A responsive double-network hydrogel and its preparation method and application

Abstract

The present invention provides a responsive double-network hydrogel and its preparation method and application, which relate to the field of nucleic acid separation and recovery. This hydrogel is composed of an agarose gel network and a poly (N-isopropylacrylamide) network. By adjusting the ratio of agarose and poly (N-isopropylacrylamide), the separation resolution of nucleic acids during electrophoresis can be enhanced, and the separation range of nucleic acids of different molecular weights can be regulated. The temperature sensitivity of poly (N-isopropylacrylamide) enables the double-network hydrogel to have thermal responsiveness. When heated above its lower critical phase transition temperature (~32°C), the double-network hydrogel can squeeze out the aqueous phase in the gel through the phase transition of poly (N-isopropylacrylamide), thereby releasing the nucleic acids therein without destroying the hydrogel network, simplifying the nucleic acid recovery operation, and improving the nucleic acid recovery efficiency and sample recovery rate. This method is suitable for the separation and recovery of various nucleic acid fragments and nucleic acid-assembled nanostructures.

Description

Technical Field

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

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Agarose gel electrophoresis is one of the core technologies for separating and identifying nucleic acid molecules. Its separation principle is based on the size and conformational differences of nucleic acid molecules. During electrophoresis, negatively charged DNA molecules migrate toward the anode under the action of the electric field, and the three-dimensional network structure formed by the agarose gel acts as a molecular sieve, allowing smaller DNA fragments to migrate faster, thereby achieving separation. Combined with fluorescent labeling of nucleic acid dyes such as ethidium bromide (EB), electrophoretic bands can be directly observed through ultraviolet excitation, and the size of the target fragment can be estimated based on standard molecular weight markers. Due to its ease of operation, low cost and high resolution, agarose gel electrophoresis has become a basic experimental method in molecular biology, genetic engineering and nucleic acid chemistry research.

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

[0005] Chemical dissolution methods typically use high concentrations of chaotropic salts (such as sodium iodide or sodium perchlorate) to dissolve agarose, followed by purification of DNA using silica gel columns or alcohol precipitation. However, this method can introduce chemical residues that can affect downstream experiments, and recovery efficiency is low, particularly when processing large fragments or complex DNA structures. Among physical disruption methods, high-temperature melting (>60°C) can rapidly dissolve standard or low-melting-point agarose, but this heat can denature DNA or disrupt its higher-order structure (such as the stability of DNA origami). Freeze-extrusion, while avoiding high temperatures, is cumbersome and prone to contamination with gel fragments, reducing the purity of the recovered product. Electroelution, which uses a reversed electric field to migrate DNA into a dialysis bag, is susceptible to DNA adsorption to the bag walls, resulting in low recovery rates. The complex procedure also hinders scalable application. Agarose enzyme digestion, a milder alternative, can enzymatically release DNA from the agarose matrix, avoiding high temperatures and chemical residues. However, complete digestion requires long incubation times and the high cost of the enzyme limits its widespread use.

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

[0007] In response to the shortcomings of the prior art, the present invention aims to provide a responsive double-network hydrogel, its preparation method, and its 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. Furthermore, the responsive double-network hydrogel is temperature-responsive, capable of releasing the aqueous phase and DNA from the gel matrix, with high DNA recovery efficiency, simple operation, and mild conditions.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] In its first aspect, the present invention provides a responsive dual-network hydrogel comprising a first rigid network formed by agarose and a second flexible network formed by polymerization of N-isopropylacrylamide monomers in the presence of a crosslinker. The agarose (AG) first rigid network provides the hydrogel with a three-dimensional network and mechanical properties, while poly (N-isopropylacrylamide) (pNIPAM) provides temperature responsiveness as a second flexible network. The combination of these two networks, with different structures and densities, effectively regulates the pore size and density of the gel, enabling high-resolution separation and efficient recovery of DNA fragments.

[0010] In some embodiments, the cross-linking agent is any one of N,N′-methylenebisacrylamide (BIS) and polyethylene glycol diacrylate (PEGDA).

