paper-based nucleic acid extraction material after PCR amplification.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention deposits a nanocellulose fiber solution onto cotton fiber paper by vacuum filtration, and the cotton fiber (CF) and the nanocellulose fiber (CNF) are entangled with each other. The pore size of the paper can be changed by changing the concentration of the added nanocellulose fiber, and the nucleic acid can be intercepted by adjusting the pore size. At the same time, a new functional group amino group is introduced by adding levodopa, so that the surface of the paper-based nucleic acid extraction material is positively charged. Because the isoelectric point of nucleic acid molecules is 4 to 4.5, the nucleic acid is positively charged when the solution pH is less than its isoelectric point, and negatively charged when it is greater than its isoelectric point. The nucleic acid in the sample extract can be negatively charged by adjusting the solution pH, so that it interacts with the positive charge on the surface of the paper-based nucleic acid extraction material to adsorb the negatively charged nucleic acid.

[0015] The paper-based nucleic acid extraction material prepared by the present invention can realize simple, rapid and efficient extraction of nucleic acid based on the synergistic effect of pore size and functional group, and can directly extract nucleic acid from various biological samples, significantly improving the extraction efficiency and purity. The paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional group obtained by the present invention has the advantages of low cost, easy to carry, simple operation, and batch production, and can quickly and efficiently extract nucleic acid from different biological samples in different paper-based nucleic acid detection platforms. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The electron micrographs (A) of the pure cotton cellulose paper prepared in the present invention and the CF / CNF paper-based material prepared by adding nanocellulose fibers, and the pore size (B) of the CF / CNF paper-based material was measured by Image J software based on the results of the scanning electron microscope; (a) in A is CF paper; (b) is CF / 1%CNF paper-based material; (c) is CF / 5%CNF paper-based material; and (d) is CF / 10%CNF paper-based material.

[0018] Figure 2 The nucleic acid adsorption standard curve diagram (a) of the CF / 5% CNF paper-based material prepared by the present invention, as well as the nucleic acid adsorption amount diagram (b) of the CF / CNF paper-based material with different concentrations and the gel electrophoresis detection result diagram (c) are shown.

[0019] Figure 3 This is an infrared image of the CF / CNF / L-DOPA paper-based material modified with nanocellulose fibers and levodopa (L-DOPA) prepared in the present invention.

[0020] Figure 4 The nucleic acid adsorption standard curve of CF / CNF / 2-L-DOPA prepared by the present invention (a), the nucleic acid adsorption amount of CF / CNF / 2-L-DOPA paper-based materials with different L-DOPA loading amounts (b) and the gel electrophoresis detection result (c). DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] It should be noted that the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices. 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 to define 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.

[0024] The method for preparing the paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional group of the present invention comprises: S1, dissolving cotton pulp in water, stirring and mixing, to obtain a mixture 1; filtering the mixture 1 into paper, to obtain CF paper; S2, using CF paper as a substrate, pouring mixture 2 on the CF paper, filtering, and drying to obtain a CF / CNF paper-based material; wherein the mixture 2 is an aqueous solution of nanocellulose fibers; S3, immersing the CF / CNF paper-based material into the mixture 3, shaking and incubating, then taking out the CF / CNF paper-based material, rinsing and drying, to obtain the CF / CNF / L-DOPA paper-based nucleic acid extraction material; wherein the mixture 3 is prepared by dissolving L-DOPA in a Tris-HCl solution.

[0025] In some embodiments of the present invention, in S1, the beating degree of the cotton pulp is 30~50° SR.

[0026] In some embodiments of the present invention, in S2, the mass of nanocellulose fibers in the CF / CNF paper-based material accounts for 1% to 10% of the mass of the CF paper.

[0027] In some embodiments of the present invention, in S3, the mass ratio of the levodopa to the mass of the CF paper is (0.03-0.09): 0.64.

[0028] The present invention can change the pore size and surface functional groups of the paper-based material by regulating the dosage of nanocellulose fibers and levodopa (L-DOPA) to further improve the nucleic acid adsorption performance of the paper-based material.

[0029] In some embodiments of the present invention, in S3, the shaking incubation temperature is room temperature, and the shaking incubation time is 10 to 12 hours.

