Preparation method and application of ultra-light high-nucleic-acid-absorbing electrospun fiber sponge material

Abstract

The application belongs to the field of functional material preparation, and particularly relates to a preparation method of a super-light high-nucleic-acid-absorption electrospun fiber sponge material and application thereof. The SiO2 electrospun fiber is used as a framework, a three-dimensional monolithic structure assembled by fragmented electrospun nanofiber membranes is used, the interconnected nanofiber cell structures of which endow the three-dimensional monolithic structure with high porosity and excellent structural flexibility and stability. Meanwhile, the amino-rich polyethylene imine and chitosan are chemically crosslinked by using epichlorohydrin as a crosslinking agent, and finally during freeze-drying, the ice crystals are partially sublimated, leaving a pore structure, and a porous sponge material is obtained. The material has a high nucleic acid adsorption capacity, and makes nucleic acid extraction more simple, rapid and sensitive.

Description

Technical Field

[0001] This invention belongs to the field of functional material preparation, specifically relating to a method for preparing an ultralight high nucleic acid adsorption electrospun fiber sponge material and its application. Background Technology

[0002] Nucleic acid detection technology is a detection method based on the molecular biological characteristics of nucleic acids. It achieves precise determination of pathogens or gene variations through the amplification and recognition mechanisms of specific sequences. Its application value is also evident in food safety monitoring, where the analysis of nucleic acid components in food samples can determine the status of microbial contamination. The rapid identification of foodborne pathogens such as Salmonella and Escherichia coli is a typical application scenario of this technology, providing results 24-72 hours earlier than traditional culture methods. The specific identification of exogenous gene fragments in genetically modified foods also relies on this technology, thus safeguarding consumers' right to choose products.

[0003] Traditional nucleic acid extraction methods include guanidinium isothiocyanate-phenol-chloroform extraction, EtBr-CsCl gradient centrifugation, CTAB extraction, silica-based material adsorption, and magnetic bead adsorption. However, when dealing with complex food samples, due to the complex food background and low bacterial concentration, the disadvantages of traditional methods become increasingly apparent. These include the large use of volatile organic solvents, time-consuming operation, reliance on expensive equipment, and cumbersome procedures. Furthermore, existing rapid detection methods are not suitable for on-site applications. Developing a material for simpler, faster, more sensitive nucleic acid extraction with high nucleic acid adsorption capacity has become an urgent problem to be solved. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for preparing an ultralight electrospun fiber sponge material with high nucleic acid adsorption.

[0005] This invention also provides the application of the above-mentioned electrospun fiber sponge material in the efficient extraction of nucleic acids.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0007] This invention provides a method for preparing an ultralight electrospun fiber sponge material with high nucleic acid adsorption, comprising the following steps:

[0008] (1) SiO2 sol solution: Add a certain mass ratio of TEOS and H3PO4 to deionized water, stir magnetically for 12 h to obtain SiO2 sol solution;

[0009] (2) PVA solution: Dissolve PVA powder in deionized water and stir magnetically to obtain a PVA solution;

[0010] (3) Spinning solution: SiO2 sol solution and PVA solution are mixed and magnetically stirred to obtain spinning solution;

[0011] (4) Electrospinning the spinning solution to obtain nanofibers, and calcining the spun nanofibers in a muffle furnace to obtain SiO2 nanofibers.

[0012] (5) Cut the SiO2 nanofibers into small pieces, transfer them to deionized water, and homogenize them to obtain a SiO2 nano suspension. Add the SiO2 nano suspension to the chitosan solution and stir magnetically until homogeneous. Then add epichlorohydrin and PEI solution and continue stirring until a homogeneous mixture is formed. Subsequently, heat the mixture, pre-freeze the product, and freeze-dry it to obtain a porous sponge material.

[0013] Preferably, in step (1), the mass ratio of TEOS, H3PO4 and deionized water is 1-2:0.025:8.

[0014] Preferably, in step (1), the mixing and stirring time is 11-13 hours.

[0015] Preferably, in step (2), the concentration of the PVA solution is 5~15 wt%;

[0016] Preferably, in step (3), the mass ratio of the SiO2 sol solution to the PVA solution is 2:1 to 1:1, and the mixing and stirring time is 7-9 hours.

[0017] Preferably, in step (4), the specific process of electrospinning is as follows: take a syringe, assemble a 20~22G stainless steel flat-mouth needle (inner diameter 0.61~0.41 mm), inject the spinning solution into the syringe and fix it on the spinneret; turn on the constant temperature environment control system and maintain the temperature at 25℃; connect the positive electrode to the needle and the negative electrode to the roller receiver, keep the distance between the needle and the roller surface at 15~20 cm, and at the same time start the roller to rotate at a constant speed of 200~300 rpm / min and apply a DC voltage of 17~20 kV; after operation, start the micro-injection pump and push the solution at a constant rate of 0.8~1.2 mL / h; then place the obtained spun membrane in a muffle furnace for calcination, and calcinate it from the ambient temperature to 600-800 ℃ at a heating rate of 5 ℃ / min for 2-4 h.

