Responsive dual-network hydrogel as well as preparation method and application thereof

Abstract

The invention provides responsive dual-network hydrogel as well as a preparation method and application thereof, and relates to the field of nucleic acid separation and recovery. The hydrogel is composed of an agarose gel network and a poly (N-isopropylacrylamide) network, the separation resolution of nucleic acid during electrophoresis can be enhanced by adjusting the proportion of agarose to poly (N-isopropylacrylamide), and the separation range of nucleic acid with different molecular weights can be regulated and controlled. The temperature sensitivity of the poly (N-isopropylacrylamide) enables the double-network hydrogel to have thermal response performance, and when the double-network hydrogel is heated to a low critical phase transition temperature (-32 DEG C) or above, the double-network hydrogel can extrude a water phase in the hydrogel through phase transition of the poly (N-isopropylacrylamide), so that nucleic acid in the hydrogel is released under the condition that a hydrogel network does not need to be destroyed, and the hydrogel can be used for preparing the hydrogel. Nucleic acid recovery operation is simplified, and nucleic acid recovery efficiency and sample recovery rate are improved. The method is suitable for separation and recovery of various nucleic acid fragments and nucleic acid assembled nanostructures.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a responsive double-network hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention and is not necessarily regarded as an admission or an indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Agarose gel electrophoresis is one of the core techniques for separating and identifying nucleic acid molecules, and its separation principle is based on the size and conformation differences of nucleic acid molecules. During electrophoresis, negatively charged DNA molecules migrate towards the anode under the action of an electric field, and the three-dimensional network structure formed by agarose gel acts as a molecular sieve, enabling smaller DNA fragments to migrate faster, thereby achieving separation. By combining fluorescence labeling with nucleic acid dyes such as ethidium bromide (EB), the electrophoresis bands can be directly observed by ultraviolet excitation, and the size of the target fragment can be estimated based on the standard molecular weight marker. Due to the simplicity, low cost, and high resolution of agarose gel electrophoresis, this method has become a basic experimental means in the research of molecular biology, genetic engineering, and nucleic acid chemistry.

[0004] With the rapid development of genetic engineering and DNA nanotechnology, the demand for efficiently recovering target DNA fragments from agarose gels is increasing. For example, in DNA self-assembly technologies (such as DNA origami), high-purity and structurally intact DNA products are required for subsequent functional applications. However, traditional agarose gel recovery methods still have significant limitations in terms of efficiency, purity, and applicability. Currently, the commonly used recovery techniques are mainly divided into three categories: chemical dissolution methods (such as chaotropic agent treatment), physical destruction methods (such as freeze-extrusion method, high-temperature melting method), and electroelution methods.

[0005] The chemical dissolution method usually uses high-concentration chaotropic salts (such as sodium iodide or sodium perchlorate) to dissolve agarose, and then purifies DNA through a silica column or alcohol precipitation. However, this method may introduce chemical residues, affecting downstream experiments, and has a low recovery efficiency, especially when dealing with large fragments or DNA with complex structures. In the physical disruption method, the high-temperature melting method (>60 °C) can quickly dissolve ordinary or low-melting-point agarose, but the high temperature may cause DNA denaturation or damage its higher-order structure (such as the stability of DNA origami); while the freezing-extrusion method avoids high temperature, but the operation is cumbersome and is prone to introducing gel fragment contamination, reducing the purity of the recovered product. The electroelution method makes DNA migrate into the dialysis bag through a reverse electric field, but DNA is easily adsorbed on the dialysis bag wall, resulting in low recovery rate, and the operation is complex and difficult to be applied on a large scale. In addition, as a mild alternative, the agarose enzyme digestion method can release DNA by enzymatically digesting the agarose matrix, avoiding high temperature and chemical residues, but complete digestion requires long-term incubation, and the enzyme cost is high, limiting its wide use.