[0011] In some embodiments, the responsive double-network hydrogel has a mass fraction of agarose ranging from 0.5 to 1.0 wt %, and a mass fraction of N-isopropylacrylamide ranging from 1 to 4 wt %. Adjusting the ratio of agarose to poly(N-isopropylacrylamide) can control the pore size of the gel network. Experiments have demonstrated that the aforementioned amounts of agarose and N-isopropylacrylamide provide excellent DNA fragment separation and high resolution.

[0012] 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 cross-linker and a buffer solution, wherein 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 the cross-linker and the initiator.

[0013] In some embodiments, the method for preparing the responsive double-network hydrogel specifically comprises the following steps:

[0014] Agarose is added to a buffer solution and heated until fully dissolved, followed by addition of N-isopropylacrylamide and a cross-linking agent. The solution is cooled to a temperature 1-2°C higher than the solidification point of the agarose gel, and then an initiator is added. The mixed solution is immediately transferred into a mold, where the agarose first forms a first rigid network through hydrogen bond interactions. The solution is then allowed to stand at 4-25°C, allowing the N-isopropylacrylamide to form a second flexible network through free radical polymerization, thereby preparing the responsive dual-network hydrogel.

[0015] In some embodiments, the agarose includes normal melting point agarose and low melting point agarose.

[0016] In some embodiments, the N-isopropylacrylamide is a commercially available N-isopropylacrylamide product that is recrystallized into an N-isopropylacrylamide monomer.

[0017] In some embodiments, the initiator is any one or a combination of ammonium persulfate (APS), potassium peroxodisulfate (KPS), N,N,N',N'-tetramethylethylenediamine (TEMED), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (photoinitiator 2959) and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (photoinitiator LAP).

[0018] In some embodiments, the cross-linking agent is any one of N,N′-methylenebisacrylamide (BIS) and polyethylene glycol diacrylate (PEGDA), and the mass ratio of the cross-linking agent to N-isopropylacrylamide is 1:10-40.

[0019] In some embodiments, the buffer comprises tris(hydroxymethyl)amine, boric acid, ethylenediaminetetraacetic acid, acetic acid, sodium ions, magnesium ions, and water. Tris(hydroxymethyl)amine is primarily used to maintain pH stability, forming a buffer pair with acetic acid or boric acid to counteract pH changes caused by electrolysis during electrophoresis and prevent nucleic acid denaturation due to pH fluctuations. EDTA chelates metal ions such as magnesium to prevent nucleic acid degradation.

[0020] In some embodiments, an initiator is added to a mixed solution containing ordinary agarose or low-melting point agarose after cooling to 25-31°C. After being transferred to a mold, the temperature of the mixed solution drops, rapidly forming a first rigid agarose network, which is then polymerized for 1-24 h at a temperature below the critical phase transition temperature of poly (N-isopropylacrylamide) to form a second flexible network.

[0021] In some embodiments, the mass ratio of agarose to N-isopropylacrylamide is 1:(1-8).

[0022] In a third aspect, the present invention provides an application 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.

[0023] In some embodiments, the application of the responsive double-network hydrogel in the field of nucleic acid separation and recovery specifically includes the following steps:

[0024] (a) The responsive double-network hydrogel was placed in an electrophoresis tank, a nucleic acid sample was added, and electrophoresis was performed at a voltage of 50-150 V for 1-180 min. After the electrophoresis, the nucleic acid was stained.

[0025] (b) Observe the nucleic acid bands in the gel under blue or UV light and cut out the target bands. The combination of agarose and poly (N-isopropylacrylamide) networks effectively controls the size and density of the gel pores, thereby achieving high-resolution separation of DNA fragments.

[0026] (c) chopping the gel block containing the target band and transferring it into a container with a filter of a set pore size;

[0027] (d) The container containing the gel block is placed in an environment at 30-40°C for 5-30 minutes. Since poly (N-isopropylacrylamide) provides temperature responsiveness, the gel block can responsively shrink under these conditions to squeeze out the aqueous phase and DNA, thereby separating the nucleic acid recovery solution.

[0028] (e) Collect the nucleic acid recovery solution and calculate the nucleic acid concentration and sample recovery rate in the recovery solution based on the UV absorbance or fluorescence intensity signal.