[0030] In some embodiments of the present invention, in S2, the drying temperature is 35-45°C; in S3, the drying temperature is 35-45°C.

[0031] The paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional group obtained by the preparation method of the present invention can be used to extract nucleic acids in biological samples. The specific application method is: the biological sample mixture after lysis is dripped onto the paper-based nucleic acid extraction material, and the paper-based nucleic acid extraction material adsorbs nucleic acids; then, the paper-based nucleic acid extraction material adsorbed with nucleic acids is used as a template for PCR amplification, and then the paper-based nucleic acid extraction material after PCR amplification is subjected to nucleic acid detection by gel electrophoresis.

[0032] Example 1 The preparation method of CF / CNF paper-based material comprises the following steps: Step 1: A trough-type beating machine is used to prepare cotton pulp with an absolute dryness of 28.4% at a beating degree of 40° SR.

[0033] Step 2: Weigh cotton pulp containing 0.64 g of cotton fiber (absolute dry mass) and dissolve it in 100 mL of deionized water, mix and stir to obtain a mixture 1, and filter the mixture 1 into paper by vacuum filtration to obtain pure cotton fiber paper (CF paper).

[0034] Step 3: According to the mass ratio of 1%, 5%, and 10%, 0.0064 g, 0.032 g, and 0.064 g CNF (absolute dry mass) were weighed and dissolved in 100 mL of deionized water, and stirred and ultrasonicated to completely dissolve them to obtain CNF solutions of different concentrations.

[0035] Step 4: Using CF paper as the substrate, slowly and evenly pour CNF solutions of different concentrations onto the prepared CF paper, compound them on the surface of the CF paper by vacuum filtration, and dry them in an oven at 35°C for 30 minutes to obtain CF / CNF paper-based materials (including CF / 1%CNF, CF / 5%CNF, and CF / 10%CNF paper-based materials). At this time, the quantitative weight of the paper-based material is 100g / m 2 .

[0036] In order to investigate the influence of the properties of paper-based materials on the nucleic acid extraction performance, the present invention observed the morphology and pore size of CF / CNF ( Figure 1 ) and nucleic acid adsorption performance ( Figure 2 ).

[0037] Figure 1 Result of A shows that after introducing CNF, the pore size of CF paper has changed significantly. The pore size of the paper-based material with added CNF has decreased significantly. As the CNF content increases from 1% to 10%, the pore size gradually becomes smaller. Figure 1 Result of B is the quantitative result of the pore size of each prepared paper-based material, which is similar to the result of SEM. The pore sizes are as follows: CF (71.203μm) > CF / 1%CNF (42.5366μm) > CF / 5%CNF (29.9603μm) > CF / 10%CNF (15.9823μm). This is because nanocellulose fibers, as a kind of nano filler, fill and even block the macroporous structure of cotton fibers, thus changing the pore size of cotton fibers.

[0038] Figure 2 In (a), a nucleic acid adsorption standard curve is made through five different DNA concentrations to calculate the amount of DNA adsorbed by the subsequent paper-based materials. From Figure 2 As can be seen from (b), the nucleic acid adsorption capacities of the prepared paper-based materials are: CNF (2.07 ± 0.18μg) < CF (5.44 ± 0.19μg) < CF / 1%CNF (6.42 ± 0.07μg) < CF / 5%CNF (7.01 ± 0.20μg) < CF / 10%CNF (7.48 ± 0.12μg). The reason for the analysis may be that as the pore size decreases, the specific surface area becomes larger and the nucleic acid adsorption amount also becomes larger. From Figure 2 By observing and comparing the pure DNA adsorption performances of different paper-based materials in (c), it is found that the amplification band of the CF / 5%CNF paper-based material is brighter and the extraction effect is better. The nucleic acid extraction efficiency of the CF / 5%CNF paper-based material is the highest. The reason may be that a higher concentration of CNF interacts with the required enzymes or other components in the nucleic acid amplification reaction system, affecting the enzyme activity or the function of DNA polymerase, thus producing an inhibitory effect. Considering the physicochemical properties and nucleic acid extraction ability of the CF / CNF paper-based material comprehensively, CF / 5%CNF is initially selected as the subsequent experimental condition. All CNF concentrations appearing later are 5%, denoted as mixture 2. Using pure cotton fiber paper as the substrate, a 5% CNF solution is deposited onto the pure cotton fiber paper by vacuum filtration to obtain the CF / CNF paper-based material.