[0006] Although agarose gel electrophoresis can separate these complex structures, it is difficult to efficiently extract the complete product. With the development of DNA nanotechnology, the demand for the recovery of high-purity and structurally intact DNA products has become increasingly prominent. The above traditional methods for separating DNA fragments can no longer meet the needs of separating and recovering more complex DNA-assembled nanostructures. Therefore, there is an urgent need to develop a method that takes into account efficiency, purity and can completely recover DNA fragments and DNA-assembled nanostructures. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a responsive double-network hydrogel and its preparation method and application. The responsive double-network hydrogel provided by the present invention can separate DNA fragments under the action of electrophoresis, with good separation effect and high resolution, and the responsive double-network hydrogel has temperature responsiveness, can release the aqueous phase and DNA in the gel matrix, and has a high DNA recovery efficiency, simple operation and mild conditions.

[0008] In order to achieve the above purpose, the technical solution of the present invention is as follows: In the first aspect, the present invention provides a responsive double-network hydrogel, which has a double-network structure in which a first rigid network formed by agarose and a second flexible network formed by the polymerization of N-isopropylacrylamide monomers in the presence of a cross-linking agent penetrate each other. The first rigid network of agarose (AG) provides the basic three-dimensional network and mechanical properties for the hydrogel, and poly-N-isopropylacrylamide (pNIPAM) as the second flexible network provides temperature-responsive properties. At the same time, the combination of two different structures and densities of networks is used to effectively regulate the pore size and density of the gel, so as to achieve high-resolution separation and efficient recovery of DNA fragments.

[0009] In some embodiments, the crosslinking agent is any one of N,N′-methylenebisacrylamide (BIS) and polyethylene glycol diacrylate (PEGDA).

[0010] In some embodiments, in the responsive double-network hydrogel, the mass fraction of agarose is 0.5-1.0 wt%, and the mass fraction of N-isopropylacrylamide is 1-4 wt%. By adjusting the ratio of agarose and poly-N-isopropylacrylamide, the pore size of the gel network can be regulated. Experiments have shown that under the above dosages of agarose and N-isopropylacrylamide, the separation effect of DNA fragments is good and the resolution is high.

[0011] In a second aspect, the present invention provides a method for preparing a responsive double-network hydrogel, which is made of agarose, N-isopropylacrylamide, an initiator, a crosslinking agent, and a buffer solution. Among them, the completely dissolved agarose solution forms a first rigid network after cooling, and N-isopropylacrylamide forms a second flexible network through free radical polymerization under the action of a crosslinking agent and an initiator.

[0012] In some embodiments, the method for preparing the responsive double-network hydrogel specifically includes the following steps: Add agarose to the buffer solution, heat it until it is fully dissolved, then add N-isopropylacrylamide and a crosslinking agent. When the temperature is cooled to 1-2 °C higher than the agarose gel freezing point, add an initiator, and immediately transfer the mixed solution into a mold. Agarose first forms a first rigid network through hydrogen bond interaction, and stand at 4-25 °C to enable N-isopropylacrylamide to form a second flexible network through free radical polymerization reaction, and the responsive double-network hydrogel is prepared.

[0013] In some embodiments, the agarose includes ordinary melting point agarose and low melting point agarose.

[0014] In some embodiments, the N-isopropylacrylamide is the N-isopropylacrylamide monomer after recrystallization of a commercially available N-isopropylacrylamide product.

[0015] In some embodiments, the initiator is any one or combination of ammonium persulfate (APS), potassium peroxydisulfate (KPS), N,N,N′,N′-tetramethylethylenediamine (TEMED), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (photoinitiator 2959), and lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (photoinitiator LAP).

[0016] In some embodiments, the crosslinking agent is any one of N,N′-methylenebisacrylamide (BIS) and polyethylene glycol diacrylate (PEGDA), and the mass ratio of the crosslinking agent to N-isopropylacrylamide is 1:10-40.