[0018] Preferably, in step (5), the concentration of the SiO2 nano-suspension is 30~50 mg / mL; the chitosan solution is prepared by dissolving 40~120 mg of chitosan in 4 mL of acetic acid solution to obtain a chitosan solution concentration of 1~3 wt%; the concentration of the PEI solution is 3~5 wt%; the mass ratio of SiO2 nanofibers to chitosan is 1:4~5:4; the mass ratio of chitosan to PEI is 2:1~2:5; and the amount of epichlorohydrin added is 200~400 μL.

[0019] Preferably, in step (5), the magnetic stirring time is 20-40 min; the stirring time is 20-40 min; the heating is placed at 50-60℃ for 4-5 h; the pre-freezing temperature is pre-freezing at -20℃ for 8-16 h; and the freeze-drying is drying at -60~-80℃ for 12-24 h.

[0020] This invention also provides an application of the sponge material prepared by the above preparation method in the efficient extraction of nucleic acids.

[0021] This invention uses electrospinning technology to prepare nanofibers;

[0022] The preparation method provided by this invention can produce nanofiber membranes with high porosity and large specific surface area, providing an ideal adsorption matrix for bioseparation. Simultaneously, in-situ chemical modification technology can significantly enhance the adsorption performance of the material by introducing functional groups. Furthermore, compared to traditional solid-phase adsorbents, polymer nanofiber membranes offer advantages due to their low cost, good flexibility, and excellent processability.

[0023] The beneficial effects of this invention are as follows:

[0024] (1) This invention uses SiO2 electrospun fibers as the skeleton and assembles a three-dimensional integral structure using fragmented electrospun nanofiber membranes. The interconnected nanofiber cell structure endows it with extremely high porosity and excellent structural flexibility and stability. At the same time, epichlorohydrin is used as a crosslinking agent to chemically crosslink amino-rich polyethyleneimine and chitosan. Finally, during freeze-drying, the ice crystals sublimate, leaving a porous structure, to obtain a porous sponge material. This material has a high nucleic acid adsorption capacity, making nucleic acid extraction simpler, faster, and more sensitive.

[0025] (2) The material prepared in this invention is modified to be rich in amino groups. Under acidic conditions, it forms a stable bond with nucleic acid molecules through electrostatic adsorption. Under alkaline conditions, a low-salt buffer solution is used to disrupt the electrostatic adsorption of nucleic acid molecules by the material, releasing the nucleic acid molecules. This material adsorbs nucleic acids through electrostatic action, combined with porous physical adsorption, which can greatly reduce the nucleic acid extraction time, thereby achieving efficient adsorption of nucleic acids and is expected to be used in the field for nucleic acid detection. Attached Figure Description

[0026] Figure 1 A flowchart for preparing sponge materials;

[0027] Figure 2 Comparison of electrospinning conditions before and after optimization; where A: residue of the film on tin foil before optimization, B: SiO2 nanofiber film after optimization, and C: residue of the film on tin foil after optimization.

[0028] Figure 3 A photograph of the PEI / CS / SiO2 sponge material prepared in Example 1;

[0029] Figure 4 The infrared spectrum of the sponge material PEI / CS / SiO2 prepared in Example 1;

[0030] Figure 5 Scanning electron microscope (SEM) images of the sponge material PEI / CS / SiO2 prepared in Example 1; where A represents the surface morphology of PEI / CS / SiO2, and B represents the surface morphology of PEI / CS / SiO2 after adsorption of nucleic acids.

[0031] Figure 6 Optimization of the adsorption pH of PEI / CS / SiO2 in the sponge material PEI / CS / SiO2 prepared in Example 1;

[0032] Figure 7 Optimization of elution pH for the sponge material PEI / CS / SiO2 prepared in Example 1;

[0033] Figure 8 The adsorption time curve of the sponge material PEI / CS / SiO2 prepared in Example 1.

[0034] Figure 9 Determination of the maximum adsorption capacity of the sponge material PEI / CS / SiO2 prepared in Example 1

[0035] Figure 10 Fitting to a quasi-first-order dynamic model;

[0036] Figure 11 Fitting to a quasi-second-order dynamic model;

[0037] Figure 12 Comparison of nucleic acid adsorption capacity of the sponge material PEI / CS / SiO2 prepared in Example 1, the ECH-free PEI / CS / SiO2 gel material prepared in Comparative Example 1, and the CS / SiO2 gel material prepared in Comparative Example 2;

[0038] Figure 13 Scanning electron microscope image of the ECH-free PEI / CS / SiO2 gel material prepared for Comparative Example 1;

[0039] Figure 14 Scanning electron microscope image of the CS / SiO2 gel material prepared for Comparative Example 2. Detailed Implementation

[0040] The technical solution of the present invention will be further explained and illustrated below through specific embodiments.