[0029] In some embodiments, the molecular weight of the standard nucleic acid that enters the gel by electrophoresis ranges from 100 to 10,000 bp.

[0030] The beneficial effects of the present invention are:

[0031] (1) The present invention utilizes agarose and poly (N-isopropylacrylamide) to construct a double-network hydrogel, which can separate DNA fragments and DNA self-assembled nanostructures under the action of electrophoresis, with good separation effect and high resolution.

[0032] (2) The double-network hydrogel of the present invention is temperature-responsive and can release the aqueous phase and DNA components in the gel matrix. It has high DNA sample recovery efficiency, simple operation, and mild conditions. It has good application prospects in the field of separation and recovery of DNA fragments and DNA self-assembled nanostructures. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 Schematic diagram of the synthesis of the responsive double-network hydrogel and the separation and recovery of DNA after gel electrophoresis in Examples 1-4; (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.

[0035] Figure 2 The figures are the gel electrophoresis patterns of the responsive double network hydrogels in Examples 1-4 and the agarose single network hydrogels in Comparative Examples 1-3; wherein, (a) is the gel electrophoresis pattern of the agarose single network hydrogel in Comparative Example 1, (b) is the gel electrophoresis pattern of the agarose single network hydrogel in Comparative Example 2, (c) is the gel electrophoresis pattern of the agarose single network hydrogel in Comparative Example 3, (d) is the electrophoresis pattern of the responsive double network hydrogel in Example 1, (e) is the electrophoresis pattern of the responsive double network hydrogel in Example 2, (f) is the electrophoresis pattern of the responsive double network hydrogel in Example 3, and (g) is the electrophoresis pattern of the responsive double network hydrogel in Example 4.

[0036] Figure 3Appearance images and diameter change histograms 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, wherein (a) is an appearance image 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; (b) is a diameter change histogram of the responsive double network hydrogel in Example 1, (c) is a diameter change histogram of the responsive double network hydrogel in Example 2, (d) is a diameter change histogram of the responsive double network hydrogel in Example 3, and (e) is a diameter change histogram of the responsive double network hydrogel in Example 4.

[0037] Figure 4 The figures are mass loss ratio graphs 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, wherein (a) is the mass loss ratio graph of Example 1, (b) is the mass loss ratio graph of Example 2, (c) is the mass loss ratio graph of Example 3, (d) is the mass loss ratio graph of Example 4, (e) is the mass loss ratio graph of Comparative Example 1, (f) is the mass loss ratio graph of Comparative Example 2, and (g) is the mass loss ratio graph of Comparative Example 3.

[0038] Figure 5 The following are histograms of DNA recovery rates for the agarose single-network hydrogels in Comparative Examples 1-3 and the responsive double-network hydrogels in Examples 1-4. (a) shows the recovery rate histogram for the 10 kbp band, (b) shows the recovery rate histogram for the 3 kbp band, (c) shows the recovery rate histogram for the 1 kbp band, (d) shows the recovery rate histogram for the 0.5 kbp band, and (e) shows the recovery rate histogram for the 0.2 kbp band.

[0039] Figure 6 The gel electrophoresis images and recovery rate histograms of the DNA self-assembled structures of 4HB and 8HB separated by the 0.5% agarose single-network hydrogel in Comparative Example 1 and the responsive double-network hydrogel in Example 3, wherein (a) is the gel electrophoresis image of Example 3, and (b) is the gel electrophoresis image of Comparative Example 1. Figure 6 (c) is the recovery rate histogram. DETAILED DESCRIPTION

[0040] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0041] The technical solution of the present invention is further described below with reference to specific embodiments.

[0042] Example 1

[0043] Preparation of responsive double-network hydrogel: A set amount of ordinary melting point agarose was added to 20 mL of buffer (1xTBE), heated until fully dissolved, and then a set amount of N-isopropylacrylamide and crosslinker BIS (with a mass ratio of 1:29) were added. When the temperature was cooled to 1.5±0.5°C higher than the freezing point of the agarose gel, 100 μL of 10 wt% APS solution and 20 μL of N,N,N',N'-tetramethylethylenediamine (TEMED) were added. The mixture was shaken and immediately transferred into a mold. The agarose first formed a first rigid network through hydrogen bond interaction. The mixture was allowed to stand at 4°C to allow N-isopropylacrylamide to form a second flexible network through free radical polymerization, thereby preparing the responsive double-network hydrogel, wherein the mass fraction of agarose was 0.5 wt% and the mass fraction of N-isopropylacrylamide was 1 wt%.