[0017] In some embodiments, the components of the buffer solution include tris(hydroxymethyl)aminomethane, boric acid, ethylenediaminetetraacetic acid, acetic acid, sodium ions, magnesium ions, and water. Tris(hydroxymethyl)aminomethane is mainly used to maintain the pH stability, form a buffer pair with acetic acid or boric acid, resist the pH change caused by electrolysis during electrophoresis, and prevent nucleic acids from denaturing due to pH fluctuations; EDTA chelates metal ions such as Mg to avoid nucleic acid degradation.

[0018] In some embodiments, an initiator is added when a mixed solution containing ordinary agarose or low melting point agarose is cooled to 25 - 31°C. After being transferred to a mold, the temperature of the mixed solution drops, and an agarose first rigid network is rapidly formed. It is polymerized for 1 - 24 h at a temperature below the critical phase transition temperature of poly(N-isopropylacrylamide) to form a second flexible network.

[0019] In some embodiments, the mass ratio of agarose to N-isopropylacrylamide is 1:(1 - 8).

[0020] In a third aspect, the present invention provides the use of the responsive double-network hydrogel described in the first aspect or the responsive double-network hydrogel prepared in the second aspect in the field of nucleic acid separation and recovery.

[0021] In some embodiments, the use of the responsive double-network hydrogel in the field of nucleic acid separation and recovery specifically includes the following steps: (a) Place the responsive double-network hydrogel into an electrophoresis tank, add a nucleic acid sample, and perform electrophoresis for 1 - 180 min at a voltage of 50 - 150 V. After electrophoresis, stain the nucleic acid. (b) Observe the nucleic acid bands in the gel under a blue light lamp or an ultraviolet lamp, and cut out the target band; the combination of the agarose network and the poly(N-isopropylacrylamide) network effectively regulates the pore size and density of the gel, thereby achieving high-resolution separation of DNA fragments. (c) Chop the gel block containing the target band and transfer it into a container with a filter screen of a set pore size. (d) Place the container containing the gel block in an environment of 30 - 40°C and keep it for 5 - 30 min. Since poly(N-isopropylacrylamide) provides temperature-responsive performance, the gel block can extrude the aqueous phase and DNA under this condition through responsive shrinkage, that is, the nucleic acid recovery solution is separated.

[0022] (e) Collect the nucleic acid recovery solution, and calculate the nucleic acid concentration and sample recovery rate in the recovery solution according to the ultraviolet absorption or fluorescence intensity signal.

[0023] In some embodiments, the standard nucleic acid molecular weight range that enters the gel through electrophoresis is 100 - 10000 bp.

[0024] The beneficial effects of the present invention are as follows: (1) The present invention constructs a double-network hydrogel using agarose and poly(N-isopropylacrylamide). This double-network hydrogel can separate DNA fragments and DNA self-assembled nanostructures under the action of electrophoresis, with good separation effect and high resolution.

[0025] (2) The double-network hydrogel in the present invention has temperature responsiveness and can release the aqueous phase and DNA components in the gel matrix. The DNA sample recovery efficiency is high, the operation is simple, and the conditions are mild. It has good application prospects in the field of separating and recovering DNA fragments and DNA self-assembled nanostructures. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0027] Figure 1 It is a schematic diagram of the synthesis of the responsive double-network hydrogel and a schematic diagram of the separation and recovery of DNA after gel electrophoresis in Examples 1-4; wherein (a) is a schematic diagram of the synthesis of the responsive double-network hydrogel, and (b) is a schematic diagram of the separation and recovery of DNA after gel electrophoresis.