[0041] Example 1

[0042] 1. Experiment and Materials

[0043] 1.1 Experimental Procedure

[0044] The overall process for preparing the sponge material according to this invention is as follows: Figure 1

[0045] 1.2 Experimental Section

[0046] 1.2.1 Reagents and Instruments

[0047] The bacterial genomic DNA extraction kit used in this experiment was purchased from Tiangen Biotech Co., Ltd.; tetraethyl orthosilicate (TEOS), polyethyleneimine (PEI, MW = 10000), and chitosan (CS, degree of deacetylation 95%) were purchased from Shanghai Aladdin Reagent Co., Ltd.; polyvinyl alcohol and epichlorohydrin were purchased from Shanghai Maclean Biotechnology Co., Ltd.; and tris(hydroxymethyl)aminomethane (Tris) was purchased from Shanghai Bioengineering Co., Ltd.

[0048] The main instruments used in this experiment are shown in Table 1.

[0049] Table 1 Main Instruments

[0050]

[0051] 1.2.2 Nucleic acid extraction and preparation

[0052] (1) Resuscitation and culture of Vibrio parahaemolyticus

[0053] Take a small amount of bacterial culture from the strain preserved in glycerol at -80℃, streak it on a TCBS nutrient agar plate, and incubate it in a 37℃ incubator for 12-16 hours. After the characteristic blue-green colony forms, use a sterile inoculation needle to pick a single colony with a clear edge and culture it in a shaker tube (the shaker tube contains LB liquid medium with 2.5% NaCl). Then, inoculate the culture from the shaker tube into LB liquid medium containing 2.5% NaCl at an inoculation ratio of 1:1000, and incubate overnight at 37℃ in a shaker for 12-16 hours to obtain a high concentration of bacterial culture for subsequent experiments.

[0054] (2) Extraction of Vibrio parahaemolyticus nucleic acid

[0055] DNA extraction: Follow the steps of the bacterial genomic DNA extraction kit. Use a centrifugal adsorption column that can specifically bind to DNA and a unique buffer system to extract bacterial genomic DNA. High-purity genomic DNA can be obtained within 1 hour.

[0056] RNA extraction: Extraction was performed using the Trizol method. Trizol is a novel RNA extraction reagent containing substances such as guanidine isothiocyanate, which can rapidly disrupt cells, inhibit the release of nucleases, and preserve the integrity of RNA. After adding chloroform and centrifuging, the sample separated into an aqueous layer and an organic layer, with RNA present in the aqueous layer. After collecting the aqueous layer, RNA could be precipitated using isopropanol.

[0057] The concentration and purity of the extracted DNA and RNA were determined using a Nanodrop ultra-micro UV spectrophotometer, and the measured samples were labeled and stored at -20℃ for subsequent experiments.

[0058] 1.2.3 Material Preparation

[0059] (1) Preparation of spinning solution

[0060] SiO2 sol solution: TEOS, H3PO4 and deionized water were mixed and stirred for 12 h at a mass ratio of 1:0.025:8 (5 g TEOS, 0.125 g H3PO4 and 40 g deionized water) to obtain SiO2 sol solution.

[0061] 10 wt% PVA solution: Dissolve 5 g of PVA powder in 45 g of deionized water under magnetic stirring at 80 °C, and stir for 1 h until the solution is homogeneous and stable.

[0062] Spinning solution: Equal weights of SiO2 sol solution and PVA solution were mixed and stirred for 8 hours (20 g of each) to obtain the spinning solution. The spinning solution was sealed and stored in a cool place for subsequent electrospinning.

[0063] (2) Preparation of SiO2 nanofibers by electrospinning

[0064] Electrospinning process parameters:

[0065] Take a 10 mL medical-grade polypropylene plastic syringe, equip it with a 22G stainless steel flat-tipped needle (0.41 mm inner diameter), inject the pre-prepared spinning solution into the syringe and fix it on the bracket of the electrospinning equipment, adjusting the needle to point towards the receiver. Turn on the constant temperature environment control system and maintain the temperature at 25℃. Connect the positive electrode to the needle and the negative electrode to the ground roller receiver, keeping the distance between the needle and the roller surface at 20 cm. Simultaneously start the roller to rotate at a constant speed of 300 rpm and apply a 17 kV DC voltage. After operation, start the micro-injection pump to advance the solution at a constant rate of 1 mL / h. During the experiment, observe the Taylor cone morphology and current stability in real time. After electrospinning, use a blade to cut the fiber membrane covered with tin foil, peel off the sample with toothless tweezers, and seal the nanofiber membrane in a sample bag for storage away from light.