[0039] Example 2 A preparation method of an efficient paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional groups, comprising the following steps: Step 1: Prepare cotton pulp with a beating degree of 40° SR using a trough beater.

[0040] Step 2: Weigh cotton pulp containing 0.64 g of cotton fiber (absolute dry mass) and dissolve it in 100 mL of deionized water, stir and mix to obtain a mixture 1, and filter the mixture 1 into paper by vacuum filtration to obtain pure cotton fiber paper (CF paper).

[0041] Step 3: Weigh 0.032 g of CNF (absolute dry mass) and dissolve it in 100 mL of deionized water to prepare a 5% nanocellulose fiber solution to obtain mixture 2. Step 4: Using CF paper as the substrate, slowly and evenly pour mixture 2 onto the prepared CF paper, compound it on the surface of the CF paper by vacuum filtration, and dry it in an oven at 35°C for 30 minutes to obtain CF / CNF paper-based material. At this time, the quantitative weight of the paper-based material is 100g / m 2 .

[0042] Step 5: Weigh 0.03 g of L-DOPA and dissolve it in 30 mL of Tris-HCl solution (15 mM, pH 8.8, 150 mM NaCl) to prepare a L-DOPA solution to obtain mixture 3.

[0043] Step 6: Immerse the CF / CNF paper-based material in mixture 3 and incubate it in a constant temperature shaker (25°C, 100 rpm) for 12 hours. Then, take out the paper-based material, rinse it with deionized water five times, and dry it at 35°C for 2 hours to obtain the CF / CNF / L-DOPA paper-based material (CF / CNF / 1-L-DOPA).

[0044] Step 7: Punch the paper-based material prepared in step 6 into a paper disc with a diameter of 4.5 mm for later use. Soak the punched paper disc in a binding solution (10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl) at pH 4 for 10 min, take it out and dry it for later use. Take 100 μL of saliva sample and 120 μL of neutral lysis solution (2 M sodium acetate, 2 M guanidine hydrochloride, 5 mM EDTA, 1% Triton X-100 and 3% PEG400) were mixed evenly to obtain a mixed solution; a paper disk was placed on a multi-layer absorbent paper, and the mixed solution was added to the paper disk. After the liquid had completely passed through, the paper disk was taken out and the excess liquid was gently pressed with absorbent paper to be removed for the next amplification test. The test was performed by gel electrophoresis. If the target band appeared between 200 and 300 bp, it indicated that nucleic acid extraction could be performed. The brighter the band, the higher the purity of the extracted nucleic acid and the less adsorption of impurities. If no target band appeared, it indicated that the nucleic acid was not extracted or the extracted nucleic acid was too little to be detected. Example 3 A method for preparing an efficient paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional groups comprises the following steps: Step 1: Prepare cotton pulp with a beating degree of 40° SR using a trough beater.

[0045] Step 2: Weigh cotton pulp containing 0.64 g of cotton fiber (absolute dry mass) and dissolve it in 100 mL of deionized water, mix and stir to obtain a mixture 1, and filter the mixture 1 into paper by vacuum filtration to obtain pure cotton fiber paper (CF paper).

[0046] Step 3: Weigh 0.032 g of CNF (absolute dry mass) and dissolve it in 100 mL of deionized water to prepare a 5% nanocellulose fiber solution to obtain mixture 2. Step 4: Using CF paper as the substrate, slowly and evenly pour mixture 2 onto the prepared CF paper, compound it on the surface of the CF paper by vacuum filtration, and dry it in an oven at 35°C for 30 minutes to obtain CF / CNF paper-based material. At this time, the quantitative weight of the paper-based material is 100g / m 2 .

[0047] Step 5: Weigh 0.06 g of levodopa and dissolve it in 30 mL of Tris-HCl solution (15 mM, pH 8.8, 150 mM NaCl) to prepare a levodopa solution to obtain a mixture 3.