[0028] Figure 2 It is a gel electrophoresis diagram of the responsive double-network hydrogel in Examples 1-4 and the agarose single-network hydrogel in Comparative Examples 1-3; wherein, (a) is the gel electrophoresis diagram of the agarose single-network hydrogel in Comparative Example 1, (b) is the gel electrophoresis diagram of the agarose single-network hydrogel in Comparative Example 2, (c) is the gel electrophoresis diagram of the agarose single-network hydrogel in Comparative Example 3, (d) is the gel electrophoresis diagram of the responsive double-network hydrogel in Example 1, (e) is the gel electrophoresis diagram of the responsive double-network hydrogel in Example 2, (f) is the gel electrophoresis diagram of the responsive double-network hydrogel in Example 3, and (g) is the gel electrophoresis diagram of the responsive double-network hydrogel in Example 4.

[0029] Figure 3 It is an appearance diagram and a histogram of diameter change of the responsive double-network hydrogel in Examples 1-4 and the agarose single-network hydrogel in Comparative Examples 1-3 with temperature change; wherein, (a) is the appearance diagram of the responsive double-network hydrogel in Examples 1-4 and the agarose single-network hydrogel in Comparative Examples 1-3 with temperature change; (b) is the histogram of diameter change of the responsive double-network hydrogel in Example 1, (c) is the histogram of diameter change of the responsive double-network hydrogel in Example 2, (d) is the histogram of diameter change of the responsive double-network hydrogel in Example 3, and (e) is the histogram of diameter change of the responsive double-network hydrogel in Example 4.

[0030] Figure 4 Mass loss ratio diagrams of the responsive double-network hydrogels in Examples 1-4 and the agarose single-network hydrogels in Comparative Examples 1-3 in response to temperature changes. Among them, (a) is the mass loss ratio diagram of Example 1, (b) is the mass loss ratio diagram of Example 2, (c) is the mass loss ratio diagram of Example 3, (d) is the mass loss ratio diagram of Example 4, (e) is the mass loss ratio diagram of Comparative Example 1, (f) is the mass loss ratio diagram of Comparative Example 2, and (g) is the mass loss ratio diagram of Comparative Example 3.

[0031] Figure 5 DNA recovery rate histograms of the agarose single-network hydrogels in Comparative Examples 1-3 and the responsive double-network hydrogels in Examples 1-4. Among them, (a) is the recovery rate histogram of the 10 kbp band, (b) is the recovery rate histogram of the 3 kbp band, (c) is the recovery rate histogram of the 1 kbp band, (d) is the recovery rate histogram of the 0.5 kbp band, and (e) is the recovery rate histogram of the 0.2 kbp band.

[0032] Figure 6 Gel electrophoresis diagrams and recovery rate histograms of the DNA self-assembly structures separating 4HB and 8HB of the 0.5% agarose single-network hydrogel in Comparative Example 1 and the responsive double-network hydrogel in Example 3. Among them, (a) is the gel electrophoresis diagram of Example 3, and (b) is the gel electrophoresis diagram of Comparative Example 1. Figure 6 Among them, (c) is the recovery rate histogram. Detailed implementation manners

[0033] It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0034] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0035] Example 1 Preparation of Responsive Double-Network Hydrogel: Add a set amount of ordinary melting-point agarose into 20 mL of buffer solution (1xTBE). After heating until it is fully dissolved, add a set amount of N-isopropylacrylamide and crosslinking agent BIS (the mass ratio of BIS to N-isopropylacrylamide is 1:29). When cooling to a temperature 1.5 ± 0.5 °C higher than the freezing point of agarose gel, add 100 μL of 10 wt% APS solution and 20 μL of N,N,N’,N’-tetramethylethylenediamine (TEMED). Shake well and immediately transfer the mixed solution into a mold. Agarose first forms the first rigid network through hydrogen bond interaction. Let it stand at 4 °C to enable N-isopropylacrylamide to form the second flexible network through free radical polymerization reaction, thus preparing the described responsive double-network hydrogel, where the mass fraction of agarose is 0.5 wt% and the mass fraction of N-isopropylacrylamide is 1 wt%.

[0036] Example 2 The difference between the preparation method of the responsive double-network hydrogel in this example and that in Example 1 is only that: the amount of N-isopropylacrylamide is 2 wt%.