[0066] Calcination: The collected nanofibers were placed in a crucible and calcined in a muffle furnace from ambient temperature to 800 ℃ at a heating rate of 5 ℃ / min for 3 h. After the muffle furnace cooled down, pure SiO2 nanofibers were obtained.

[0067] (3) Crosslinking of PEI and CS

[0068] 40 mg of calcined SiO2 nanofibers were shredded and transferred to 1 mL of deionized water. A SiO2 nanofiber suspension was obtained using a high-speed homogenizer. 80 mg of chitosan was dissolved in 4 mL of 0.35 mol / L acetic acid solution, and the SiO2 nanofiber suspension was added. The mixture was magnetically stirred for 30 minutes. When the mixture became homogeneous and viscous, 310 μL of epichlorohydrin and 2 mL of 4 wt% PEI solution were added, and stirring continued for 30 minutes until a homogeneous mixture was formed. The mixture was then placed in a 60°C oven for 4 hours to obtain high cross-linking stability. It was then pre-frozen at -20°C for 12 hours, and further freeze-dried at -80°C for 24 hours to obtain a porous sponge material.

[0069] 1.2.4 Characterization Analysis of Materials

[0070] (1) Fourier transform infrared spectroscopy analysis

[0071] To verify whether the material possesses amino groups and whether PEI and CS undergo chemical cross-linking, we performed infrared spectroscopy analysis on the material. First, dried potassium bromide powder was compressed into tablets using a tablet press, followed by blank background sampling. Then, 1 mg of the material was mixed with 100 mg of potassium bromide and ground into a fine powder in a mortar. After thorough mixing, the mixture was placed into a mold and compressed into tablets using a tablet press. Fourier transform infrared spectroscopy (FT-IR) was used to analyze the material in the range of 500–4000 cm⁻¹. −1 Within the wavenumber range, at 0.4 cm −1 The resolution is used to analyze the infrared spectrum of the sample.

[0072] (2) Scanning electron microscopy analysis

[0073] To observe the surface morphology and analyze pore size of the material, scanning electron microscopy (SEM) analysis was performed on the sample. Before measurement, the sample needed to be dried in a 60°C oven for 6 hours to prevent moisture from affecting the SEM observation. The scanning sample needed to be conductive; non-conductive samples required gold sputtering to ensure stability and clarity during observation. After gold sputtering, the sample was placed on the prepared stage, the vacuum pump was turned on, the electron gun was activated, and appropriate parameters were adjusted for observation.

[0074] 1.2.5 Adsorption capacity assessment

[0075] (1) pH optimization of adsorption conditions

[0076] To optimize adsorption conditions and investigate the optimal pH for nucleic acid adsorption, Tris-HCl buffer solutions of different pH values ​​were mixed with DNA solutions, setting a pH gradient of 4-8, and maintaining the DNA solution concentration at 300 ng / μL. For each experiment, 0.004 g of material and 200 μL of DNA solution were used. After adsorption for a period of time, the solution in the material was squeezed out, and the remaining solution was vortexed to mix it evenly. Finally, the concentration of the remaining nucleic acid was measured using a Nanodrop spectrophotometer. Each experiment was repeated three times. The adsorption capacity (1) and adsorption efficiency (2) at different pH values ​​were calculated using the following formulas.

[0077] Adsorption capacity:

[0078]

[0079] in and V0 and m are the final and initial DNA concentrations in the adsorption medium, respectively (ng / μL), V0 is the volume of the solution (mL), and m is the mass of the material (mg).

[0080] Adsorption efficiency:

[0081]

[0082] in and These represent the final and initial DNA concentrations (ng / μL) in the adsorption medium, respectively.

[0083] (2) pH optimization of elution conditions

[0084] The material was immersed in a 300 ng / μL DNA solution. After a period of time, the concentration of the remaining DNA in the solution was measured to ensure that the material had reached adsorption equilibrium. The material was washed twice with a washing buffer of pH=6. An elution buffer with a pH of 8-11 was prepared using Tris-HCl buffer. The material of each experiment was incubated in the elution buffer for 20 minutes, and the process was repeated 3 times. The concentration of nucleic acid in the solution was measured using a Nanodrop spectrophotometer, and the elution efficiency was calculated according to the following formula (3).

[0085] Elution efficiency:

[0086]

[0087] in and These represent the final and initial DNA concentrations (ng / μL) in the adsorption medium, respectively. This is the DNA concentration in the elution buffer (ng / μL).