[0044] Example 2

[0045] The only difference between the preparation method of the responsive double-network hydrogel in this embodiment and that in Example 1 is that the amount of N-isopropylacrylamide is 2 wt %.

[0046] Example 3

[0047] The only difference between the preparation method of the responsive double-network hydrogel in this embodiment and that in Example 1 is that the amount of N-isopropylacrylamide is 3 wt %.

[0048] Example 4

[0049] The only difference between the preparation method of the responsive double-network hydrogel in this embodiment and that in Example 1 is that the amount of N-isopropylacrylamide is 4 wt %.

[0050] The schematic diagrams of the synthesis of the responsive double network hydrogel and the separation and recovery of DNA by gel electrophoresis in Examples 1-4 are shown in FIG. Figure 1 (a) and Figure 1 As shown in (b).

[0051] Comparative Example 1

[0052] Preparation of agarose single network hydrogel: Add a set amount of ordinary melting point agarose to a buffer solution, heat to fully dissolve it, and cool to form an agarose gel, wherein the mass fraction of agarose is 0.5 wt%.

[0053] Comparative Example 2

[0054] Preparation of agarose single network hydrogel: A set amount of ordinary melting point agarose was added to a buffer solution, heated to fully dissolve it, and cooled to form an agarose gel, wherein the mass fraction of agarose was 0.7 wt%.

[0055] Comparative Example 3

[0056] Preparation of agarose single network hydrogel: Add a set amount of ordinary melting point agarose to a buffer solution, heat to fully dissolve it, and cool to form an agarose gel, wherein the mass fraction of agarose is 1.0 wt%.

[0057] Performance Analysis

[0058] (1) Electrophoresis was performed on the agarose-poly N-isopropylacrylamide (AG-pNIPAM) double network hydrogel prepared in Example 1-4 and the agarose (AG) single network hydrogel prepared in Comparative Example 1-3. The samples were 500-10000 bp DNA ladder and 100-1517 bp DNA ladder. The electrophoresis conditions were 60V and 120min. The results are shown in Figure 2. Figure 2 As shown. Among them, Figure 2 (a) is the gel electrophoresis image of the agarose single network hydrogel in Comparative Example 1, (b) is the gel electrophoresis image of the agarose single network hydrogel in Comparative Example 2, (c) is the gel electrophoresis image of the agarose single network hydrogel in Comparative Example 3, (d) is the electrophoresis image of the responsive double network hydrogel in Example 1, (e) is the electrophoresis image of the responsive double network hydrogel in Example 2, (f) is the electrophoresis image of the responsive double network hydrogel in Example 3, and (g) is the electrophoresis image of the responsive double network hydrogel in Example 4. The left lane of each electropherogram, from top to bottom, is the DNA bands with standard molecular weights of 10k bp, 8k bp, 6k bp, 5k bp, 4k bp, 3k bp, 2kbp, 1.5k bp, 1k bp, and 0.5k bp. The right lane, from top to bottom, is the DNA bands with standard molecular weights 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, and 0.1k bp.

[0059] from Figure 2 It can be seen that the range and size of DNA sample separation are controlled by the density and size of the gel network. Double-network hydrogels with 1-4% pNIPAM additions can allow DNA fragments ranging from 100 to 10,000 bp to migrate into the gel under the action of current. Compared to agarose (AG) single-network hydrogels, 0.5% AG-2% pNIPAM (Example 2) and 0.5% AG-3% pNIPAM (Example 3) achieve higher resolution for DNA fragments ranging from 100 to 1517 bp, with clearer band gradients.