[0037] Example 3 The difference between the preparation method of the responsive double-network hydrogel in this example and that in Example 1 is only that: the amount of N-isopropylacrylamide is 3 wt%.

[0038] Example 4 The difference between the preparation method of the responsive double-network hydrogel in this example and that in Example 1 is only that: the amount of N-isopropylacrylamide is 4 wt%.

[0039] The synthesis schematic diagrams of the responsive double-network hydrogels in Examples 1 - 4 and the separation and recovery schematic diagrams of DNA gel electrophoresis are respectively as shown in Figure 1 (a) in Figure 1 and (b) in

[0040] Comparative Example 1 Preparation of agarose single-network hydrogel: Add a set amount of ordinary melting-point agarose into the buffer solution, heat it until it is fully dissolved, and cool to form agarose gel, where the mass fraction of agarose is 0.5 wt%.

[0041] Comparative Example 2 Preparation of agarose single-network hydrogel: Add a set amount of ordinary melting-point agarose into the buffer solution, heat it until it is fully dissolved, and cool to form agarose gel, where the mass fraction of agarose is 0.7 wt%.

[0042] Comparative Example 3 Preparation of agarose single-network hydrogel: A set amount of ordinary melting point agarose was added to the buffer solution, heated to dissolve it fully, and then cooled to form an agarose gel, where the mass fraction of agarose was 1.0 wt%.

[0043] Performance analysis (1) Electrophoresis tests were carried out on the agarose-poly(N-isopropylacrylamide) (AG-pNIPAM) double-network hydrogels prepared in Examples 1-4 and the agarose (AG) single-network hydrogels prepared in Comparative Examples 1-3. The samples were 500-10000 bp DNA ladder and 100-1517 bp DNA ladder. The electrophoresis conditions were all 60 v and 120 min. The results are as Figure 2 shown. Among them, Figure 2 (a) is the gel electrophoresis diagram of the agarose single-network hydrogel in Comparative Example 1, (b) is the gel electrophoresis diagram of the agarose single-network hydrogel in Comparative Example 2, (c) is the gel electrophoresis diagram of the agarose single-network hydrogel in Comparative Example 3, (d) is the electrophoresis diagram of the responsive double-network hydrogel in Example 1, (e) is the electrophoresis diagram of the responsive double-network hydrogel in Example 2, (f) is the electrophoresis diagram of the responsive double-network hydrogel in Example 3, and (g) is the electrophoresis diagram of the responsive double-network hydrogel in Example 4. In each electrophoresis diagram, the left lane from top to bottom is the DNA band of the standard molecular weight of 10k bp, 8k bp, 6k bp, 5k bp, 4k bp, 3k bp, 2kbp, 1.5k bp, 1k bp, 0.5k bp in turn, and the right lane from top to bottom is the DNA band of the standard molecular weight of 1.517kbp, 1.2k bp, 1k bp, 0.9k bp, 0.8k bp, 0.7k bp, 0.6k bp, 0.5 / 0.517bp, 0.4k bp,0.3k bp, 0.2k bp, 0.1k bp in turn.

[0044] From Figure 2 it can be seen that the range and size of DNA sample separation are regulated by the gel network density and size. The double-network hydrogels added with 1-4% pNIPAM can all make the DNA fragments of 100-10000 bp migrate into the gel interior under the action of current. Compared with the agarose (AG) single-network hydrogel, 0.5% AG-2% pNIPAM (Example 2) and 0.5% AG-3% pNIPAM (Example 3) have higher resolution for the 100-1517 bp DNA fragment and the band gradient is clearer.