[0088] (3) Adsorption time curve

[0089] 0.004 g of the material was immersed in a DNA solution with a pH of 6 and a concentration of 450 ng / μL. The concentration of the remaining nucleic acid was measured every 10 minutes over a period of 60 minutes. Subsequently, the concentration of the remaining nucleic acid was measured at 90 minutes and 120 minutes, and the adsorption capacity of the material was calculated. The trend of the adsorption-time curve was analyzed by plotting time on the x-axis and adsorption capacity on the y-axis.

[0090] (4) Determination of maximum adsorption capacity

[0091] To investigate the maximum capacity of the material for nucleic acid adsorption, a concentration of 200 ng / μL was prepared. 、 300 ng / μL 、 400ng / μL 、 0.004 g of material was immersed in RNA solutions of 500 ng / μL and 600 ng / μL. After adsorption for a period of time, the solution in the material was squeezed out, and the remaining solution was vortexed to mix it evenly. Finally, the concentration of the remaining nucleic acid was measured using a Nanodrop spectrophotometer, and the adsorption capacity and adsorption efficiency were calculated.

[0092] 2. Results and Discussion

[0093] 2.1 Optimization of preparation process and synthesis of materials

[0094] 2.1.1 Optimization of electrospinning process

[0095] Electrospinning was performed according to the process parameters in 1.2.3(2). The resulting nanofiber membrane was relatively thin, and some of it remained on the tin foil (e.g. Figure 2 As shown in A), this results in low yield.

[0096] To make the SiO2 nanofiber membrane easier to remove from the foil and improve yield, based on the influence of electrospinning parameters on membrane thickness, the present invention optimizes the process parameters in 1.2.3(2) as follows: First, the voltage parameter is optimized. Appropriately increasing the voltage can increase the deposition rate and density of the fibers, thereby increasing the membrane thickness. Therefore, we set the voltage to 20kV. In addition, we also appropriately shortened the distance between the needle and the receiver. Too far a distance may cause the fibers to be overstretched during flight, becoming thinner and thus reducing the membrane thickness. Therefore, we shortened the distance to 15cm.

[0097] Electrospinning was performed using the optimized process to obtain SiO2 nanofiber membranes (such as...). Figure 2 (As shown in B) The thickness is more uniform and it is easier to remove from the foil. The amount remaining on the foil (such as...) Figure 2 (As shown in C) fewer.

[0098] 2.1.2 Synthesis of PEI / CS / SiO2

[0099] The PEI / CS / SiO2 material prepared by this invention is as follows: Figure 3 (As shown). Through cutting and extrusion, the material exhibits a certain degree of elasticity, while its surface has numerous pores, indicating that the obtained PEI / CS / SiO2 possesses the physical properties of a sponge material, consistent with the expected material's physical characteristics.

[0100] 2.2 Characterization analysis of PEI / CS / SiO2

[0101] 2.2.1 Fourier Transform Infrared Spectroscopy Analysis

[0102] This invention successfully prepared PEI / CS / SiO2 materials using electrospinning, chemical crosslinking, and freeze-drying techniques. First, its surface structure was characterized using Fourier transform infrared spectroscopy (FT-IR), and the results are as follows: Figure 4 As shown. By analyzing the characteristic vibrational peaks, the chemical bonding information of each component in the composite system can be obtained. High wavenumber region (3000-3500 cm⁻¹) -1The broadband absorption characteristic peaks of PEI exhibit multi-component superposition, mainly due to the NH stretching vibrations of primary amines (-NH2) and secondary amines (-NH-) in the PEI molecular chain. Furthermore, at 1633 cm⁻¹... -1 The characteristic peak at this location corresponds to the in-plane bending vibration mode of NH. The presence of this peak confirms that no significant protonation or chemical transformation of the amino group occurred in the composite system, providing structural evidence for the binding of the amino group in PEI and CS to nucleic acids through electrostatic interactions. 1074 cm⁻¹ -1 The sharp absorption peak at the point exhibits a dual-component superposition characteristic, representing the antisymmetric stretching vibration of the COC ether bond, reflecting the crosslinking of PEI and CS under the action of the crosslinking agent epichlorohydrin. Simultaneously, it also represents the asymmetric stretching vibration of the Si-O-Si bond in the SiO2 network, indicating that SiO2 has been successfully introduced into the composite system as a framework. FT-IR spectroscopy results verify the successful preparation of the PEI / CS / SiO2 composite material.

[0103] 2.2.2 Scanning electron microscopy analysis

[0104] Figure 5 A shows the morphology of the material at 200 μm, revealing a high porosity and relatively uniform pore size, approximately 100 μm. During the freeze-drying stage, water molecules in the material freeze into ice crystals, while the solute is displaced into the gaps between the ice crystals, forming an isolated solid network. Subsequently, in the sublimation stage, the ice crystals sublimate directly into a gaseous state without passing through a liquid phase. The spaces previously occupied by the ice crystals become pores as the gas escapes, while the solute and solid framework remain as the skeleton of the porous structure. The abundant pores significantly increase the specific surface area of ​​the material, providing more active sites, making it easier for nucleic acid molecules to contact and be captured on the material surface.