[0060] (2) Circular gel blocks with a diameter of 1.5 cm were taken from the AG-pNIPAM double network hydrogel prepared in Example 1-4 and the agarose single network hydrogel prepared in Comparative Example 1-3, and their 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 histogram of each gel block is shown in Figure 2. Figure 3 As shown. Among them, Figure 3 (a) is an appearance diagram of the responsive double-network hydrogel in Example 1-4 and the agarose single-network hydrogel in Comparative Example 1-3 in response to temperature changes; Figure 3 (b) is the histogram of the diameter change of the responsive double network hydrogel in Example 1, (c) is the histogram of the diameter change of the responsive double network hydrogel in Example 2, (d) is the histogram of the diameter change of the responsive double network hydrogel in Example 3, and (e) is the histogram of the diameter change of the responsive double network hydrogel in Example 4. Figure 3 In (a), 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; numbers 5-7 are 0.5% AG, 0.7% AG, and 1% AG, respectively.

[0061] from Figure 3 As can be seen from the results of Comparative Examples 1-3, the agarose single network hydrogels prepared showed no significant changes at the above temperatures. The temperature responsiveness of the AG-pNIPAM double network hydrogels with 1-4% pNIPAM added increased with increasing pNIPAM content.

[0062] (3) A set mass of gel blocks were removed from the AG-pNIPAM double network hydrogel prepared in Example 1-4 and the agarose single network hydrogel prepared in Comparative Example 1-3, chopped, and placed in a centrifuge tube containing a 20 μm pore size filter. The centrifuge tubes with gel blocks were kept at room temperature, 30°C, 35°C, and 40°C for a set time to perform temperature response. The tubes were removed every 5 minutes, and the mass of the tubes was weighed after low-speed centrifugation. The cumulative response time was 30 minutes. The mass loss rate of the gel after temperature response was calculated as follows: Figure 4 The results are shown as mean ± standard error. Figure 4 (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, and (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.

[0063] from Figure 4 As can be seen from the figure, temperature has little effect on agarose single-network hydrogels. The mass loss rate is primarily related to water loss from the gel surface. For AG-pNIPAM dual-network hydrogels, at room temperature, the mass loss rate is related to water loss from the gel surface and decreases with increasing gel network density. However, as temperature increases, the temperature responsiveness increases, manifesting as a decrease in gel volume and an increase in water phase extrusion, i.e., an increase in mass loss rate.

[0064] (4) DNA bands were recovered by gel excision after electrophoresis of the AG-pNIPAM double network hydrogel prepared in Example 1-4 and the agarose single network hydrogel prepared in Comparative Example 1-3. Bands of 10k bp, 3k bp, 1k bp, 0.5k bp and 0.2k bp were selected to compare the DNA recovery rates between the gels. The DNA recovery rates were as follows: Figure 5 The results are shown as mean ± standard error. Figure 5 (a) is the recovery rate histogram of the 10k bp band, (b) is the recovery rate histogram of the 3k bp band, (c) is the recovery rate histogram of the 1k bp band, (d) is the recovery rate histogram of the 0.5k bp band, and (e) is the recovery rate histogram of the 0.2k bp band. Figure 5It can be seen that the DNA recovery rate of AG-pNIPAM double network hydrogel is improved to varying degrees compared with 0.5%, 0.7% and 1% AG single network hydrogels. For a 10 k bp DNA fragment, the DNA recovery rates of 0.5% AG (Comparative Example 1) and 0.5% AG-1% pNIPAM hydrogel (Example 1) were 17.20 ± 1.59% and 24.07 ± 1.97%, respectively. For a 3 k bp 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) were 19.53 ± 4.68%, 25.84 ± 3.11%, 19.99 ± 1.90% and 32.78 ± 1.94%, respectively. The DNA recovery rates of pNIPAM (Example 2) and 0.5% AG-3% pNIPAM (Example 3) hydrogels were 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 a DNA fragment of 0.5 k bp, 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% The DNA recovery rates of the pNIPAM hydrogel (Example 4) were 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 a 0.2 kbp DNA fragment, the DNA recovery rates of the 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) were 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 significantly improved the recovery of DNA fragments.