[0048] Step 6: Immerse the CF / CNF paper-based material in mixture 3 and incubate it in a constant temperature shaker (25°C, 100 rpm) for 12 hours. Then, take out the paper-based material, rinse it with deionized water five times, and dry it at 35°C for 2 hours to obtain CF / CNF / L-DOPA paper-based material (CF / CNF / 2-L-DOPA).

[0049] Step 7: Punch the paper-based material prepared in step 6 into a paper disc with a diameter of 4.5 mm for later use. Soak the punched paper disc in a binding solution (10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCL) with a pH of 4 for 10 min, take it out and dry it for later use. Take 100 μL of saliva sample and mix it evenly with 120 μL of neutral lysis solution (2M sodium acetate, 2M guanidine hydrochloride, 5mM EDTA, 1% Triton X-100 and 3% PEG400) to obtain a mixed solution; place the paper disc on a multi-layer absorbent paper, add the mixed solution to the paper disc, and after the liquid has completely passed through, take out the paper disc and use absorbent paper to gently press to remove excess liquid for the next step of amplification detection.

[0050] Example 4 Step 1: Prepare cotton pulp with a beating degree of 40° SR using a trough beater.

[0051] Step 2: Weigh cotton pulp containing 0.64 g of cotton fiber (absolute dry mass) and dissolve it in 100 mL of deionized water, mix and stir to obtain a mixture 1, and filter the mixture 1 into paper by vacuum filtration to obtain pure cotton fiber paper (CF paper).

[0052] Step 3: Weigh 0.032 g of CNF (absolute dry mass) and dissolve it in 100 mL of deionized water to prepare a 5% nanocellulose fiber solution to obtain mixture 2. Step 4: Using CF paper as the substrate, slowly and evenly pour mixture 2 onto the prepared CF paper, compound it on the surface of the CF paper by vacuum filtration, and dry it in an oven at 35°C for 30 minutes to obtain CF / CNF paper-based material. At this time, the quantitative weight of the paper-based material is 100g / m 2 .

[0053] Step 5: Weigh 0.09 g of levodopa and dissolve it in 30 mL of Tris-HCl solution (15 mM, pH 8.8, 150 mM NaCl) to prepare a levodopa solution to obtain a mixture 3.

[0054] Step 6: Immerse the CF / CNF paper-based material in mixture 3 and incubate it in a constant temperature shaker (25°C, 100 rpm) for 12 hours. Then, take out the paper-based material, rinse it with deionized water five times, and dry it at 35°C for 2 hours to obtain CF / CNF / L-DOPA paper-based material (CF / CNF / 3-L-DOPA).

[0055] Step 7: Punch the paper-based material prepared in step 5 into a paper disc with a diameter of 4.5 mm for later use. Soak the punched paper disc in a binding solution (10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCL) with a pH of 4 for 10 min, take it out and dry it for later use. Take 100 μL of saliva sample and mix it evenly with 120 μL of neutral lysis solution (2M sodium acetate, 2M guanidine hydrochloride, 5mM EDTA, 1% Triton X-100 and 3% PEG400) to obtain a mixed solution; place the paper disc on a multi-layer absorbent paper, add the mixed solution to the paper disc, and after the liquid has completely passed through, take out the material and use absorbent paper to gently press to remove excess liquid for the next step of amplification detection.

[0056] In order to investigate the effect of paper-based material properties on nucleic acid extraction performance, the chemical properties of CF / CNF / L-DOPA were observed. Figure 3 ) and nucleic acid adsorption performance ( Figure 4 ).

[0057] In order to verify the successful preparation of CF / CNF / L-DOPA, Figure 3The surface functional groups of pure CF and the CF / CNF / L-DOPA paper-based material prepared in Example 2 were observed by FTIR results. It was found that after the composite of L-DOPA, the band at 3300~3500 cm -1 became wider and deeper. This is due to the Fermi resonance effect. When the frequency of one vibration mode is close to the fundamental frequency of another vibration, a strong absorption band is generated due to their interaction. This is also a kind of vibration coupling, which belongs to the overlapping vibration peaks of N-H and -OH. The characteristic peak at 1760 cm -1 corresponds to the stretching vibration of -COOH of CNF, and the absorption peak at 1650 cm -1 corresponds to the vibration of C=O. Generally speaking, compared with the CF paper, the newly emerged N-H and C=O in CF / CNF / L-DOPA indicate the successful introduction of L-DOPA.