[0105] Figure 5 B is a scanning electron microscope image of the material after adsorbing nucleic acids. At a scale of 2 μm, it can be observed that after adsorbing nucleic acids, many linear structures appear on the surface of some pores of the material. The linear parts are the nucleic acid molecules adsorbed by the material. These changes also prove that the material has a good adsorption capacity for nucleic acids.

[0106] 2.3 pH Optimization of PEI / CS / SiO2 Adsorption and Elution

[0107] like Figure 6As shown, the nucleic acid adsorption performance of this material in the pH range of 4-8 was investigated. Quantitative analysis was performed using batch adsorption experiments combined with a Nanodrop spectrophotometer. The results showed that the adsorption capacity and efficiency of the material exhibited a significant dependence on the solution pH. Specifically, the adsorption capacity showed a trend of first increasing and then decreasing with pH: it increased from pH 4-6, decreased from pH 6-8, and reached its highest value of 16495 μg / g at pH 6. The adsorption efficiency followed a similar trend, reaching its highest value of 98% at pH 6. This phenomenon is related to the charge state of the material surface. In a weakly acidic environment, the amino groups on the material surface carry a large amount of positive charge through protonation, which efficiently binds to negatively charged nucleic acid molecules (with a negatively charged phosphate backbone) through electrostatic attraction, significantly improving the adsorption efficiency. However, as the pH gradually increases, the concentration of hydroxyl ions in the solution increases, prompting the amino groups to undergo deprotonation, leading to a decrease in the positive charge density on the material surface and a weakening of the electrostatic attraction to nucleic acids. The experiment ultimately determined that Tris-HCl buffer solution with pH=6 was the optimal adsorption medium. This system can maintain the charge complementarity between the material and nucleic acid at the molecular level, thereby improving the nucleic acid extraction efficiency to near saturation and effectively enhancing the nucleic acid extraction effect.

[0108] like Figure 7 The figure shows the elution efficiency of the adsorbed nucleic acid material in Tris-HCl elution buffer with pH values ​​ranging from 8.0 to 11.0. The elution efficiency exhibits a clear pattern of first increasing and then decreasing with pH. Specifically, the elution efficiency increases significantly between pH 8.0 and 9.5, while as the pH increases and the alkalinity of the elution buffer increases, the elution efficiency shows a slow decreasing trend. The highest elution efficiency, approximately 21%, is reached at around pH 9.5. Under these conditions, the positive charge density on the material surface decreases, weakening the electrostatic attraction to nucleic acids. Using a buffer solution with pH 9.5 as the elution buffer and appropriately increasing the ionic strength of the elution buffer can effectively improve the elution efficiency.

[0109] 2.4 Adsorption time curve

[0110] To further investigate the effect of adsorption time on adsorption capacity, the concentration of remaining nucleic acid was measured using a Nanodrop spectrophotometer at certain time intervals, and the corresponding adsorption capacity was calculated. Figure 8As shown, the adsorption time curve is divided into two stages: a rising phase and a plateau phase. Specifically, within 30 minutes, the material's adsorption capacity increases significantly, reaching 22059 μg / g. After 30 minutes, the increase in adsorption capacity slows down, entering a plateau phase. This typical "rapid adsorption-gradual saturation" kinetic behavior can be reasonably explained by the Langmuir adsorption model: In the initial stage, due to the abundant surface active sites and strong adsorption driving forces (such as electrostatic potential difference), DNA molecules rapidly occupy high-affinity sites on the surface through diffusion; as the adsorption process progresses, the remaining sites gradually distribute into deep pores or low-energy binding sites, while the steric hindrance effect generated by the adsorbed DNA molecules inhibits the approach of subsequent molecules, leading to a significant decrease in the adsorption rate; when the surface adsorption and desorption rates reach dynamic equilibrium, the adsorption capacity exhibits a plateau characteristic.