[0045] (2) Circular gel blocks with a diameter of 1.5 cm were taken from the AG-pNIPAM double-network hydrogels prepared in Examples 1-4 and the agarose single-network hydrogels prepared in Comparative Examples 1-3, and the diameter changes at different temperatures were compared to characterize the temperature responsiveness. Each group of gel blocks was kept at room temperature, 30 °C, 35 °C, and 40 °C for 10 min, and the diameters of the gel blocks were measured and statistically analyzed. The results were expressed as mean ± standard error. The appearance and diameter change histograms of each gel block are shown as Figure 3 shown. Among them, Figure 3 in (a) are the appearance diagrams of the responsive double-network hydrogels in Examples 1-4 and the agarose single-network hydrogels in Comparative Examples 1-3 in response to temperature changes; Figure 3 in (b) is the diameter change histogram of the responsive double-network hydrogel in Example 1, (c) is the diameter change histogram of the responsive double-network hydrogel in Example 2, (d) is the diameter change histogram of the responsive double-network hydrogel in Example 3, and (e) is the diameter change histogram of the responsive double-network hydrogel in Example 4. Figure 3 In (a), the numbers 1-4 are 0.5% AG-1% pNIPAM, 0.5% AG-2% pNIPAM, 0.5% AG-3% pNIPAM, and 0.5% AG-4% pNIPAM respectively; the numbers 5-7 are 0.5% AG, 0.7% AG, and 1% AG respectively.

[0046] It can be seen from Figure 3 that the agarose single-network hydrogels prepared in Comparative Examples 1-3 did not show obvious changes at the above temperatures. The temperature responsiveness of the AG-pNIPAM double-network hydrogels added with 1-4% pNIPAM increased with the increase in the content of pNIPAM.

[0047] (3) Gel blocks with a set mass were taken from the AG-pNIPAM double-network hydrogels prepared in Examples 1-4 and the agarose single-network hydrogels prepared in Comparative Examples 1-3, chopped, and placed in a centrifuge tube with a 20 μm pore size filter. The centrifuge tubes with the gel blocks were kept at room temperature, 30 °C, 35 °C, and 40 °C for a set time for temperature response. The small tubes were taken out every 5 min, centrifuged at low speed, and the mass of the small tubes was weighed. The cumulative response time was 30 min. The mass loss rate of the gel after temperature response was calculated as shown in Figure 4 shown, and the results were expressed as mean ± standard error. Among them, Figure 4 in (a) is the mass loss ratio diagram of Example 1, (b) is the mass loss ratio diagram of Example 2, (c) is the mass loss ratio diagram of Example 3, (d) is the mass loss ratio diagram of Example 4, (e) is the mass loss ratio diagram of Comparative Example 1, (f) is the mass loss ratio diagram of Comparative Example 2, and (g) is the mass loss ratio diagram of Comparative Example 3.

[0048] It can be seen from Figure 4 that for the agarose single-network hydrogel, the temperature has little effect on it. The mass loss rate is mainly related to the loss of water on the gel surface. For the AG-pNIPAM double-network hydrogel, at room temperature, the mass loss rate is related to the loss of water on the gel surface and decreases with the increase in the gel network density; as the temperature increases, the temperature responsiveness increases, manifested as the shrinkage of the gel volume and the increase in the extruded aqueous phase, that is, the mass loss rate increases.