[0065] (5) DNA bands were recovered by gel cutting after electrophoresis of the AG-pNIPAM double network hydrogel prepared in Example 3 and the agarose single network hydrogel prepared in Comparative Example 1. The DNA recovery rates between gels were compared for the DNA self-assembled nanostructures of 4 helix bundles (4HB) and 8 helix bundles (8HB). The DNA bands and recovery rates were shown in Figure 2. Figure 6 The results are shown as mean ± standard error. Figure 6 (a) is the gel electrophoresis image of Example 3, and (b) is the gel electrophoresis image of Comparative Example 1, wherein the gel electrophoresis bands numbered 1 and 3 are the gel electrophoresis bands of the DNA self-assembly structure of 4HB, and the gel electrophoresis bands numbered 2 and 4 are the gel electrophoresis bands of the DNA self-assembly structure of 8HB; Figure 6 (c) is the recovery rate histogram.

[0066] from Figure 6 As can be seen, both the 0.5% AG single-network hydrogel (Comparative Example 1) and the 0.5% AG-3% pNIPAM double-network hydrogel (Example 3) exhibit clear bands. The 0.5% AG single-network hydrogel exhibited recoveries of 17.24±0.83% and 23.21±1.03% for 4HB and 8HB, respectively, while the 0.5% AG-3% pNIPAM double-network hydrogel exhibited recoveries of 24.75±0.69% and 31.38±1.58% for 4HB and 8HB, respectively. Therefore, the AG-pNIPAM double-network hydrogel significantly improves the recovery of DNA self-assembled structures.

[0067] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Application of a responsive double-network hydrogel in the field of nucleic acid separation and recovery, characterized in that: The invention has a double network structure in which a first rigid network formed by agarose and a second flexible network formed by polymerization of N-isopropylacrylamide monomers in the presence of a cross-linking agent interpenetrate 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 cross-linking agent is any one of N,N'-methylenebisacrylamide and polyethylene glycol diacrylate; and the mass ratio of the cross-linking agent to N-isopropylacrylamide is 1:10-40.

2. The use according to claim 1, characterized in that The specific steps include: (a) The responsive double-network hydrogel is placed in an electrophoresis tank, a nucleic acid sample is added, and electrophoresis is performed at a voltage of 50-150 V for 1-180 min. After the electrophoresis, the nucleic acid is stained. (b) Observe the nucleic acid bands in the gel under blue light or UV light and cut out the target band; (c) chopping the gel block containing the target band and transferring it into a container with a filter of a set pore size; (d) Place the container containing the gel block at 30-40°C for 5-30 minutes. The liquid squeezed out by the gel block's responsive shrinkage 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 based on the UV absorbance or fluorescence intensity signal.

3. The use according to claim 1, characterized in that The responsive double-network hydrogel is made of agarose, N-isopropylacrylamide, an initiator, a cross-linking agent and a buffer. The completely dissolved agarose solution forms a first rigid network after cooling, and the N-isopropylacrylamide forms a second flexible network through free radical polymerization under the action of the cross-linking agent and the initiator.

4. The use according to claim 3, characterized in that The preparation method of the responsive double-network hydrogel specifically includes the following steps: adding agarose to a buffer solution, heating it until it is fully dissolved, adding N-isopropylacrylamide and a cross-linking agent, cooling it to a temperature 1-2°C higher than the solidification point of the agarose gel, adding an initiator, and transferring the mixed solution into a mold. The agarose first forms a first rigid network through hydrogen bond interaction, and the mixture is allowed to stand at 4-25°C to allow the N-isopropylacrylamide to form a second flexible network through a free radical polymerization reaction, thereby preparing the responsive double-network hydrogel.

5. The use according to claim 3, characterized in that The initiator is any one or a combination of ammonium persulfate, potassium peroxodisulfate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.

6. The use according to claim 3, characterized in that The components of the buffer solution include aminotrihydroxymethylmethane, 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 use according to claim 4, characterized in that The agarose includes common melting point agarose and low melting point agarose.

8. The use according to claim 7, characterized in that The initiator is added to a mixed solution containing ordinary agarose or low-melting-point agarose after cooling to 25-31°C. After being transferred to a mold, the temperature of the mixed solution drops to form a first rigid agarose network, which is then polymerized for 1-24 hours at a temperature lower than the critical phase transition temperature of poly (N-isopropylacrylamide) to form a second flexible network.

Images