[0111] 2.5 Determination of maximum adsorption capacity

[0112] like Figure 9 As shown, the adsorption efficiency of the material exhibits a partial negative correlation with the initial nucleic acid concentration, and a plateau phase is observed. When the nucleic acid concentration is between 200-400 ng / μL and 500-600 ng / μL, the adsorption efficiency decreases. However, at 400-500 ng / μL, the adsorption efficiency remains roughly the same, around 45%. This concentration-dependent phenomenon may stem from the charge neutralization effect on the material surface. When low-concentration nucleic acids (<400 ng / μL) come into contact with the material, the amino groups on the surface rapidly capture negatively charged nucleic acid molecules through electrostatic interactions. However, when the concentration exceeds 500 ng / μL, the active sites may be gradually neutralized by the negative charge of the nucleic acid phosphate backbone, leading to a sharp decrease in adsorption rate. Despite the decreased adsorption efficiency, the adsorption capacity of the material still shows a trend of first increasing and then decreasing with increasing nucleic acid concentration. When the nucleic acid concentration is below 500 ng / μL, the adsorption capacity of the material increases; above 500 ng / μL, it decreases. At an initial nucleic acid concentration of 500 ng / μL, the adsorption capacity reached 17693 μg / g. Compared to novel paper-based nucleic acid extraction materials for on-site testing, PEI / CS / SiO2 material, due to its excellent porosity and surface chemical properties (rich in amino groups), exhibits significantly higher adsorption capacity and is better suited for on-site testing.

[0113] 2.6 Adsorption Kinetics Analysis

[0114] To better understand the mechanism of nucleic acid adsorption in materials, the concentration of remaining nucleic acid was measured by varying the time, and the kinetics of nucleic acid adsorption were analyzed based on pseudo-first-order (4) and pseudo-second-order (5) kinetic models. Figure 3-7As shown, a rise and plateau phases are clearly observed during the material adsorption process. This is likely due to the abundance of adsorption sites on the material surface and the concentration gradient from the nucleic acid solution, which drive the process. The adsorption capacity then gradually decreases until equilibrium is reached after 30 minutes. This implies that the diffusion resistance of nucleic acid molecules on the material surface increases over time. With prolonged contact time, the number of active sites for nucleic acid molecules to be adsorbed also decreases. Finally, adsorption equilibrium is reached when all available adsorption sites are occupied. Analysis of the adsorption behavior using an intraparticle diffusion model reveals that adsorption is actually a coherent but segmented sequence. Specifically, the first stage is the adsorption and diffusion of substances on the surface of the adsorbent material; the second stage reveals the intraparticle diffusion process of substances entering the interior of the adsorbent material; and the final stage represents the relative equilibrium between adsorption and desorption, forming a dynamic equilibrium.

[0115] The quasi-first-order dynamic equation is:

[0116]

[0117] The quasi-second-order dynamic equation is:

[0118] in This indicates the adsorption capacity (mg / g) of the material when it reaches equilibrium with nucleic acid adsorption. represents the amount of material adsorbed within time t (mg / g); t represents time (min); k1 represents the rate constant of the quasi-first-order kinetic equation (min). -1 k2 represents the rate constant (g·mg) of the quasi-second-order kinetic equation. -1 ·min -1 ).

[0119] The experimental data were fitted using pseudo-first-order and pseudo-second-order dynamic models. The fitted images and dynamic parameters are shown below.

[0120] Table 2 Kinetic parameters of nucleic acid adsorption process of PEI / CS / SiO2

[0121]

[0122] contrast Figure 10 , Figure 11 Based on the linear fitting results and the kinetic parameter analysis in Table 2, the correlation coefficient of the pseudo-second-order kinetic model is higher than that of the pseudo-first-order kinetic model for the adsorption process of this material. This indicates that the pseudo-second-order kinetic model can better describe the adsorption behavior of PEI / CS / SiO2 material.

[0123] 3. Conclusion

[0124] This invention successfully prepared a PEI / CS / SiO2 material by crosslinking SiO2 electrospun fibers as a backbone with polyethyleneimine (PEI) and chitosan (CS). Characterization analysis confirmed the successful crosslinking of amino-rich PEI and CS, and the presence of relatively uniform pores on the material surface, significantly increasing the specific surface area and adsorption efficiency during nucleic acid adsorption. In evaluating the material's adsorption capacity for nucleic acids, Tris-HCl buffer at pH 6 was determined to be the optimal adsorption medium, and Tris-HCl buffer at pH 9.5 was the optimal elution medium. Furthermore, the material rapidly reached adsorption equilibrium within 30 minutes, with a maximum adsorption capacity of 17693 μg / g, significantly superior to traditional nucleic acid adsorption materials.

[0125] Based on a comprehensive analysis of various indicators, PEI / CS / SiO2 materials exhibit significant advantages, including greater adsorption capacity, higher extraction efficiency, and wider application scenarios. This study combines electrospinning, chemical cross-linking, and freeze-drying techniques to achieve more comprehensive material performance. This not only provides new ideas and methods for the development and application of nucleic acid extraction materials but also offers reliable experimental data and theoretical basis for more comprehensive optimization and innovation of existing nucleic acid extraction methods.

[0126] Comparative Example 1

[0127] Based on the original Example 1, without the crosslinking agent epichlorohydrin. After the mixture was mixed evenly, it was placed in an oven at 60°C for 4 hours, then transferred to a freezer at -20°C for pre-freezing for 12 hours, and then further freeze-dried at -80°C for 24 hours to obtain the porous gel material PEI / CS / SiO2 (without ECH).