[0049] (4)The DNA bands after electrophoresis of the AG-pNIPAM double-network hydrogels prepared in Examples 1-4 and the agarose single-network hydrogels prepared in Comparative Examples 1-3 were excised and recovered. 10 kbp, 3 kbp, 1 kbp, 0.5 kbp and 0.2 kbp bands were respectively selected for comparison of the DNA recovery rates between the gels. The DNA recovery rates are as Figure 5 shown, and the results are expressed as the mean ± standard error. Among them, Figure 5 in (a) is the histogram of the recovery rate of the 10 kbp band, (b) is the histogram of the recovery rate of the 3 kbp band, (c) is the histogram of the recovery rate of the 1 kbp band, (d) is the histogram of the recovery rate of the 0.5 kbp band, and (e) is the histogram of the recovery rate of the 0.2 kbp band. It can be seen from Figure 5It can be seen that compared with the 0.5%, 0.7% and 1% AG single-network hydrogels, the DNA recovery rates of the AG-pNIPAM double-network hydrogels have been improved to varying degrees. For the 10 kbp DNA fragment, the DNA recovery rates of 0.5% AG (Comparative Example 1) and 0.5% AG-1% pNIPAM hydrogel (Example 1) are 17.20±1.59% and 24.07±1.97% respectively. For the 3 kbp DNA fragment, the DNA recovery rates of 0.5% AG (Comparative Example 1), 0.7% AG (Comparative Example 2), 1% AG (Comparative Example 3) and 0.5% AG-1% pNIPAM hydrogel (Example 1) are 19.53±4.68%, 25.84±3.11%, 19.99±1.90% and 32.78±1.94% respectively. For the 1 kbp DNA fragment, the DNA recovery rates of 0.5% AG (Comparative Example 1), 0.7% AG (Comparative Example 2), 1% AG (Comparative Example 3), 0.5% AG-1% pNIPAM (Example 1), 0.5% AG-2% pNIPAM (Example 2) and 0.5% AG-3% pNIPAM (Example 3) hydrogels are 29.60±4.42%, 29.16±1.45%, 18.73±2.58%, 36.42±2.66%, 34.70±1.29% and 53.32±5.46% respectively. For the 0.5 kbp DNA fragment, the DNA recovery rates of 0.7% AG (Comparative Example 2), 1% AG (Comparative Example 3), 0.5% AG-1% pNIPAM (Example 1), 0.5% AG-2% pNIPAM (Example 2), 0.5% AG-3% pNIPAM (Example 3) and 0.5% AG-4% pNIPAM hydrogel (Example 4) are 26.14±1.25%, 25.54±1.36%, 37.36±4.45%, 52.66±1.88%, 74.09±4.76% and 73.47±1.76% respectively. For the 0.2 kbp DNA fragment, the DNA recovery rates of 0.5% AG-1% pNIPAM (Example 1), 0.5% AG-2% pNIPAM (Example 2), 0.5% AG-3% pNIPAM (Example 3) and 0.5% AG-4% pNIPAM hydrogel (Example 4) are 38.17±5.19%, 45.39±2.02%, 63.22±5.54% and 75.06±0.77% respectively. Therefore, the AG-pNIPAM double-network hydrogel has a significantly improved recovery effect on DNA fragments.

[0050] (5) The DNA bands after electrophoresis of the AG-pNIPAM double-network hydrogel prepared in Example 3 and the agarose single-network hydrogel prepared in Comparative Example 1 were excised and recovered. The DNA recovery rates between gels of the DNA self-assembled nanostructures of 4-helix bundle (4HB) and 8-helix bundle (8HB) were compared. The DNA bands and recovery rates are as Figure 6 shown, and the results are expressed as mean ± standard error. Among them, Figure 6 in (a) is the gel electrophoresis diagram of Example 3, and (b) is the gel electrophoresis diagram of Comparative Example 1. Among them, the numbers 1 and 3 are the gel electrophoresis bands of the DNA self-assembled structure of 4HB, and the numbers 2 and 4 are the gel electrophoresis bands of the DNA self-assembled structure of 8HB; Figure 6 in (c) is the histogram of the recovery rate.

[0051] From Figure 6 it can be seen that both the 0.5% AG single-network hydrogel (Comparative Example 1) and the 0.5% AG-3% pNIPAM double-network hydrogel (Example 3) can present clear bands. The recovery rates of the 0.5% AG single-network hydrogel for 4HB and 8HB are 17.24 ± 0.83% and 23.21 ± 1.03% respectively, while the recovery rates of the 0.5% AG-3% pNIPAM double-network hydrogel for 4HB and 8HB are 24.75 ± 0.69% and 31.38 ± 1.58% respectively. Therefore, the AG-pNIPAM double-network hydrogel has a significantly improved recovery effect on the DNA self-assembled structure.