[0058] Figure 4 In (a), the nucleic acid adsorption standard curve was made through five different DNA concentrations to calculate the amount of DNA adsorbed by the subsequent paper-based materials. From Figure 4 in (b), it can be seen that the nucleic acid adsorption capacity of the paper-based materials is CF / 5%CNF (5.44±0.19 μg) < CF / CNF / 1-L-DOPA (6.53 ± 0.12 μg) < CF / CNF / 3-L-DOPA (7.58 ± 0.15 μg) < CF / CNF / 2-L-DOPA (7.69±0.10 μg). By comparing the pure DNA adsorption performance of different papers, it can be seen that the nucleic acid adsorption performance of the paper-based materials can be improved after loading L-DOPA. At the same time, it can also be found that the amplified band of CF / CNF / 2-L-DOPA paper is brighter and the extraction effect is better. Figure 4 In (c), the nucleic acid gel electrophoresis results all show that the nucleic acid extraction effect of CF / CNF / 2-L-DOPA is the best.

[0059] In summary, the present invention has successfully prepared a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional groups. By regulating the pore size and introducing new functional groups, this material realizes the efficient extraction of nucleic acid molecules. This design not only enhances the adsorption and retention ability of the material for nucleic acid from the physical factor aspect, but also improves the adsorption ability of the material for nucleic acid from the chemical factor aspect. The present invention not only provides an efficient nucleic acid extraction method, but also provides new ideas for the design and application of paper-based biological materials, showing significant advantages in improving extraction efficiency and reducing costs, and providing new possibilities for the future biological detection and molecular diagnosis fields.

Claims

1. A method for preparing a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional group, characterized in that: include: S1, dissolving cotton pulp in water, stirring and mixing, to obtain a mixture 1; filtering the mixture 1 into paper, to obtain CF paper; S2, using CF paper as a substrate, pouring mixture 2 on the CF paper, filtering, and drying to obtain a CF / CNF paper-based material; wherein the mixture 2 is an aqueous solution of nanocellulose fibers; S3, immersing the CF / CNF paper-based material into the mixture 3, shaking and incubating, then taking out the CF / CNF paper-based material, rinsing and drying, to obtain the CF / CNF / L-DOPA paper-based nucleic acid extraction material; wherein the mixture 3 is prepared by dissolving L-DOPA in a Tris-HCl solution.

2. The method for preparing a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional groups according to claim 1, characterized in that: In S1, the beating degree of the cotton pulp is 30~50° SR.

3. The method for preparing a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional groups according to claim 1, characterized in that: In S2, the mass of nanocellulose fibers in the CF / CNF paper-based material accounts for 1% to 10% of the mass of CF paper.

4. The method for preparing a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional groups according to claim 1, characterized in that: In S3, the mass ratio of the levodopa to the mass ratio of the CF paper is (0.03-0.09):0.

64.

5. The method for preparing a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional groups according to claim 1, characterized in that: In S3, the shaking incubation temperature is room temperature.

6. The method for preparing a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional groups according to claim 1, characterized in that: In S3, the shaking incubation time is 10~12h.

7. The method for preparing a paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional groups according to claim 1, characterized in that: In S2, the drying temperature is 35-45°C; in S3, the drying temperature is 35-45°C.

8. A paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional group obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the paper-based nucleic acid extraction material based on the synergistic effect of pore size and functional group as claimed in claim 8 in extracting nucleic acids from biological samples.

10. The use according to claim 9, characterized in that: The lysed biological sample mixture is dripped onto the paper-based nucleic acid extraction material, and the paper-based nucleic acid extraction material adsorbs nucleic acid; then, the paper-based nucleic acid extraction material adsorbed with nucleic acid is used as a template for PCR amplification, and then the paper-based nucleic acid extraction material after PCR amplification is subjected to nucleic acid detection by gel electrophoresis.

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