[0128] Comparative Example 2

[0129] Based on the original Example 1, PEI was not added. After the mixture was mixed evenly, it was transferred to an oven at 60°C and placed for 4 hours, then transferred to a freezer at -20°C for pre-freezing for 12 hours, and then further freeze-dried at -80°C for 24 hours to obtain the porous gel material CS / SiO2.

[0130] The maximum nucleic acid adsorption capacity of the above-mentioned PEI / CS / SiO2 (without ECH) and CS / SiO2 gel materials was determined according to the steps in Example 1. The results are as follows. Figure 12 As shown.

[0131] Simultaneously, scanning electron microscopy was used to observe and analyze the surface morphology and pore size of PEI / CS / SiO2 (without ECH) and CS / SiO2 gel materials. Figure 13 , 14The images show the morphological characteristics of PEI / CS / SiO2 (without ECH) and CS / SiO2 gels at 500 μm and 200 μm, respectively. Figure 13 It can be seen that the gel is relatively loose, exhibiting an overall sheet-like and layered structure. Lacking the cross-linking agent ECH, CS and PEI cannot cross-link to form a porous network structure, resulting in poor overall elasticity and mechanical properties of the gel, and low nucleic acid adsorption capacity. However, due to… Figure 14 It can be seen that CS can crosslink to form a porous network structure under the action of ECH. However, the electron microscopy characterization results show that the pore size of the gel prepared by adding only CS is larger, and the nucleic acid adsorption effect is not as good as that of the gel with both CS and PEI added.

Claims

1. A method for preparing an ultralight electrospun fiber sponge material with high nucleic acid adsorption, characterized in that, Includes the following steps: (1) SiO2 sol solution: Add a certain mass ratio of TEOS and H3PO4 to deionized water, stir magnetically for 12 h to obtain SiO2 sol solution; (2) PVA solution: Dissolve PVA powder in deionized water and stir magnetically to obtain a PVA solution; (3) Spinning solution: SiO2 sol solution and PVA solution are mixed and magnetically stirred to obtain spinning solution; (4) Electrospinning the spinning solution to obtain nanofibers, and calcining the spun nanofibers in a muffle furnace to obtain SiO2 nanofibers. (5) Cut the SiO2 nanofibers into small pieces, transfer them to deionized water, and homogenize them to obtain a SiO2 nano suspension; add the SiO2 nano suspension to the chitosan solution and stir magnetically until uniform; then add epichlorohydrin and PEI solution and continue stirring until a uniform mixture is formed; then heat the mixture, pre-freeze the product, and freeze-dry it to obtain a porous sponge material. In step (5), the concentration of the SiO2 nano-suspension is 30-50 mg / mL; the chitosan solution is prepared by dissolving 40-120 mg of chitosan in 4 mL of acetic acid solution to obtain a chitosan solution concentration of 1-3 wt%; the concentration of the PEI solution is 3-5 wt%; the mass ratio of SiO2 nanofibers to chitosan is 1:4-5:4; the mass ratio of chitosan to PEI is 2:1-2:5; and the amount of epichlorohydrin added is 200-400 μL.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of TEOS, H3PO4 and deionized water is 1-2:0.025:

8.

3. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the PVA solution is 5~15 wt%.

4. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of the SiO2 sol solution to the PVA solution is 2:1 to 1:1, and the mixing and stirring time is 7-9 hours.

5. The preparation method according to claim 1, characterized in that, In step (4), the specific process of electrospinning is as follows: Take a syringe, assemble a 20~22G stainless steel flat-mouth needle with an inner diameter of 0.61~0.41 mm, inject the spinning solution into the syringe and fix it on the spinneret; turn on the constant temperature environment control system and maintain the temperature at 25℃; connect the positive electrode to the needle and the negative electrode to the roller receiver, keep the distance between the needle and the roller surface at 15~20 cm, and start the roller to rotate at a constant speed of 200~300 rpm / min and apply a DC voltage of 17~20 kV; after operation, start the micro-injection pump and push the solution at a constant rate of 0.8~1.2 mL / h; then place the obtained spun membrane in a muffle furnace for calcination, and calcinate it from the ambient temperature to 600-800 ℃ at a heating rate of 5 ℃ / min for 2-4 h.

6. The preparation method according to claim 1 or 5, characterized in that, In step (5), the magnetic stirring time is 20-40 min; the stirring time is 20-40 min; the heating is placed at 50-60℃ for 4-5 h; the pre-freezing temperature is pre-freezing at -20℃ for 8-16 h; and the freeze drying is drying at -60~-80℃ for 12-24 h.

7. The application of a sponge material prepared by the preparation method according to any one of claims 1-6 in the efficient extraction of nucleic acids.

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