[0052] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A responsive double-network hydrogel, characterized in that It has a double-network structure in which a first rigid network formed by agarose and a second flexible network formed by the polymerization of N-isopropylacrylamide monomers in the presence of a crosslinking agent penetrate each other; wherein, the mass fraction of agarose is 0.5-1 wt%, and the mass fraction of N-isopropylacrylamide is 1-4 wt%; the crosslinking agent is any one of N,N'-methylenebisacrylamide and polyethylene glycol diacrylate.

2. The preparation method of the responsive double-network hydrogel according to claim 1, characterized in that The responsive double-network hydrogel is made of agarose, N-isopropylacrylamide, an initiator, a crosslinking agent and a buffer solution. Among them, the completely dissolved agarose solution forms a first rigid network after cooling, and N-isopropylacrylamide forms a second flexible network through free radical polymerization under the action of a crosslinking agent and an initiator; the crosslinking agent is any one of N,N'-methylenebisacrylamide and polyethylene glycol diacrylate.

3. The preparation method according to claim 2, characterized in that, Specifically, it includes the following steps: Add agarose to the buffer solution, heat it until it is fully dissolved, then add N-isopropylacrylamide and a crosslinking agent. When the temperature is cooled to 1-2°C higher than the agarose gel freezing point, add an initiator, transfer the mixed solution into a mold. Agarose first forms a first rigid network through hydrogen bond interaction, and stands at 4-25°C to make N-isopropylacrylamide form a second flexible network through free radical polymerization reaction, and the responsive double-network hydrogel is prepared.

4. The preparation method according to claim 2, characterized in that, The initiator is any one or combination of ammonium persulfate, N,N,N',N'-tetramethylethylenediamine, potassium peroxydisulfate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and lithium phenyl(2,4,6-trimethylbenzoyl)phosphate.

5. The preparation method according to claim 2, characterized in that, The mass ratio of the crosslinking agent to N-isopropylacrylamide is 1:10-40.

6. The preparation method according to claim 2, wherein The components of the buffer solution include tris(hydroxymethyl)aminomethane, boric acid, ethylenediaminetetraacetic acid, acetic acid, sodium ions, magnesium ions and water; the mass ratio of agarose to N-isopropylacrylamide is 1:(1-8).

7. The preparation method according to claim 3, characterized in that, The agarose includes ordinary melting point agarose and low melting point agarose.

8. The preparation method according to claim 7, wherein When the mixed solution containing ordinary agarose or low melting point agarose is cooled to 25-31°C, add an initiator. After transferring to a mold, the temperature of the mixed solution drops to form a first rigid network of agarose, and polymerize at a temperature lower than the critical phase transition temperature of poly-N-isopropylacrylamide for 1-24 h to form a second flexible network.

9. The application of the responsive double-network hydrogel described in claim 1 or the responsive double-network hydrogel prepared by the preparation method described in any one of claims 2-8 in the field of nucleic acid separation and recovery.

10. The application according to claim 9, wherein, Specifically, it includes the following steps: (a) Put the responsive double-network hydrogel into an electrophoresis tank, add a nucleic acid sample, and perform electrophoresis at a voltage of 50-150V for 1-180 min. After electrophoresis, stain the nucleic acid. (b) Observe the nucleic acid bands in the gel under a blue light lamp or an ultraviolet lamp, and cut out the target band. (c) Chop the gel block containing the target band and transfer it into a container with a filter screen of a set pore size. (d) Place the container containing the gel block in an environment of 30 - 40 °C for 5 - 30 min. The liquid extruded due to the responsive contraction of the gel block is the nucleic acid recovery solution; (e) Collect the nucleic acid recovery solution, and calculate the nucleic acid concentration and sample recovery rate in the recovery solution according to the ultraviolet absorption or fluorescence intensity signal.

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